Vanderbilt Institute of Chemical Biology

 

 






2015 VICB
Student Research
Symposium

 

 

 

 

 

2015 VICB Student Research Symposium

 



Photographs

 

Click here to view photographs of the 2015 VICB Student Symposium held August 13th at Cheekwood's Botanical Hall!


Symposium Summary

 

The 11th Annual VICB Student Research Symposium was held on August 13th at Cheekwood’s Botanical Hall. Students from Vanderbilt and other universities (Notre Dame, Minnesota, Wisconsin, Indiana, Northwestern, Kentucky, Illinois, and Brown) participated in a full day of oral presentations and poster sessions.

 

VICB Director and Associate Vice Chancellor for Research Larry Marnett kicked off the day by presenting Nichole Lareau (McLean Lab) with the inaugural Richard N. Armstrong Prize in Chemical Biology. Wilfred van der Donk, University of Illinois-Urbana/Champaign, then presented a keynote talk on the “Evolution of peptide dehydratases involved in natural product biosynthesis.”

 

Awards for top oral presentations went to Connor Lamberson (Porter Lab), Clifford Gee (University of Minnesota), and Jessica Moore (Skaar Lab). Poster award recipients included Jonathan Hempel (Hong Lab), Charles Williams (Hong Lab), Allison Eberly (Hadjifrangiskou Lab), Daniel Sprague (Johnston Lab), Matthew Surdel (Skaar Lab), and Jaime Wenke (Schey Lab). The iPad Air 2 raffle prize winner was Diana Chavez (Eichman Lab).

 

Special recognition goes out to the CBAS (Chemical Biology Association of Students) Organizing Committee members and others who planned this event: Kim Fong, Jeannie Camarillo, James Galligan, Jennifer Benoy, James Poland, Thomas Struble, and Sarah Stow, Reese Knippel, Jacob Choby, and Wes Bauer.

 

Guest Speaker


Wilfred van der Donk
University of Illinois-Urbana/Champaign

 

 

 

Awards

 

Richard N. Armstrong Prize in Chemical Biology:

Nichole M. Lareau (view larger image)

 

 

 

 

Oral Presentation Awards (view larger image)


 

 

 

Poster Awards (view larger image)

 

 

Agenda

 

MORNING SESSION

Coffee, Poster Hanging
8:00 A.M. – 8:45 A.M.
Welcome:
8:45 A.M. – 9:00 A.M.

 

Keynote Speaker
9:00 A.M. – 10:00 A.M.

Wilfred van der Donk: "Evolution of peptide dehydratases involved in natural product biosynthesis"

 

 

Poster Session A
10:00 A.M. – 11:00 A.M.

 

Oral Presentations
11:00 A.M. – 12:00 P.M.
Erin Shockley
Michelle Mitchener
Jessica Moore

 

Lunch/Poster Viewing
12:00 P.M. – 1:00 P.M.

 

AFTERNOON SESSION

 

Oral Presentations
1:00 P.M. - 2:00 P.M.
Joseph Zackular
Connor Lamberson
Clifford Gee, U of Minnesota

 

Poster Session B
2:00 P.M. – 3:00 P.M.

Oral Presentations
3:00 P.M. - 4:00 P.M.
Laura Woods, U of Notre Dame
Nichole Lareau, VICB Prize winner

 

Reception, Student Awards & Raffle Prizes
(must be present to win)
4:00 P.M. – 5:00 P.M.


 

Raffle Prize

 

iPad Air 2 16 GB

Winner: Diana Chavez (view larger image)


 

 

Contact

 

For additional information, contact the VICB office at 322-0907.


 
 2015 Keynote Video 2015 Oral Presentations 2015 Poster Session

 

 

Oral & Poster Presentations Abstracts

 

Oral Presentation Abtracts

 

1. "Efficiently Fitting Signaling Models to Experimental Data," Erin M. Shockley, Michelle M. Mitchener, James C. Pino, Lawrence J. Marnett, Carlos F. Lopez


The ErbB family of receptors bind multiple ligands and transmit signals involved in proliferation, growth, and survival. Dysregulation of ErbB signaling occurs frequently in cancer and is commonly pharmaceutically targeted. Since experimentally testing all combination drug therapies would be a monumental task, testing these systems computationally to probe network response to multiple perturbations is an attractive prospect. I have encoded ErbB-driven proliferative, apoptotic, and crosstalk signaling events as a large (~1500 chemical species) model using the mass-action kinetics formalism. However, both the size of such a model and the large number of experimental measurements available for model calibration outpace the currently available model calibration algorithms. To overcome this limitation I have developed and implemented novel calibration methods to efficiently fit large models to high-throughput experimental data within a high-performance computing environment. I present the successful application of the Differential Evolution Adaptive Metropolis (DREAM) algorithm to both a relatively simple model of COX-2 enzyme substrate interactions and a more complex model of extrinsic apoptosis signaling. In both cases, DREAM makes it possible to quickly determine distributions of kinetic rates consistent with experimental data; this paves the way for future calibration of the large ErbB apoptosis-proliferation model.

 

2. "Competition and Allostery Govern Substrate Selectivity of Cyclooxygenase-2," Michelle Mitchener, Daniel J. Hermanson, Erin M. Shockley, H. Alex Brown, Craig W. Lindsley, Jeff Reese, Carol A. Rouzer, Carlos F. Lopez, and Lawrence J. Marnett

The cyclooxygenase enzymes perform the initial steps in the synthesis of a wide array of potent signaling molecules with diverse physiological functions. Cyclooxygenase-2 (COX-2) can oxygenate both arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. While the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages and bone marrow-derived macrophages by complementary chemical, pharmacologic, or genetic techniques uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based analysis of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG whereas 2-AG binding at the allosteric site increases enzyme efficiency toward AA. These results provide the first evidence of fatty acid-based modulation of COX-2 activity in a cellular setting and demonstrate the complexity of the interaction of multiple substrates with COX-2 that leads to preferential oxygenation of AA.

 

3. "MALDI Imaging Mass Spectrometry: Providing Molecular Insight at the Pathogen-Host Interface," Jessica L. Moore, Neal D. Hammer, Jeffrey M. Spraggins, Boone M. Prentice, Jeremy L. Norris, Richard M. Caprioli, Eric P. Skaar


Bacterial pathogens are gaining resistance to existing antimicrobial therapeutics at an alarming rate, undermining the ability of modern medicine to treat microbial infections.  In light of this, the need to identify and to study novel targets for antimicrobial intervention using emerging analytical technologies is paramount for the future successful treatment of infectious diseases.

Imaging Mass Spectrometry (IMS) is an analytical technology that provides molecular information directly from samples while preserving spatial information. As analytical instrumentation has advanced, high spatial and high mass resolution IMS has emerged. High spatial resolution IMS, when applied to infected tissue, provides molecular information at the pathogen-host interface – the exact location where host immune proteins encounter the invading bacterial pathogens.  This provides multiplexed molecular definition of histological features without a priori knowledge of analytes.

High mass resolution IMS using a MALDI FTICR MS allows for isotopic resolution of protein species and post-translationally modified (PTM) epitopes in infectious lesions. In pathogenesis, this allows not only for the determination of what PTMs are present but also the elucidation of their location within a lesion. Such analyses enable the study of infectious diseases with unprecedented resolution.

 

4. "Dietary Zinc Alters the Gut Microbiome and Increases Susceptibility to Clostridium difficile Infection," Joseph P. Zackular, Jessica Moore, Lillian Juttukonda, Ashley T. Jordan, and Eric P. Skaar

 

Clostridium difficile is a gram-positive, spore-forming bacterium that infects the colon, causing a wide range of disorders that vary in severity from mild diarrhea to fulminant colitis and/or death. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased significantly. Difficulties in treating infections with conventional antibiotics, increasing rates of recurrent infection, and the emergence of hyper-virulent strains underscore the need for investigating new preventative and therapeutic strategies. Initiation of CDIis facilitated by disruption of the gut microbiome, most commonly mediated by antimicrobial treatment, which enables C. difficile colonization and outgrowth. However, the rate of non-antibiotic associated CDI cases are well documented and rapidly on the rise. This suggests that unexplored environmental, nutrient, and genetic factors likely influence the gut microbiome and susceptibility to CDI.  


During infection, access to nutrient metals profoundly impacts bacterial replication and virulence factor production. To exploit this, vertebrates produce factors that limit metal availability in a process termed nutritional immunity. One such protein factor, calprotectin (CP), has strong antimicrobial properties mediated by its ability to tightly bind zinc (Zn) and manganese. Surprisingly, little work has been done to characterize the contribution of CP-mediated Zn starvation during CDI. Another factor likely impacting Zn availability during infection is diet. Altered dietary Zn levels have been associated with decreased immune system function and increased susceptibility to various infections; however, there is a paucity of data on how altered dietary metal levels affect the gut microbiome. Furthermore, the impact of dietary Zn on susceptibility to CDI has yet to be elucidated. We hypothesized that (i) alterations in dietary Zn levels profoundly impact the composition of the gut microbiome and this remodeling affects susceptibility to C. difficile, (ii) CP-mediated metal sequestration is essential for limiting growth, pathogenesis, and persistence of C. difficile. To test this, we custom synthesized diets with altered levels of Zn and examined their impact on the gut microbiome and CDI. We observed that increased levels of dietary Zn significantly altered structure and diversity of the gut microbiome, while other metals had no effect. Utilizing a murine model of CDI, we next demonstrated that high levels of dietary Zn significantly reduced the threshold for antibiotic-dependent susceptibility to C. difficile. Mice fed diets with low or normal levels of Zn were completely resistant to CDI following low-level antibiotic treatment, however mice fed a diet high in Zn became highly susceptible. Furthermore, mice fed a high Zn diet showed increased C. difficile associated disease, with significant pseudomembranous colitis, epithelial damage, and inflammation. Next, to examine the impact of host-mediated metal limitation during CDI, we infected CP-deficient mice with C. difficile. Mice lacking CP showed a significant decrease in survival and an increase in disease pathology. Remarkably, this increase in disease was largely ablated when CP-deficient mice were fed a diet lacking Zn, strongly suggesting that CP-mediated Zn restriction is essential for combating CDI. Together, these data demonstrate that dietary Zn is an important mediator of gut microbiome community structure and Zn profoundly impacts susceptibility to CDI . Additionally, these data have revealed an essential role for CP-mediated Zn limitation during CDI. These discoveries will lay the groundwork for the development of novel preventative and therapeutics strategies for CDI.

 

5. "Unusual Kinetic Isotope Effects of Deuterium Reinforced Polyunsaturated Fatty Acids in Tocopherol-mediated Free Radical Chain Oxidations," C. Lamberson, L. Xu, J. R. Montenegro-Burke, H. Muchalski, V. Shmanai, A. Bekish, J. McLean, C. Clarke, M. Shchepinov, N. Porter

           
The bis-allylic –CH2- groups in polyunsaturated fatty acids (PUFAs) are susceptible to hydrogen atom abstraction, making PUFAs prone to free radical-mediated peroxidation.  A recent strategy to diminish lipid peroxidation in vivo is based on substituting bis-allylic hydrogen atoms with deuterium.  Previous studies have shown these deuterated PUFAs (D-PUFAs) undergo peroxidation in solution with propagation rate constants some 10-fold less than the natural fatty acids.  This isotope effect falls outside of the range of kH/kD (<7) which has been reported for other H(D) atom transfers from carbon to peroxyl radicals.  Recently, we have carried out tocopherol-mediated oxidations of several D-PUFAs and measured isotope effects ranging from 23 to 36 (substrate dependent) using LC-MS and HRMS techniques.  The H-atom transfer from the bis-allylic –CH2- center to the tocopheryl radical is the rate-determining step in tocopherol-mediated peroxidation (TMP) of lipids in human low-density lipoproteins (LDL), a process which has been linked to coronary artery disease.  These unexpectedly large kinetic isotope effects for the tocopherol-mediated oxidation of linoleic and linolenic acid suggest that H-atom tunneling makes this process favorable and plays a significant role in the oxidative modification of human LDL.

 

6. "Protein Observed 19F NMR Spectroscopy: An Effective Molecular Discovery Approach for Protein Targets," Clifford Gee, William C.K. Pomerantz, and Gunda I. Georg, University of Minnesota

 

Protein-protein interactions (PPIs) play a key role in many biological processes. The ability to selectively and effectively target these interactions is important due to the links between their dysregulation and various disease states including cancer and neurological disorders. PPIs have historically been considered to be “undruggable.” However, recent discoveries have challenged this assertion. Screening of low-complexity molecules, termed fragments, has emerged as an effective small molecule discovery strategy for targeting such interactions. Herein we describe the use of protein-observed 19F (PrOF) NMR as a powerful screening tool for PPIs, using the KIX binding domain of the transcriptional coactivator CREB Binding Protein (CBP) as a model system.

PrOF NMR possesses several advantages as a biophysical characterization tool including its ability to detect small changes in chemical environment, consistent with ligand binding, and provide useful protein structural information. In addition, PrOF NMR mitigates several challenges inherent in other biophysical techniques based on its tolerance to a wide range of solution conditions and additives while effectively detecting low-affinity binders. While PrOF NMR has been used to study several proteins, our lab was the first to utilize it in a fragment screening capacity. From screening 508 fragments against KIX, four molecules were verified as hits. Through studying the structure-activity relationships (SAR) of these molecules, three additional ligands were identified, including the commercially available drug flurbiprofen. These aryl-acetic and phenyl-acetic acid-containing compounds represent a new class of KIX ligands to be developed. Efforts to optimize and develop these compounds into high affinity ligands are underway. Simultaneously, a second ligand discovery project is being pursued for BrdT, a member of the BET family of bromodomains and a newly identified protein target for male contraception, as further demonstration of the utility and efficacy of PrOF NMR for aiding in molecular discovery efforts.

 

7. "Combinatorial Therapy with Epothilone and Aurora Kinase Inhibitors Induces a Novel Form of Cell Death," Woods, Laura M., Taylor, Richard E., Vaughan, Kevin, University of Notre Dame

 

Microtubule stabilizing agents (MSAs) are an important class of therapeutic compounds used for the treatment of multiple cancers. Subtle differences in the affect of MSAs can provide an opportunity to increase the susceptibility of specific protein targets critical to the cell cycle.
The epothilones are a relatively new class of microtubule-stabilizing agents effective for the treatment of Taxol® resistant breast cancer and the basis for the drug, Ixempra®. Using live cell analysis, we have shown that epothilones are effective at arresting cell division specifically at prometaphase. At this same stage of cell division, Aurora B kinases are involved in chromosomal separation and inhibition of these kinases causes a frozen phenotype. When combining epothilone and Aurora kinase inhibitors (AKI), we observed a highly-penetrant mitotic defect for inhibitors of AurB that induced cell death. This defect was distinct from those caused by treatment with epothilone or AKI alone. Studies examining this new model in which the combination of epothilone and AKI shifts the specificity of Aurora B from prometaphase substrates to telophase substrates will be presented. This outcome for combinatorial drug treatment provides a novel mechanism of action with broad potential.

 

8. Richard N. Armstrong Award in Chemical Biology Prize Winner! "Development of Ion Mobility and Mass Spectrometry Strategies in Support of Integrated Omics and Systems Biology," Nichole M. Lareau, Sarah M. Stow, Jody C. May, John A. McLean

 

Systems biology greatly enhances the study of complex biological processes by expanding on traditional reductionist approaches, where individual components are targeted (i.e. glycomics).  Systems biology strategies allow for the comprehensive analysis of biological samples as a whole.  To support these systems analyses strategies, we have developed ion mobility-mass spectrometry (IM-MS) techniques to study biological systems in the gas phase through class specific structural separations. Proteins, lipids, and carbohydrates, which exhibit overlapping signals in a 1-D mass spectrum, are separated in IM-MS because each biomolecular class occupies a unique region of conformational space. Thus, IM-MS analysis is able to differentiate molecules present in complex biological samples with minimal sample purification, which greatly improves upon current methodologies. IM-MS provides broad scale biological structural descriptors, which can be further honed to describe subclass and multiclass descriptors.

 

High throughput chip-based HPLC-IM-MS technologiesare well suited for metabolite analysis and lead target prioritization. As many metabolites of interest are decorated with carbohydrate and peptide moieties, parallel studies aim to develop technologies to address the challenges associated with the analysis of these moieties. Here, we describe a new LC-IM-MS method to simultaneously analyze carbohydrates and peptidic molecules. Novel sequencing workflows were developed that utilize fragmentation techniques to obtain finer structural detail of glycoconjugate and glycoprotein complexes. The IM-MS configuration allows for radical driven fragmentation by electron transfer dissociation (ETD) and subsequent vibrational activated collision induced dissociation (CID) fragmentation as complementary techniques in support of structural assignment.  These techniques were applied to a carcinoembryonic antigen and show great promise as a more comprehensive sequencing strategy. Combining broad and fine structural studies in this manner creates a toolbox for extensive analysis of metabolomics, glycomics and more generally, integrated omics at large.

 

 

Poster Presentation Abstracts

 

1. "Bridging the gap between target-based and phenotypic screens to understand drug mechanism of action against Plasmodium falciparum," Kim Fong

 

Malaria is an entirely preventable and treatable disease, yet it is endemic in nearly half of the countries around the world.  One of the most important drug targets for this disease is the hemozoin formation pathway, a heme detoxification process found in the malaria parasite, Plasmodium falciparum.  In the search for novel hemozoin inhibitors, we developed an in vitro target-based assay that closely mimics the parasiteenvironment and tested established antimalarials including chloroquine, pyrimethamine, artemisinin, and atovaquone, each with a unique mechanism of action.  These drugs were then tested in a secondary phenotypic screen for antiplasmodial activity against drug-sensitive and –resistant strains of P. falciparum.  Despite the results from both the target-based assay and phenotypic assay, there is a disconnect in fully understanding the in vivo mechanism of action and predicting the unexpected biology of a particular drug candidate.  Therefore, we developed an assay to validate this pathway through analysis of the three types of parasitic heme: hemoglobin, intracellular free heme, and hemozoin.  Chloroquine, a known hemozoin inhibitor, exhibited a rise in free intravacuolar heme levels with increasing drug concentration.  This correlated with decreased levels of hemozoin and parasite survival.  The other tested antimalarials resulted in unchanged levels of free heme, confirming that they, in fact, target alternative modes of action within the parasite.  Combining these screens with flow cytometry, we can further understand mechanism of action through determining the life cycle stage following treatment.  In drug discovery, it is not only important to find molecules that possess in vitro antimalarial activity, but also to understand the mechanism of the drug target pathway within a parasite culture in order to aid in optimization of lead compounds, ultimately allowing for greater success. 

 

2. "Discovery and Development of EGM1, a Downstream of Smoothened Hedgehog Signaling Inhibitor," Jonathan Hempel

 

The Hedgehog (Hh) signaling pathway is essential for vertebrate developmental patterning, but its mutation-driven dysregulation is recognized as an essential driver of basal cell carcinoma, medulloblastoma, and numerous other cancers. Thus, the FDA approval of the Smoothened (Smo) antagonist vismodegib for treatment of advanced basal cell carcinoma represented an important step forward in the treatment of Hh-dependent malignancies; however, downstream somatic mutations and inhibitor-driven resistance have limited its widespread clinical implementation. Therefore, identification of downstream of Smo Hh inhibitors represents the next frontier for treatment of Hh-driven cancers. An unbiased zebrafish in vivo chemical genetic screen for small molecule developmental patterning modulators identified EGM1, which phenocopied the loss of Hh zebrafish mutant. In vitro, EGM1 inhibited Hh target gene transcription downstream of Smo and epistatic to the Gli transcription factor regulator Suppressor of Fused (Sufu). Thus, phenotypic hit to lead development utilized a cell-based Hh gene transcription readout for Hh pathway inhibition, and secondary evaluation of pathway activity in Sufu-null cells was employed to confirm mode of action retention. To be presented are the results of our efforts to identify a lead EGM1 analog for PK/PD optimization and in vivo evaluation in the context of Hh-dependent malignancies.

 

3. “Nanoelectrode Array Designed to Monitor Cellular Bioenergetics and Evaporation for Organ-on-a-Chip Systems," Anna N. Davis, John P. Wikswo, David E. Cliffel

 

Organ-on-a-chip (OoC) systems are designed to more realistically mimic in vivo cellular responses than traditional two dimensional tissue culturing platforms (such as well plates and culture flasks). The Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE), in collaboration with other institutions across the country, have been working to develop different OoCs to monitor responses to pharmaceuticals or environmental toxins. For meaningful information to be determined from long term studies on OoCs, the health of the cells needs to be monitored to observe real-time toxicological events and ensure the viability of additional results produced. Previous work has been done in the Cliffel lab using modified screen printed electrodes in a 26µL chamber to detect changes in cellular glucose metabolism; however, the volume of effluent produced from smaller OoCs require lower sample volumes for real-time analysis of cellular energetics. Additionally, gas permeable materials used to produce OoCs introduce the possibility of evaporation from circulating media, which could result in hypertonic stress causing damage to cellular processes or apoptosis. As water evaporates from the media, the concentrations of ions increase, resulting in increased solution conductivity which can be measured using electrochemical impedance techniques. A nanoelectrode array has been fabricated using electron beam deposition and soft lithography and modified with electrodeposition and ink-jet printing techniques. The modified sensor was utilized to perform electrochemical detection of glucose, lactate, oxygen, conductivity and acidification in a submicroliter multichannel PDMS sample chamber.  Once developed the nanoelectrode array will be used to make automated offline measurements from small volume OoCs.

 

4. "Photosensitization by a small molecule activator of Gram-positive coproporphyrinogen oxidase," Matthew C. Surdel, Lisa J. Lojek, Brendan F. Dutter, Devin L. Stauff, Gary A. Sulikowski, and Eric P. Skaar

Staphylococcus aureus is a pathogen contributing to significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for numerous critical processes. To satisfy these requirements, S. aureus imports host heme through dedicated systems, while retaining the ability to synthesize heme de novo. Although heme is essential for growth, excess heme is toxic. S. aureus utilizes a two component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for pathogenesis, yet the mechanism of heme sensing remains unknown. Small molecule ‘8882 was identified in a high-throughput screen as an activator of staphylococcal HssRS. ‘8882 increases endogenous heme synthesis through a previously unidentified mechanism. By utilizing a suicide strain containing a Phrt-driven relE construct, we identified suppressor mutations preventing sensing of ‘8882 and heme. This uncovered numerous residues within HssRS and the hrt promoter required for hrtAB activation. Importantly, three strains have been identified that are unresponsive to ‘8882 and do not contain mutations in hssRS or Phrt.  Whole genome sequencing and follow up studies have identified that ‘8882 specifically interacts with HemY, an enzyme involved in the terminal steps of heme biosynthesis.  Recently it has been uncovered that Gram-positive pathogens utilize a distinct heme biosynthesis pathway in which HemY is a coproporphyrinogen III oxidase, as opposed to the classical protoporphyrinogen IX oxidase. These findings implicate Gram-positive HemY as a new potential drug target for antibacterial strategies. Importantly, accumulation of the product of HemY, coproporphyrin III, sensitizes bacteria to light. In keeping with this, we have shown that ‘8882 induces photosensitization in S. aureus, and therefore represents a lead compound for the development of light-based antimicrobial therapies. As HemY is conserved in Gram-positive pathogens, ‘8882 has been used to induce photosensitivity in many pathogens of the skin, including P. acnes, S. epidermidis and B. anthracis. Taken together, these data identify activators of HemY as a new strategy for the development of antimicrobial therapies.

5. "MALDI imaging mass spectrometry reveals age-related deamidation and truncation of human lens insoluble proteins," Jamie L Wenke, Kristie L Rose, Jeffrey M Spraggins, Kevin L Schey
Introduction
The human lens contains long-lived proteins that are extensively modified with age. For example, the water channel Aquaporin 0 (AQP0) is truncated, lipidated, and deamidated over time; modifications that may affect water permeability. Understanding the spatial distribution of modified forms of AQP0 can shed light on its function in the aging lens. MALDI imaging mass spectrometry (MALDI IMS) has provided valuable insight into the distribution of AQP0 and other lens proteins. However, resolving small mass shifts due to deamidation can be challenging at the intact protein level. We performed in situ digestion directly on lens sections to generate spatially-localized tryptic peptides. Using a high mass resolution FTICR instrument, we imaged deamidated and truncated peptides of insoluble proteins in human lenses.
Methods
Human lenses were cryosectioned to 20µm thickness and methanol soft-landed onto slides. Tissue was washed with water and 1% formic acid to remove soluble proteins. Sinapinic acid matrix was applied by Portrait acoustic spotter (Labcyte Inc.) at 200µm final spacing. Protein images were acquired on a Bruker Autoflex Speed TOF. For peptide imaging, tissue was sprayed with trypsin via TM sprayer (HTX Technologies, LLC) and incubated in a humidified chamber for in situ digestion. CHCA matrix was sprayed via TM sprayer before analysis on a Bruker SolariX 15T FTICR. For peptide identification, microextraction was performed using 20% ACN using a gel-loading pipet tip. Extracts were analyzed by LC-MS/MS on a Thermo OrbiVelos and data searched against a human protein database.
Preliminary data
In the lens, new fiber cells are added to the outer cortex atop older existing fiber cells. Thus, cell age can be inferred by distance into the lens. By tailoring tissue washes to remove soluble proteins, we could detect membrane proteins as well as other proteins that become insoluble over time. Intact protein MALDI images of washed human lenses (4 month to 56 year) show progressive C-terminal truncation of the membrane protein AQP0 in all but the youngest lens. Lipid-modified AQP0 is localized to the lens cortex, suggesting lipid modification occurs in young fiber cells.
In situ digestion enabled analysis of small m/z posttranslational modifications on peptides from insoluble proteins. The C-terminal tryptic peptide of AQP0 (239-263, m/z 2550.241), which contains residues critical for protein function, can be deamidated at two asparagine residues. Deamidation is a common protein modification, considered a molecular clock that reflects protein age. This small mass shift from asparagine to aspartic acid (+0.9847 Da) can be resolved from the first 13C isotope (+1.0034 Da) by FTICR mass spectrometry. The undeamidated 239-263 AQP0 peptide was only detected in the outer cortex of a 4 month human lens, whereas singly and doubly deamidated peptides were localized to older fiber cells. Overlaid ion images suggest that deamidation occurs before truncation at residue 259. These unprecedented MALDI images show rapid and progressive deamidation of AQP0. Furthermore, we detected multiple crystallin proteins that become increasingly modified and therefore less soluble with age. Older lens fiber cells contained mostly deamidated αA crystallin and βB2 crystallin peptides, while these undeamidated peptides were only detected in the lens outer cortex. This work highlights the power of high mass resolution FTICR instruments for imaging age-related changes in biological tissues. Future work will include cataract lens imaging to investigate insoluble proteins and posttranslational modifications.
Novel aspect
In situ digestion and high mass resolution FTICR mass spectrometry for imaging deamidated tryptic peptides in tissue sections.

 

6. "Dietary Zinc Alters the Gut Microbiome and Increases Susceptibility to Clostridium difficile Infection," Joseph P. Zachular, Jessica Moore, Lillian Juttukonda, Ashley T. Jordan, and Eric P. Skaar

 

Clostridium difficile is a gram-positive, spore-forming bacterium that infects the colon, causing a wide range of disorders that vary in severity from mild diarrhea to fulminant colitis and/or death. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased significantly. Difficulties in treating infections with conventional antibiotics, increasing rates of recurrent infection, and the emergence of hyper-virulent strains underscore the need for investigating new preventative and therapeutic strategies. Initiation of CDIis facilitated by disruption of the gut microbiome, most commonly mediated by antimicrobial treatment, which enables C. difficile colonization and outgrowth. However, the rate of non-antibiotic associated CDI cases are well documented and rapidly on the rise. This suggests that unexplored environmental, nutrient, and genetic factors likely influence the gut microbiome and susceptibility to CDI. During infection, access to nutrient metals profoundly impacts bacterial replication and virulence factor production. To exploit this, vertebrates produce factors that limit metal availability in a process termed nutritional immunity. One such protein factor, calprotectin (CP), has strong antimicrobial properties mediated by its ability to tightly bind zinc (Zn) and manganese. Surprisingly, little work has been done to characterize the contribution of CP-mediated Zn starvation during CDI. Another factor likely impacting Zn availability during infection is diet. Altered dietary Zn levels have been associated with decreased immune system function and increased susceptibility to various infections; however, there is a paucity of data on how altered dietary metal levels affect the gut microbiome. Furthermore, the impact of dietary Zn on susceptibility to CDI has yet to be elucidated. We hypothesized that (i) alterations in dietary Zn levels profoundly impact the composition of the gut microbiome and this remodeling affects susceptibility to C. difficile, (ii) CP-mediated metal sequestration is essential for limiting growth, pathogenesis, and persistence of C. difficile. To test this, we custom synthesized diets with altered levels of Zn and examined their impact on the gut microbiome and CDI. We observed that increased levels of dietary Zn significantly altered structure and diversity of the gut microbiome, while other metals had no effect. Utilizing a murine model of CDI, we next demonstrated that high levels of dietary Zn significantly reduced the threshold for antibiotic-dependent susceptibility to C. difficile. Mice fed diets with low or normal levels of Zn were completely resistant to CDI following low-level antibiotic treatment, however mice fed a diet high in Zn became highly susceptible. Furthermore, mice fed a high Zn diet showed increased C. difficile associated disease, with significant pseudomembranous colitis, epithelial damage, and inflammation. Next, to examine the impact of host-mediated metal limitation during CDI, we infected CP-deficient mice with C. difficile. Mice lacking CP showed a significant decrease in survival and an increase in disease pathology. Remarkably, this increase in disease was largely ablated when CP-deficient mice were fed a diet lacking Zn, strongly suggesting that CP-mediated Zn restriction is essential for combating CDI. Together, these data demonstrate that dietary Zn is an important mediator of gut microbiome community structure and Zn profoundly impacts susceptibility to CDI. Additionally, these data have revealed an essential role for CP-mediated Zn limitation during CDI. These discoveries will lay the groundwork for the development of novel preventative and therapeutics strategies for CDI.

 

7. "Quantitative analysis of hemiketal eicosanoid formation in human leukocytes," Juan Antonio Gimenez Bastida, Katie Connors Sprinkel, Claus Schneider

 

Background and aims: 5-Lipoxygenase (5-LOX) and cyclooxygenase 2 (COX-2) are responsible for the synthesis of leukotrienes and prostaglandins. 5S-Hydroxyeicosatetraenoic acid (5S-HETE), a product of the oxygenation of arachidonic acid (AA) by 5-LOX, can serve as a substrate for COX-2 giving rise to 5,11-diHETE and 5,15-diHETE as minor and a di-endoperoxide as the major product. The latter compound is in turn rearranged into two novel eicosanoids identified as hemiketals D2 and E2 (HKD2 and HKE2). Since the hemiketals could play a role in inflammation, it is important to determine: i) the level of HKs produced by the cells involved in their biosynthesis, and ii) the role of 5-LOX and COX-2 in the synthesis of these compounds. The aim of this study is to quantify the levels of hemiketals produced by 5-LOX and COX-2 expressing cells isolated from human blood in response to inflammatory stimulation.
Material and Methods: Human peripheral leukocytes were isolated and exposed to LPS and calcium ionophore (A23187) to stimulate COX-2 and 5-LOX activity, respectively. 5-LOX and COX-2 inhibitors were used to investigate the role of these enzymes in the production of HKs. The eicosanoids were analyzed by LC-MS/MS using a Thermo TSQ Vantage triple quadrupole mass spectrometer interfaced to a Waters Acquity UPLC. In addition to direct analysis in the negative ion mode, we evaluated a derivatization approach to increase the sensitivity of the LC-MS analysis. Samples were derivatized by converting the carboxylic acid moiety to a stable cationic N-(4-amino-methylphenyl)pyridinium-amide (AMPP derivatization).
Results: The levels of eicosanoids produced by 5-LOX (5S-HETE and LTB4) and COX-2 (PGE2, 5,11-diHETE, 5,15-diHETE, and HKE2) were higher in activated than non-activated leukocytes. HKD2 was not detected in both stimulated and non-stimulated leukocytes in non-derivatized samples. Derivatization improved the signal-to-noise of all compounds analyzed. Furthermore, unlike in underivatized samples, HKD2 was detectable. Specific inhibitors of 5-LOX and COX-2 inhibited the synthesis of the eicosanoids in the expected manner, indicating the key role of these two enzymes in the synthesis of the HKs.
Conclusion: Our results show that HKs are formed by human leukocytes ex vivo after stimulation with LPS (COX-2) and A23187 (5-LOX), and formation is blocked by specific inhibitors of these two enzymes. In addition, AMPP derivatization improved the detection of eicosanoids by LC-MS/MS allowing the detection of compounds such as HKD2 which was not detected in underivatized samples.

 

8. "Modulators of GIRK2-containing, G protein-regulated, Inwardly-rectifying Potassium (GIRK) Channels," Krystian A. Kozek and C. David Weaver

 

Opioids are prescribed to patients for severe temporary pain and to treat neuropathic pain, which affects millions of people worldwide and responds poorly to treatment with pharmaceuticals. Unfortunately, these potent analgesic medications are highly addictive. Decoupling the addictive and analgesic effects of opioids would be beneficial to the healthcare industry and society in general. The mechanisms by which opioids induce analgesia and addiction have not yet been defined, but research suggests involvement of downstream signaling that modulates G protein-regulated inwardly-rectifying potassium (GIRK) channels. GIRKs are ligand-activated, G protein-regulated, and plasma membrane localized proteins. They regulate resting membrane potentials via potassium (K+) efflux and limit cell excitability in neurons. GIRKs are hetero- and homotetrameric proteins composed of one or two types of subunits; the four GIRK subunits are GIRK1, 2, 3, and 4, and specific GIRK tetramer expression varies throughout the body. GIRK1/2 tetramers are expressed throughout the central nervous system, while GIRK2/3 tetramers are expressed in fewer brain regions, such as the ventral tegmental area (VTA). The VTA is one brain region involved in development of drug addiction. To study the effects of GIRK channels on the behaviors controlled by different brain regions, our lab is interested in pharmacologically modulating each specific GIRK tetramer. We conduct high-throughput drug screening in search of such compounds. ML297, a compound discovered by our laboratory, was the first selective small-molecule activator of GIRK1-containing channels (GIRK1/x) and displayed in vivo efficacy in behavioral animal studies. A screen of a molecular library conducted by our lab led to the discovery a novel modulator of GIRK2/x. This product and its family of related compounds are capable of activating non-GIRK1/x. I have explored the in vitro activity of this new modulator family on a variety of GIRK tetramers in an effort to characterize GIRK channel selectivity and the structure-activity relationship of these molecules. Development of GIRK2/x modulators will expand the pharmacological toolset available to study GIRK involvement in opioid-mediated analgesia and addiction.

 

9. "Fragment Screening and Ligand Discovery for Protein Targets via Protein-Observed 19F NMR Spectroscopy," Clifford Gee, Edward J. Koleski, Andi Wisniewski, Jin Cai, Xi Zong, William C.K. Pomerantz, and Gunda I. Georg


Protein-protein interactions (PPIs) play a vital role in many biological processes. The ability to selectively and effectively target these interactions is an important challenge due to the links between PPI dysregulation and various disease states including cancer and neurological disorders. PPIs have historically been considered to be “undruggable.” However, recent progress has challenged this perception. Screening of low-complexity molecules, termed fragments, has emerged as a viable small molecule discovery method for targeting such interactions. Herein we describe the use of protein-observed 19F (PrOF) NMR as a powerful screening tool for PPIs. PrOF NMR possesses several advantages as a biophysical characterization tool including its ability to detect small changes in chemical environment and provide useful structural information as well as mitigating challenges found in other methods based on its tolerance to a wide variety of solution conditions and additives while possessing the ability to detect low-affinity binders. By analyzing fluorine-labeled proteins via PrOF NMR, information regarding structural changes and ligand interactions can be quickly obtained. While PrOF NMR has been used to study protein dynamics and conformational changes, our lab was the first to utilize PrOF NMR as a fragment screening tool. From screening 508 fragments against the KIX protein as a model system, four molecules were verified as hits. Through structure-activity relationships (SAR) studies, three additional ligands were identified, including the commercially available drug flurbiprofen. These aryl-acetic and phenyl-acetic acid-containing compounds represent a new class of KIX ligands. Further development of these ligands is currently underway. In parallel, a second ligand discovery project is also being explored for the bromodomain BrdT, a validated protein target for male contraception, to further demonstrate of the utility and efficacy of PrOF NMR as a molecular discovery tool.

 

10. "Molecular tools for the investigation of G-Protein regulated Inward Rectifying Potassium (GIRK) channels," Susan J. Ramos-Hunter, Craig W. Lindsley, C. David Weaver, and Gary Sulikowski.

 

The design and synthesis of small molecule probes serve to provide valuable information on various cellular processes, such as the physiological role of ion channels. G-protein regulated inward rectifying potassium (GIRK) ion channels are a part of a larger family inward rectifying potassium channels that regulate process as diverse as solute balance in the kidney to cardiac and neuron excitability. Neuronal GIRKs have demonstrated links to schizophrenia, pain perception, drug addiction and epilepsy while cardiac GIRKs are linked to arrhythmias and mutations of GIRK in the adrenal gland is linked to adrenal carcinoma1. Recently, a high throughput screen identified a series of small molecule GIRK modulators leading to the development of ML297 (EC50 = 160 nM)2, a diaryl urea. Further structure-activity relationship (SAR) studies identified remarkably selective GIRK1/4 (heart) inhibitors as well as selective GIRK1/2 (brain) activators. Our current efforts are directed towards generating more selective and potent urea probes and radioligand derivatives for binding affinity assay. Achieving a better understanding of GIRK channels through small molecule probes and improved fluorescent dyes will help us to develop superior molecular probes that may help reveal the therapeutic potential of GIRK channel modulation.

 

11. "Novel Methods for Modeling G-Protein Coupled Receptors and Their Small Molecule Partners," Darwin Fu, Max Stumvoll, Greg Sliwoski, Jens Meiler

 

G-protein coupled receptors (GPCR) are integral membrane proteins that serve as targets for nearly half of all available drugs. However, only a fraction of known GPCR-small molecule complexes are well-characterized structurally, hindering progress towards structure-based drug design. Computational modeling of the protein-ligand interface can alleviate some of these obstacles and aid in identifying targetable molecular interactions. To this end, we’ve developed protocols for comparative modeling GPCRs and subsequently docking small molecule modulator. The novel methods take advantage of experimental data from multiple crystal structures and structure-activity relationships. In collaboration with other VICB labs, we’ve applied the new technologies toward a better understanding of small molecule modulation for protease-activated receptors (PAR) and metabotropic glutamate receptors (mGluR).

 

12. "Chemical genetic screen in zebrafish identifies novel role of proton sensing GPCR GPR68 in modulation of migration and metabolism in human melanoma,"Charles Williams

Increased glycolysis resulting in local acidification is a hallmark of cancer. However, the mechanisms by which these changes affect cellular behaviors such as migration. We report here the discovery of Ogremorphin (OGM) a first in class inhibitor of GPR68 through an unbiased phenotypic chemical genetic zebrafish screen. The target of OGM was identified through a combination of cheminformatics and   receptor profiling. We further confirm genetically with knock down technology that GPR68 plays a critical role in neural crest development during zebrafish development, and pheno-copies chemical treatment. Ovarian cancer G protein-coupled Receptor 1 (OGR1/GPR68) is proton sensitive in the pH range 6.8-7.8 and is expressed in melanoma cell lines. We show melanoma, which are highly glycolytic and therefore secrete protons and acidify their environment, are more motile in acidic media.  We show that the increased migratory capacity is attenuated by OGM, which dampens the actin dynamics but does not alter cells to be more epithelia. Given that melanoma and neural crest share many molecular mechanisms that govern migration, this data suggests that there neural crest cells respond to an acidic signal that promotes their migratory capacity in vivo during development.  

 

13. "Comparing Modes of Catalytic Inactivation in Human Pin1," Brendan J. Mahoney, Meiling Zhang, and Jeffrey W. Peng

Pin1 is a modular signaling isomerase that is known to interact with many signaling phosphoproteins, including tumor suppressor protein TP53 as well as the cell-cycle phosphatase CDC25C. Pin1 is often overexpressed in cancer cells, and as such has been a putative target for therapeutic treatment. It has been known that phosphorylation of a particular residue, S71, near the catalytic site completely inactivates Pin1. We have been seeking to determine the atomic details of this inactivation using a phosphomimetic mutation, S71E, using nuclear magnetic resonance. Recently, a publication by Wei et al showed that all-trans retinoic acid (ATRA) can also inactivate Pin1 by binding at the active site, and it was suggested that it acted in a similar fashion to S71 phosphorylation. By comparing our NMR results of S71E and ATRA-bound Pin1 studies, we will compare the modes of Pin1 inhibition.

 

14. "Combinatorial Therapy with Epothilone and Aurora Kinase Inhibitors Induces a Novel Form of Cell Death," Woods, Laura M., Taylor, Richard E., Vaughan, Kevin

 

Microtubule stabilizing agents (MSAs) are an important class of therapeutic compounds used for the treatment of multiple cancers. Subtle differences in the affect of MSAs can provide an opportunity to increase the susceptibility of specific protein targets critical to the cell cycle.
The epothilones are a relatively new class of microtubule-stabilizing agents effective for the treatment of Taxol® resistant breast cancer and the basis for the drug, Ixempra®. Using live cell analysis, we have shown that epothilones are effective at arresting cell division specifically at prometaphase. At this same stage of cell division, Aurora B kinases are involved in chromosomal separation and inhibition of these kinases causes a frozen phenotype. When combining epothilone and Aurora kinase inhibitors (AKI), we observed a highly-penetrant mitotic defect for inhibitors of AurB that induced cell death. This defect was distinct from those caused by treatment with epothilone or AKI alone. Studies examining this new model in which the combination of epothilone and AKI shifts the specificity of Aurora B from prometaphase substrates to telophase substrates will be presented. This outcome for combinatorial drug treatment provides a novel mechanism of action with broad potential.

 

15. "Bacterial hydrocarbon biosynthesis: Elucidating the role of Glu117β in the mechanism of OleA," Matthew R. Jensen, James K. Christenson, Lawrence P. Wackett, Carrie M. Wilmot


Rising energy costs and environmental concerns have prompted efforts to reduce petroleum-based chemical production and increase research in renewable microbial production of specialty hydrocarbons.  These hydrocarbons are used as industrial lubricants, chemical synthons, commodity chemicals, and replacement biofuels.  The bacterial biosynthesis of long-chain olefins (C23-33) represents a potential platform for production of high-energy hydrocarbons.  Over 70 divergent bacteria have been shown to express four cytosolic proteins—OleA,­­ B, C, and D—responsible for olefin production.  Efforts to understand the mechanism by which olefins are biosynthesized have utilized the x-ray crystallographic technique of in crystallo intermediate trapping to map the active site chemistries for the first enzyme in the pathway, OleA.  OleA catalyzes the head-to-head condensation of two fatty acyl-CoA substrates, and wild type and mutant structures have been solved.  The role of the active site residue, Glu117β, has been investigated through functional assays and co-crystallizing E117A and E117Q mutants with myristoyl-CoA substrate.  These data suggest the involvement of Glu117β in proton abstraction from C2 of the second fatty acyl-CoA substrate.  This activation is believed to lead to the nucleophilic attack on the thioester of the first substrate, forming a carbon-carbon bond between the two acyl chains.

 

16. "Dye-Doped Nanoparticles for Tumor Imaging and Photothermal Therapy,"Felicia Roland

           

Despite substantial advances in cancer therapy, effective treatment remains a significant clinical challenge due to the diversity of tumor size, cell type and progression that occurs from patient to patient. There is an ongoing need for improved strategies that can deliver and rapidly monitor the efficacy of cancer treatment. Recent efforts have focused on the development of individualized patient treatment regimens, which combine a therapeutic agent with a diagnostic agent into a single platform, giving birth to the term “theranostics”. Presented here, is a theranostic nanoplatform that contain a novel near infrared (NIR)-absorbing, heat generating croconaine dye as a therapeutic agent for photothermal therapy and a deep-red fluorescent dye as a diagnostic agent. Using a single-step fabrication method, croconaine dye and/or fluorescent dye are loaded into a nanoparticle core of poly(DL-lactide-co-glycolide) (PLGA) and coated with 1,2-Distearoyl-phosphatidyl ethanolamine-methyl-polyethyleneglycol (DSPE-MPEG-2000) to produce dye-doped nanoparticles (PEG-PLGA-Cr). The nanoparticle formulation has been optimized and tested in cell and small animal cancer models. Whole-body fluorescence imaging was used to monitor nanoparticle biodistribution, with subsequent photothermal treatment for tumor ablation. In vitro laser-induced cell death studies in the presence of the PEG-PLGA-Cr nanoparticles indicates effective photothermal killing of cancer cells (808-nm diode laser, 1.0 W/cm2 power density, 10 min duration). Photothermal tumor ablation using the PEG-PLGA-Cr nanoparticles was further examined in nude mice harboring EMT-6 mammary carcinoma tumors.  Nanoparticles were intratumorally injected (n=4) and subsequently laser-irradiated (808-nm diode laser, 2 W/cm2, 10 min duration). Tumor growth was monitored over time by measuring the tumor volume using a digital caliper. Laser-irradiated tumors were compared to a control cohort (n=4) that received an intratumoral nanoparticle injection but no laser irradiation. Results indicate: i) laser treatment using this PEG-PLGA-Cr nanoparticles for 10 minutes at 2 W/cm2 suppresses tumor growth over 10 days and ii) maximum heating temperature plays an important role in achieving tumor ablation. In vivo fluorescence imaging of a rat model bearing prostate tumor  (n=3) that had been dosed with fluorescently labeled PEG-PLGA-Cr nanoparticles showed accumulation in the tumor, presumably due to the enhanced permeation and retention effect. The ability to image the location of the nanoparticles will greatly facilitate ongoing studies to evaluate the efficacy of various photothermal regimens.

 

17. "Phosphorous Mediated Cycloadditions Towards the Synthesis of Spirooxindole Alkaloids for the Treatment of Breast Cancer with Brain Metastasis," Kevin Rodriquez, Miranda Burnette, Erin N. Howe, Siyuan Zhang, Jeremiah Zartman, Brandon Ashfeld

 

While the incidence of breast cancer patients developing brain metastasis is increasing, treatment options are severely limited. A primary cause for the lack of treatment options is the drug impermeable nature of the blood brain barrier (BBB), which hinders the development of novel adjuvant therapies for brain metastases. Based on physiochemical properties indicative of BBB permeability, we are synthesizing small (<600Da) natural agents that are lipophilic, and non-polar. More specifically, spirooxindoles, which are members of the Strychnos family of indole alkaloids, inhibit mitosis, and have recently been shown to reduce proliferation of glioma and neuroblastoma cell lines. We hypothesize that the design and synthesis of novel spirooxindole alkaloid derivatives will yield compounds effective against breast cancer brain metastases. To test this hypothesis, we have a developed a general synthetic strategy that allow rapid access of various spirocyclic oxindoles. More specifically, our phosphine-mediated cycloaddition approach allows direct access to various heterocyclic oxindoles. With this rapid approach in mind, we are screening novel spirooxindoles in both an in vitro model of breast cancer, and a Drosophila model of tumorigenesis and metastasis. The in vitro model utilizes two brain trophic breast cancer cell lines, MDA-MB-Br and BT474-Br, and we have identified multiple compounds with promising EC50 values. The whole organism assay relies on a Drosophila genetic model in which the larval eye neural epithelium has been engineered to overexpress RasV12, while down regulating the polarity gene discs large (dlg). These cells also express GFP, enabling visual tracking of tumor development and metastasis. Using this model we are analyzing the effect of these compounds on primary tumor formation, metastasis, and larval health. Functional data from these screens is used to identify effective structural components, which in turn are used to design the next round of chemical synthesis. This collaborative approach is essential for the rational design of novel therapeutics.

 

18. "Photothermal Manipulation of Membranes," Shaw, S. K.; Smith, B. D.


There is emerging evidence that highly controlled nanoscale heating processes can be used to manipulate biological membrane events such as pore formation, bilayer translocation, and fusion. These processes may have diverse applications in triggered drug release, gene transfection, and hybridoma formation. The goal of this work is to devise new methods of producing nanoscale heating and to exploit this phenomenon as a way to manipulate biomembrane structure and function. Our lab has developed organic dyes and dye-loaded nanoparticles that generate heat upon absorption of light at near-infrared (NIR) wavelengths, making them ideal for in vivo applications.1 These photothermal agents were incorporated into cells and artificial membranes, and heat was generated in a controlled process with precise spatiotemporal control afforded by a laser. Membranes sensitive to temperature were shown to release encapsulated contents and to have increased bilayer translocation rates upon irradiation to temperatures above their phase transition. Recent work with artificial temperature insensitive membranes and cell membranes is also discussed.

 

19. "Development of Rapid Immunoassays for Improved Point-of-Care Malaria Diagnostics," Keersten Davis


Delivery of diagnostic tools to low-resource settings under the burden of the malaria epidemic faces numerous challenges. These underdeveloped areas are often characterized by poverty, absent or intermittent electricity, hot and humid environmental conditions as well as a lack of skilled clinicians. Rapid diagnostic tests (RDTs) were developed to circumvent these challenges in the form of a low-cost, rapid, easy to use test. Despite the many advantages of RDTs, the changing climate of infectious disease education, prevention and treatment has brought to light the areas in which these tests can be improved to detect asymptomatic patients. This work outlines the development of two parallel magneto-immunoassays for detection of asymptomatic malaria. First, Ni(II)NTA magnetic particles were employed to extract, purify, and concentrate the most common malarial biomarker, Plasmodium falciparum Histidine Rich Protein II (pfHRPII), from a blood sample, in less than 30 minutes.  Application of this concentrated protein to commercially available RDTs afforded a 4-fold enhancement in the performance of the tests, into the single parasite/µL detection regime. While integration of new sample preparation methods with existing technologies can represent one approach toward infectious disease elimination in low-resource areas, a unique, rapid immunomagnetic on-bead ELISA for pfHRPII detection was also developed, as an alternative strategy for malaria diagnosis.  In less than 30 minutes, a full sandwich ELISA was complete for the detection of single parasites/µL. Effecting a change in low-resource diagnostics and global healthcare may not necessarily require a complete reworking of the system, but simply using innovation to make the existing constructs work better.

 

20. "Chiral Proton Catalyzed Diastero- and Enantioselective Synthesis of α-Substituted anti-α,ß-Diamino Acids: Application to the Synthesis of a Human Proteasome Inhibitor," Daniel J. Sprague, Thomas J. Struble, Anand Singh, and Jeffrey N. Johnston

 

α,ß-Diamino acids are a nonproteinogenic group of chiral, non-racemic 1,2-diamines which exist in nature in their free form or as motifs within complex molecules. These atypical amino acids are of interest in biochemistry and drug discovery due to their ability to modify the physical and chemical properties of compounds which contain them. Substitution at the α-position, generating a quaternary carbon, imparts resistance towards chemical and enzymatic degradation in peptides as well as induces helix formation. The synthesis of such residues is a sought after transformation due to both their biological activity and the difficulty in the stereoselective production of a fully substituted carbon center. Herein, we present a chiral Bis(AMidine) catalyzed addition of α-substituted nitroacetates to aryl aldimines, resulting in differentially protected anti-α,ß-diamino acid derivatives. The resulting products are obtained in good yields with excellent diastereo- and enantioselectivity (up to >20:1 d.r. and 99% e.e.). These products can be unmasked in a sequential fashion for further regioselective transformations. Interestingly, we find small changes in catalyst structure result in complete reversal of diastereoselectivity, and a model for this stereoselection will be presented. Finally, the first catalytic, stereoselective synthesis of an imidazoline based human protease inhibitor, developed by Tepe and coworkers, is achieved.

 

21. "The Investigation of the Chemical Mechanism and Inhibition of Microsomal Prostaglandin E2 Synthase 1 (MPGES1)," Michael C. Goodman* and Richard N. Armstrong

 

Prostaglandins function as signaling molecules involved in pain, fever, and many diseases associated with chronic inflammation. The most common therapeutic treatment of inflammation involves the inhibition of the COX enzymes by nonsteroidal anti-inflammatory drugs (NSAIDs) or COX-2 selective inhibitors (coxibs). The inhibition of the COX enzymes prevents the catalytic formation of the intermediate endoperoxide compound, PGH2. With the inhibition of PGH2, the production of other downstream prostaglandins is also inhibited. Therefore, COX inhibition can result in adverse gastrointestinal and cardiovascular side effects because of the subsequent low level of various prostanoids. Microsomal Prostaglandin E2 synthase 1 (MPGES1), a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily, is the most prominent PGE synthase expressed during inflammation and is the terminal enzyme in the PGE2 synthesis pathway. It is a promising therapeutic target for the treatment of chronic inflammation and potential drug candidates have been pursued by the pharmaceutical industry in recent years. However, little is known about the actual chemical mechanism of the enzyme. Glutathione (GSH) appears to participate as a cofactor in the reaction, though there hasn’t been any evidence to support this. In order to investigate GSH as a cofactor in the reaction, it is necessary to detect the thiolate of GSH in the active site and determine its exchange rate off of the enzyme surface. One equivalent of the thiolate is observable by difference spectroscopy. However, simple difference spectroscopy isn’t possible since the enzyme is unstable in the absence of GSH. Therefore, a solution of the enzyme in complex with GSH can be rapidly mixed with the serine analogue of GSH, γ-L-glutamyl-L-serylglycine (GOH), or the alanine analogue γ-L-glutamyl-L-alanylglycine (GH) and the kinetics of the loss in absorbance from the replacement of GS- with GO- or GH would confirm the presence of the thiolate in the complex and its rate of dissociation from the enzyme can be determined. In order to further study the chemical mechanism of the enzyme and verify if the thiolate of glutathione is needed for the isomerization of the substrate to the enzyme, a technique known as Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) can be implemented. This particular method can investigate the conformational dynamics of an enzyme based on the solvent exchange rate of amide hydrogens in the protein backbone. The exchange rate of these amide hydrogens for deuterium can vary depending on various factors including hydrogen bonding within secondary structural elements. Other factors including temperature, pH, and nearby amino acid side chain residues can affect amide HDX kinetics as well. It is known that the MPGES1 apoenzyme is unstable in the absence of its cofactor glutathione. However, preliminary experiments have confirmed that the enzyme is stable in the presence of the two aforementioned analogues, GOH and GH. HDX-MS can be utilized to compare the conformational dynamics of the enzyme in the presence of its natural cofactor, GSH, in comparison to the GOH and GH analogues. If the conformational dynamics are similar and the enzyme has higher activity with GSH only, it is reasonable to assume that the thiolate of glutathione is necessary as a cofactor for the isomerization of the substrate to be catalyzed.

 

22. "Oxidation of Salicylic Acid and Aspirin by Human Cytochromes P450," Carl Sedgeman
           

Aspirin (acetylsalicylic acid) is a commonly used analgesic. It is rapidly deacetylated both enzymatically and non-enzymatically to salicylic acid. Salicylic acid can then be further metabolized by conjugation with glycine or glucuronides or by hydroxylation to form 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). This oxidation has been suspected to occur from oxygen radicals via Fenton reactions as well as by cytochrome P450 enzymes. We analyzed the formation of 2,3-DHBA and 2,5-DHBA in the presence of human liver microsomes by HPLC with fluorescence detection. We were able to determine that microsomal oxidation was much faster for salicylic acid than aspirin. 2,3-DHBA was found to be the major oxidative product formed from salicylic acid, whereas aspirin could detect only 2,5-DHBA. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies were able to confirm that all six of these enzymes played a role in the formation of 2,3- and 2,5-DHBA, as well as found that 2A6 and 2B6 played a role in the hydroxylation to 2,5-DHBA. It was also shown in the inhibitor studies that P450 2E1 was the major enzyme contributing to the in both 3- and 5-hydroxylations.

 

23. "Stabilized Alpha-Helical Mimetics used in Modulating Protein-Protein Interactions," Gabriella Perell, William C. K. Pomerantz


Protein-Protein interactions (PPIs) play a fundamental role in nearly all biological processes in both health and disease. With over an estimated 100,000 protein -protein interactions predicted to form in the human interactome, discovery of chemical probes that can disrupt or enhance the affinity of such interactions has become highly sought after. Modulating protein-protein interactions using peptidomimetics and stabilized α-helices has emerged as a viable strategy to meet the challenge. This poster describes our approach for stabilizing α-helices as potential new probes for disrupting PPIs involving the protein interaction domain KIX, of coactivator CBP. Several of these interactions are known to be upregulated in the disease, AML motivating our studies. Crosslinked peptides have been shown to stabilize short helical peptides for the disruption of disease-causing PPIs. We have synthesized thioether-bridged peptides that vary in bridge length, polarity, and position of a mixed lineage leukemia (MLL) peptide, a known ligand of KIX. MLL has been previously shown to both bind to KIX as well as enhance the affinity of a second protein interaction with CREB. Optimization of the peptide analogs is assessed on synthetic conversion as well as a variety of biophysical methods such as % helicity, protease stability, and site-specific affinity. This poster describes our progress in the synthesis of several unnatural sulfur containing amino acids, peptide cross linking interactions, and biophysical characterization of peptide analogs. We anticipate stabilized peptide constructs as useful tools for the modulation and disruption of PPIs.

 

24. "Nuclear oxidation of a major peroxidation DNA adduct, M1dG, in the genome,"

Orrette R. Wauchope, William N. Beavers, James J. Galligan, Michelle M. Mitchener, Philip J. Kingsley, and Lawrence J. Marnett

 

There is extensive evidence in the literature linking chronic inflammation and carcinogenesis. Chronic inflammation can produce reactive oxygen species (ROS) that oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-b-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one (M1dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair system. We report that M1dG is present at significant levels in a variety of human and murine cell types and that it is oxidized in intact DNA to a single oxidation product, 6-oxo-M1dG. This is the first report of the production of 6-oxo-M1dG in genomic DNA and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis and repair. 6-Oxo-M1dG appears to be one of the most abundant adducts in genomic DNA yet little is known of its formation or mutagenic potential.

 

25. "Synthesis of Vicinal Diamines via Hypervalent Iodine: Oxidative Inter/Intermolecular Diamination of Hydroxy Styrenes and Inter/Intramolecular Annulative Diamination of Vinyl Pyridines," Michael Danneman

The vicinal diamine motif is ubiquitously present in a wealth of natural products, including non-proteinogenic amino acids and alkaloids. As a general approach to vicinal diamines, the diamination of alkenes is particularly expedient. Although alkene diamination has been dominatined by transition-metal-based protocols, metal-free methods are becoming more broadly effective. In particular, hypervalent iodine reagents have been employed to promote olefin diamination with non-nucleophilic amine sources, such as sulfonamides or sulfonimides. Conversely, the use of electron-rich amine sources in doubly intermolecular carbon-nitrogen bond formations without metal coordination is rare. Herein, doubly intermolecular alkene diamination is achieved with electron-rich, terminal alkenes through the use of a hypervalent iodine (PhI(OAc)2) reagent, iodide, and electron-rich amines. Mono- and disubstituted amines combine with electron-rich alkenes, particularly ortho-hydroxystyrenes, to achieve the greatest level of generality. A key novelty of this diamination is that commercially available electron-rich amines can coexist as both a nucleophile and an electrophile without conventional transition-metal-based activation. Mechanistically, this reaction is unique in the sense that alkene attack of an activated haloamine may lead to an ortho-quinone methide intermediate, a suitable electrophile for subsequent nucleophilic attack of amine en route to homodiamination. Additionally, the same oxidant additive combination (PhI(OAc)2-KI) is employed to achieve an operationally straightforward, transition-metal-free, inter/intramolecular diamination of vinyl aminopyridines. Electron-rich mono- and disubstituted amines combine with these vinyl pyridines to arrive at their corresponding aza-3-aminoindolines, achieving a high degree of generality. Since indoline and azaindoline scaffolds are present in many bioactive alkaloids and pharmaceuticals, this annulative diamination of vinyl pyridines is of high importance as it can serve as a unified approach in order to access all four azaindoline heterocyclic families.

 

26. "Regulation of HemA, the initial enzyme in Staphylococcus aureus heme synthesis," Jacob E. Choby and Eric P. Skaar

 

Heme, an Fe2+ containing tetrapyrrole, is a crucial cofactor for humans and the bacterial pathogens that invade them. During infection of the host, bacteria including Staphylococcus aureus utilize heme for energy generation by the electron transport chain and for catalase to defend against the innate immune system. De novo heme synthesis is therefore essential for pathogenesis. S. aureus is also capable of acquiring heme from human hemoglobin and incorporating it into hemoproteins or degrading heme to liberate iron.  Because excess heme is toxic, S. aureus must balance endogenous heme synthesis and exogenous heme acquisition to ensure proper amounts of intracellular heme without reaching toxic levels. We have found that S. aureus regulates levels of the enzyme HemA, a glutamyl-tRNA reductase that catalyzes the formation of glutamate 1-semialdehyde from NADH and glutamyl-tRNAGlu, the first step in heme synthesis. A mutant in hemX, the gene encoded directly downstream of hemA, has high levels of HemA relative to wildtype in heme-replete conditions, indicating that HemX is required for restricting HemA levels.  HemX is a putative integral membrane protein with potential for heme-binding. The mechanism by which HemX regulates HemA levels and the effects of HemX–mediated regulation are being investigated. HemX offers one of the first insights into the regulation of heme biosynthesis in S. aureus; little is known concerning the integration of heme synthesis and acquisition systems. Additionally, insight into the regulation of HemA will impact ongoing efforts to develop therapeutics targeting S. aureus heme synthesis.

 

27. "Hypoxia sensitive fluorescence probes for in vivo imaging of vascular dysfunction," Md. Imam Uddin, Stephanie M. Evans, Jason R. Craft, Lawrence J. Marnett, Md. Jashim Uddin, Ashwarth Jayagopal, John S. Penn


Purpose: Hypoxia is associated with insufficient supply of oxygen within inner retinal region and choroid, preventing drug delivery and treatment options in various retinal diseases including age-related macular degeneration (AMD), diabetic retinopathy and retinopathy of prematurity (ROP). Molecular imaging of hypoxia could provide vital information to hypoxic mechanisms during retinal development and adulthood, which might be targeted by therapeutic approaches to prevent irreversible vision loss.
Methods: Hypoxia-sensitive activatable probes have been synthesized conjugating fluorescence dye to a hypoxia sensitive moiety. To evaluate the utility of this contrast agent for imaging hypoxia, in vitro assays using retinal cells (R28) and in vivo studies in oxygen-induced retinopathy (OIR) or laser-induced choroidal neovascularization (LCNV) models were used to determine the hypoxia-associated sensitivity and specificity of the probe. Fluorescence measurements were performed on normoxia- or hypoxia-conditioned R28 cells exposed to the new probe to examine the ability of the probe to activate within hypoxic region. Pimonidazole hydrochloride immunostaining was utilized to confirm specificity of the contrast agent. OIR and LCNV animal models were intravenously or intraocularly-injected with the agent and analyzed by ex vivo retinal fluorescence imaging technique to determine specificity and sensitivity of the new imaging probe.
Results: R28 cells conditioned under hypoxia for 4 hours exhibited dose-dependent fluorescence enhancement due to hypoxia-selective activation of the activatable probe. The level of hypoxia in the conditioned cells was confirmed by pimonidazole hydrochloride immunostaining. In LCNV and OIR animal models intravenously or intraocularly-injected followed by ex vivo imaging could localized with hypoxic region, and was confirmed by pimonidazole hydrochloride immunostaining co-localization.
Conclusions: We have developed a new hypoxia sensitive activatable molecular imaging technique to detect level of hypoxia in retinopathy disorders. These studies will support to detect hypoxic tissue in vivo using targeted agents in conjunction with readily available retinal fluorescence imaging equipment. The hypoxia-sensitive contrast agent might be useful for early detection of retinal vascular diseases with clinical applications.

 

28. "Structural and functional effects of heme binding in rcPfHRP2: Implications for malaria diagnosis," Anna Bitting

 

PfHRP2 is a histidine-rich protein produced by the malaria parasite Plasmodium falciparum. It is used as a biomarker for antibody-based malaria rapid diagnostic tests (RDTs). However, protein sequence variation and manufacturing issues can make these tests unreliable. Part of our lab’s work is focused on developing a standard with which to rate the effectiveness of current RDTs. This standard must be a recombinant protein in order to be feasible, but we must first study how recombinant proteins behave in matrices similar to an infected patient’s blood. Past studies have indicated that PfHRP2 may play a role in the parasite’s heme detoxification process by binding free heme and promoting its crystallization into hemozoin. We hypothesize that heme-bound protein may adopt a different conformation from free protein, and that this conformation change may affect protein binding to antibodies on an RDT. This is especially a concern since native protein will be exposed to heme in blood, but purified recombinant protein used for industrial antibody production will not. This work investigates the conformational changes of rcHRP2 using circular dichroism (CD), and investigates the effects of heme on HRP2 detection using ELISA and RDT formats.

 

29. "Comprehensive Access to Apoptolidin Derived Chemical Probes to Study Cancer Cell Metabolism," Katherine Chong, Robert Davis, Nalin Leelatian, Jonathan Irish, Gary Sulikowski

 

Natural products contain novel scaffolds and further our understanding of biological phenomena through their use as selective chemical probes. In fact, three fourths of small molecule anticancer drugs are natural product based, with almost half of those molecules being natural products themselves or derived therefrom. Despite their vast utility, recent efforts have moved away from the use of natural products in drug screenings due to their challenging total syntheses and subsequent derivation. As typical “low hanging fruit” is scooped by current drug discovery methods, we begin to traverse an area of pharmaceutical research in which we must move into new chemical space. The demand for new scaffolds is ever increasing, to produce selective therapeutic agents in drug discovery—the “holy grail” in the industry being the ability to discriminate disease states from healthy cells. To do so, requires an in-depth understanding of fundamental biological phenomena. Glycosylated natural products have long been explored for their excellent antitumor activity. We have found apoptolidin to have sub-nanomolar activity against tumor cells when glycosylated. When the sugars are removed, the aglycone loses activity (> 10 μM, H292 cells, human lung cancer). Accessing variants of apoptolidin as a function of glycosylation state will enable us to examine the role deoxy sugars play in the cytotoxicity profile of the apoptolidins against varying cancer cell types. As each cancer cell type displays a unique metabolic profile, and notably distinct from healthy cells, we hope to use our toolbox of apoptolidin glycovariants in the quantification of cellular uptake, localization, and subsequent activity as a function of glycosylation state of the apoptolidins and metabolic state of each cancer line. To access our apoptolidin glycovariants, we aim to combine techniques utilizing chemical synthesis, precursor directed biosynthesis, and biosynthesis. Herein describes our efforts toward the chemical synthesis of apoptolidinone C and preliminary results utilizing phospho-specific flow cytometry to measure cellular uptake and response of the apoptolidin glycovariants.

 

30. "Fundamental studies for Drift Tube and Traveling Wave Ion Mobility-Mass Spectrometers:
Comparing Resolving Power and Resolution," James N. Dodds; Katrina L. Leaptrot; Jody C. May; John. A. McLean

While significant strides have been achieved utilizing modern mass spectrometry techniques in analyzing complex biological samples, further degrees of separation may be desired for those mixtures involving isomeric systems.  Here we investigate the nominal metrics for peak efficiency (resolving power) and discuss the potential separation capabilities of both traveling wave and drift tube ion mobility – mass spectrometers.  For drift tube ion mobility (Agilent 6560) we investigated the effects of drift voltage upon peak shape, and to a lesser extent gate width.  For the traveling wave instrument, (Waters Synapt G2) wave height and wave velocity were optimized for a model known system.  Isomeric systems of reverse peptides and carbohydrate standards were run in order to examine potential separation capabilities for each ion mobility technique.

 

31. "Base-displaced intercalated structure of the DNA adduct N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone," Dustin Politica

 

The suspected carcinogen 3-nitrobenzanthrone (3-NBA) is an environmental contaminant of concern, due in part to its high mutagenic potential coupled with likely human exposures as a component of diesel exhaust. Metabolism of 3-NBA in vivo forms an electrophilic nitrenium ion resulting in aminobenzanthrone (ABA) DNA adducts. The major product, N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA), occurs at the C8 position of guanine. We report structural and thermodynamic properties of the C8-dG-ABA adduct in the dodecamer 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA. Molecular dynamics calculations restrained by interproton distances derived from NMR NOE data revealed that the C8-dG-ABA adduct adopted a base-displaced intercalated structure. The adducted guanine maintained the syn conformation about the glycosidic bond. The ABA moiety was intercalated into the duplex displacing the cytosine opposite the lesion into the major groove of the DNA. The pattern of NOEs between the amino and imino protons of the bases showed that Watson-Crick base pairing interactions were maintained adjacent to the lesion site. A single set of NOE cross peaks indicated that one conformation was present. An 11 ºC decrease in melting temperature (Tm) of the modified as compared to the unmodified duplex indicated that the C8-dG-ABA adduct reduced the thermodynamic stability of the duplex. Supported by NIH grants R01 CA 55678 (M.P.S), R01 ES-009127 (A.B.), and R01 ES-021762 (A.B.)

 

32. "Biosynthetic Investigations into the Everninomicins," Audrey E. Yñigez-Gutierrez, Emilianne M. Limbrick, and Brian O. Bachmann


The emergence of antibiotic resistant illnesses presents a growing threat to human health and threatens to usher in a post-antibiotic era. As a result, there is renewed interest in discovering new antibiotics and revitalizing previously abandoned antibiotics. Everninomicins are complex polysaccharides produced by Micromonospora carbonacea and contain a number of unique structural features such as orthoester linkages, a methylenedioxy bridge, and a nitro sugar. Everninomicins are active against gram-positive bacteria, including antibiotic resistant strains. Despite this activity, pharmaceutical development of everninomicin A was discontinued due to pharmacological issues. However, the everninomicins still present a clear opportunity to develop potent antibiotics. The biosynthetic mechanism of everninomicins is not completely understood and robust structure activity relationship studies were not performed to optimize antimicrobial activity and pharmacological properties. Therefore, we propose to take a dual approach to revitalizing everninomicin. Previously, the Bachmann group has utilized genetic knockouts and complementation to investigate the biosynthesis of some of the most unique components of everninomicin. We intend to continue these studies to further elucidate the biosynthesis of everninomicins. We plan to take advantage of our increasing understanding of the bacterial machinery to develop analogs of everninomicin using emerging genetic manipulation techniques. Everninomicin analogs will be evaluated for enhanced antimicrobial and pharmacological properties.

 

33. "Utilizing Ion Mobility-Mass Spectrometry for the Metabolomic Analysis of Lung Epithelial Cells," James C. Poland, M. Ray Keller, Stacy D. Sherrod, John A. Mclean

 

Mass spectrometry based metabolomics seeks to identify the inventory of small molecules (i.e., <1000 Da) that are the by-products of, or directly involved in, biological processes. Owning to the chemical diversity represented by metabolites, comprehensive molecular analysis is challenging for human-based studies. In this work, multidimensional chemical analysis based on liquid chromatography coupled to ion mobility-mass spectrometry (LC-IM-MS) is utilized in order to increase the detection and characterization of small molecule. A rapid, multi-tier validation workflow has been applied to identify metabolites of interest utilizing combined information from retention time (RT), drift time (DT), mass-to-charge ratio (m/z), and tandem MS/MS spectra. Intercellular metabolites were extracted from human epithelial lung cells treated with different types of perturbations (e.g., toxins, metals, etc.). Various sample preparation procedures were evaluated using the control lung cells in an effort to enhance the metabolomics molecular breadth. These experiments determined that methanol/water and methanol/chloroform preferentially extracted polar metabolites and fatty acids, respectively. In this set of experiments, molecular coverage differences between these two extraction methods were greater than 200 unique metabolites. In an effort to study the intracellular polar metabolites in control and perturbed lung cells, following experiments consisted of only utilizing the 80:20 methanol: water (v:v) sample preparation procedure. After sample preparation and LC-IM-MS acquisition, the datasets were analyzed statistically to determine the differences in control and treated A549 cells. Briefly, pairwise comparisons (student's t-test) for each treatment type (control vs. treated) were applied to the metabolomics datasets. Using this multidimensional metabolomics workflow, we have observed over 2,000 metabolites between control and exposed lung cells, with approximately 20% of these metabolites being differentiated between the different sample sets (control vs. treated) as indicated with a P-value of less than 0.05. Candidate identities were obtained through searching the METLIN database against intact and fragmentation mass data obtained under accurate mass conditions (<5 ppm). Validation was achieved using a five-tier scoring system similar to a concept utilized in plant metabolomics studies.1 Putative identifications for this data suggest that histidine, fatty acid, cysteine and methionine metabolisms were altered. For metal-exposed lung cells, about 3,000 metabolites are observed using this workflow, with over 80% of these being differentiated from untreated (control) samples under the same criterion (p<0.05).

 

34. "I can’t believe it’s not glucose! Uropathogenic E. coli utilizes two-component systems to sense and respond to serine and pyruvate levels," Allison Eberly

 

Uropathogenic Escherichia coli (UPEC) is an extra-intestinal Gram negative facultative anaerobe that causes the majority of urinary tract infections (UTIs). UPEC attach to the bladder surface using adhesive fibers and form biofilms both on the surface of and within bladder cells during infection. In diabetic populations, the risk of developing a UTI is double of that of non-diabetics. This increased risk is not strongly associated with higher glucose levels in the urine of diabetics. In addition to glucose, diabetics also excrete higher levels of branched chain amino acids, as well as serine and pyruvate in their urine. UPEC have sensory devices that respond to serine and pyruvate. These two-component systems (TCSs) are YehUT (senses serine) and YpdAB (senses pyruvate). We also have preliminary data that pathogenic E. coli do not respond to glucose as previously described for non-pathogenic E. coli and the basis of this difference remains unknown. We hypothesize that UPEC sense and take advantage of increased metabolites during infection using modified regulatory networks. Our aims are to delineate the altered glucose response in pathogenic E. coli and to define the role of the pyruvate/serine-sensing TCSs. We have created a panel of YehUT and YpdAB mutants and tested the fitness of the resulting strains for their ability to sense and respond to serine and pyruvate changes. Our results indicate significant differences in phenotypes between UPEC and commensal E. coli, suggesting the presence of regulatory mechanisms that are distinct to pathogenic E. coli. Additionally, we observe that the presence of glucose does not repress the expression of YjiY, the YehUT reporter, in the pathogens. There is a strong repression of YjiY expression in the commensal E. coli MG1655 in the presence of glucose. Our future studies will determine the minimum metabolite requirements UPEC needs to cause infection, ultimately guiding us to understand the implications of increased UTIs in the diabetic population.

 

35. "Synthesis of oligonucleotides containing Fapy-dG and N5-(2-oxoethyl)Fapy dG adducts,"Chanchal K. Malik and Carmelo J. Rizzo


Formamidopyrimidines (Fapy) are DNA lesions that are of chemical and biological interest. Fapy-dG is a product of oxidative damage and is associated with progression of age related diseases and cancer. They are substrates for the base excision repair pathway. Fapy-dG is formed in greater amount than the 8-oxopurine lesion from γ-radiolysis. Investigations of the effects of Fapy-dG on polymerases and repair enzyme activity have lagged due to the absence of a method for chemically synthesizing oligonucleotides containing this lesion. Fapy-dG lesions can also arise from initial N7-alkylation of deoxyguanosine.  Examples of such Fapy-dG lesions include those from methylating agents, nitrogen mustards, aziridine, and chlorooxirane.  We have synthesized oligonucleotides containing the Fapy-dG and N5-(2-oxoethyl)-Fapy-dG.  The N5-(2-oxoethyl)-Fapy-dG is derived from chlorooxirane, which is derived from metabolic activation of the industrial carcinogen vinyl chloride.  Further, we developed a strategy to convert the N5-(2-oxoethyl)-Fapy-dG into other N5-substituted Fapy-dG lesions, such as those derived from aziridine and nitrogen mustards. 

 

 

 

 

 


 

36. "Unusual Kinetic Isotope Effects of Deuterium Reinforced Polyunsaturated Fatty Acids in Tocopherol-mediated Free Radical Chain Oxidations," C. Lamberson, L. Xu, J. R. Montenegro-Burke, H. Muchalski, V. Shmanai, A. Bekish, J. McLean, C. Clarke, M. Shchepinov, N. Porter

 

The bis-allylic –CH2- groups in polyunsaturated fatty acids (PUFAs) are susceptible to hydrogen atom abstraction, making PUFAs prone to free radical-mediated peroxidation.  A recent strategy to diminish lipid peroxidation in vivo is based on substituting bis-allylic hydrogen atoms with deuterium.  Previous studies have shown these deuterated PUFAs (D-PUFAs) undergo peroxidation in solution with propagation rate constants some 10-fold less than the natural fatty acids.  This isotope effect falls outside of the range of kH/kD (<7) which has been reported for other H(D) atom transfers from carbon to peroxyl radicals.  Recently, we have carried out tocopherol-mediated oxidations of several D-PUFAs and measured isotope effects ranging from 23 to 36 (substrate dependent) using LC-MS and HRMS techniques.  The H-atom transfer from the bis-allylic –CH2- center to the tocopheryl radical is the rate-determining step in tocopherol-mediated peroxidation (TMP) of lipids in human low-density lipoproteins (LDL), a process which has been linked to coronary artery disease.  These unexpectedly large kinetic isotope effects for the tocopherol-mediated oxidation of linoleic and linolenic acid suggest that H-atom tunneling makes this process favorable and plays a significant role in the oxidative modification of human LDL.

 

37. "Incorporation of Unnatural Amino Acids into the Glucosyltransferase Domain of Clostridium Difficile TcdB to Probe Binding to Rac1," Jonathan P. Davies, Heather K. Kroh, Joe Alvin, and D. Borden Lacy
 

Clostridium difficile is an opportunistic bacterium that causes pseudomembranous colitis and severe diarrhea in infected hosts. A common nosocomial infection, C. difficile presents an enormous burden on hospitals - where there is a dense population using antibiotics – with 450,000 infections and 29,300 related deaths in 2011. C. difficile secretes an exotoxin, TcdB, containing a glucosyltransferase domain (GTD) that glucosylates host GTPases in epithelial cells, leading to the disruption of downstream signaling pathways and cytoskeleton regulation. However, their exact interactions are unknown due to the low binding affinity between the two proteins. We have incorporated photoactive unnatural amino acids (p-azido-L-phenylalanine and p-benzoyl-L-phenylalanine) into the E341 residue site on TcdB GTD using amber codon suppression. Exposing the modified protein to UV light crosslinked the GTPase Rac1 to TcdB GTD. A future aim of this project is to analyze the crosslinked complex using mass spectrometry and x-ray crystallography. Armed with this technique, we hope to identify the binding region between TcdB GTD and Rac1. If successful, we would like to use this technique to investigate other parts of the TcdB mechanism, allowing researchers to probe the exact host protein interactions throughout the toxin’s timeline.

 

38. "Development of fluorescent sensors for chemoselective visualization of endogenous formaldehyde," Aaron Roth, Hao Li, Chelsea Anomra, Jefferson Chan

 

Formaldehyde (FA) has canonically been viewed as a potent neurotoxin owing to its propensity to irreversibly crosslink protein and DNA in biological systems. Indeed, it has been demonstrated in Alzheimer’s disease models, that elevated FA plays a role in the development of toxic senile plaques via this crosslinking mechanism. Interestingly, all neurons produce and maintain FA (in the 0.2-1.0 mM range), prompting one to ask whether it serves an intrinsic and beneficial biological function. Recently, it has been shown that low grade FA is essential to long-term memory formation and retention; however, the molecular details have yet to be elucidated owing to a dearth of suitable methods to monitor FA dynamics in real-time. As such, the development of chemical tools with the ability to chemoselectively detect FA with high spatio and temporal resolution would enable us to probe its role in memory formation, as well as discover new functions pertaining to neurodegenerative diseases and neurotransmission. Herein, we describe the design, synthesis, and evaluation of novel fluorescent sensors for FA using a robust Aza-Cope reaction cascade.

 

39. "Making Gd(III) Diamagnetic: Magnetic Exchange Coupling as a Tool for Reducing T1e of Gd(III)," Laura Lilley, Kang Du, David Harris, Thomas J. Meade


The inherent lack of sensitivity of Gd(III) contrast agents is a significant barrier for the wide spread implementation of magnetic resonance imaging (MRI) for molecular imaging applications. A primary goal of molecular imaging is to visualize anatomical and biochemical events in vivo and correlate these findings to molecular processes. MRI affords high spatiotemporal resolution, in addition to unlimited depth penetration. However, visualization of biochemical processes requires rationally designed Gd(III) contrast agents. Gd(III) accelerated the T1 relaxation rate, increasing the signal from nearby water protons and making the voxel appear brighter in the resulting image. Typical bioactivated contrast agents have two states, an “off” state that precludes water from interacting with the Gd(III) center, and an “on” state where water is allowed to interact with Gd(III) that results in enhanced image brightness. All bioactivated Gd(III) contrast agents possess high background signal (up to 40%) due to second- and outer-sphere water relaxation in the “off” state. To overcome this limitation, we are developing bimetallic complexes to shorten the electron relaxation time (T1e) of Gd(III) and subsequently reduce background signal of the agent prior to activation. These preliminary studies investigate magnetic coupling of Gd(III) to first-row transition metals with short T1e values (Co(II), Cu(II)). Magnetic exchange coupling results in an agent with a shortened T1e and concomitantly low relaxivity (r1 mM-1s-1). Ultimately, we will employ this methodology towards developing zero-background Gd(III) molecular imaging MR probes.

 

40. "Developing Faster Approaches for Conformational Space Sampling in Support of Structural Ion Mobility-Mass Spectrometry," Sarah M. Stow, Nichole M. Lareau, Terry P. Lybrand, David M. Hercules, and John A. McLean

 

Ion mobility-mass spectrometry (IM-MS) allows the separation of ionized molecules based on structural properties such as size and shape, in addition to mass-to-charge ratio. The drift time data that is obtained from IM-MS experiments can be used to calculate the collision cross section (CCS) of the ionized molecule. This CCS value is representative of the ion’s gas phase conformation and can be thought of as a rotationally averaged surface area.  Combining conformational space studies with CCS values derived from IM-MS experiments provides more detailed structural insight into the gas phase structure of the ions of interest. Various algorithms exist for sampling conformational space and for calculating theoretical CCS values for the computationally generated conformations.

 

One obstacle with these computational strategies is the time that is required to perform these calculations. IM-MS experiments occur on the order of milliseconds and microseconds respectively, whereas conformational space studies on larger molecules can take weeks or months to complete with traditional molecular dynamics strategies. Distance geometry is a much less computationally expensive method for sampling conformational space. Results from applying this conformational sampling algorithm to small molecules (i.e. metabolites) and larger systems (i.e. polymers) will be discussed. Although molecular dynamics tends to scale better for larger systems, distance geometry may prove useful for the polymer systems. Theoretical CCS values will be calculated with existing strategies and the results will be compared with experimental CCS values. Using less computationally expensive methods to perform these theoretical calculations will allow these strategies to be more useful in enhancing the structural insight from IM-MS experiments.

 

41. "Holding on for dear life: Environmental cues dictate expression of adhesive type 1 pili in uropathogenic Escherichia coli," Kyle A. Floyd,  Allison R. Eberly, Richard M. Caprioli, Maria Hadjifrangiskou

 

Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections (UTIs), utilize a sophisticated pathogenic cascade to establish infection within the host. This cascade is dependent upon the ability of UPEC to form large multi-cellular bacterial communities, known as biofilms, within the urinary tract. During biofilm development, production of a self-produced extracellular matrix (ECM), and generation of subpopulations with distinct expression profiles complicates treatment and enables persistence within the host. Therefore, a deeper understanding of the underlying role and regulation of UPEC biofilm formation in UTI is crucial for identifying ways in which to attenuate UPEC pathogenesis. In previous studies we used a MALDI-TOF imaging mass spectrometry (IMS) approach to dissect the spatial proteome of intact surface-associated UPEC biofilms. Those studies revealed that expression of adhesive type 1 pili (fim) was unique to subpopulations comprising the air-exposed region of the biofilm under the growth conditions studied. Type 1 pili mediate adherence to bladder epithelial cells, aid in maintaining biofilm infrastructure, and are essential for establishment of acute UTI. Expression of fim is partially controlled by the orientation of the fimS promoter, which is flanked by invertible repeats recognized by three site-specific recombinases, FimB, FimE and FimX. The observed distinct localization of type 1 pili to the air-exposed region of the biofilm led us to the hypothesis that oxygen-tension regulates expression of fim in UPEC. We thus analyzed the phase-state of the fimS promoter and discovered two novel regulatory mechanisms that suppress fim expression under oxygen-deplete conditions: one that promotes switching of the fimS promoter element to the OFF orientation (Mechanism 1), and another mechanism that functions independent of the promoter switch (Mechanism 2). We analyzed the basis of these two mechanisms and investigated the effects of alternative electron acceptors in regulation of type 1 pili. Preliminary analyses suggest that alternative terminal electron acceptors only partially support pilus production under oxygen-deplete conditions, when bacteria seeding oxygen-deplete cultures started with fim in the ON orientation. These findings suggest that respiration products impact pilus regulation. To identify which recombinase is responsible for switching fimS OFF under oxygen-deplete conditions, we created and tested mutants lacking the FimB, FimE and FimX recombinases. Deletion of fimE, which preferably recombines fimS to the OFF orientation exhibited higher (but not wild-type) levels of type 1 pili expression under oxygen-deplete conditions. This suggests that Mechanism 1 is partially exerted through the function of FimE. We are in the process of identifying how depletion of oxygen is transduced to FimE and whether this signal transduction event occurs on the transcriptional or post-transcriptional level.

 

42. "Development of Global Activity Based Proteomic Strategies for Two-Component System Profiling," Raleigh Godsey, Advisor: Dr. Erin Carlson, University of Minnesota Department of Chemistry


Two-component systems (TCSs) are signaling pathways involved in adaptive bacterial responses associated with increased fitness and virulence, making them interesting targets for therapeutic development. TCSs are composed of a protein pair that interacts and transmits chemical signals downstream for eventual gene regulation. Histidine kinases (HKs), the first protein in these pathways, sense external stimuli such as temperature change or nutrient availability and autophosphorylate a conserved histidine residue. The phosphoryl group is then transferred to the second component in this system, the response regulator (RR), which typically influences gene expression. A bulk of the previous research has focused on a single cognate protein pair (i.e. one HK and an RR partner). While informative, these strategies do not profile the global response of an organism to a stimulus. Global profiling methods are lacking, likely due to the instability of the phosphohistidine intermediate. To address this issue, our proposed method replaces the natural substrate of HKs, adenosine triphosphate (ATP), with a more stable, thiophosphorylated analogue. This alternative substrate yields an intermediate with an increased lifetime and can therefore be more readily studied. We also intend to exploit the nucleophilicity of the sulfur to selectively label with electrophilic reagents. Thiols within a cell (e.g., cysteine) need to be capped to enable selective labeling of the thiophosphate. We propose that selective thiol capping can be accomplished through oxidation of thiols with nitroxyl radicals without compromising the nucleophilicity of thiophosphate. A second proposed method of analysis involves the use of ATP-based click probes to help elucidate mechanisms relevant to TCSs. We propose that a general procedure, such as the one described here, will be applicable to the study of many TCSs simultaneously. 

 

43. "Progress Towards the Chemical Synthesis of Prostaglandin Metabolite PGD-M," Jennifer Benoy

 

Prostaglandin D2 (PGD2) levels affect smooth muscle contraction, sleep, platelet aggregation, anaphylaxis and many diseases, such as mastocytosis.  Interestingly, PGD2 has three major metabolites: PGD-M, tetranor PGDM, and 15-deoxyD 12, 14 – PGJ2. While their respective routes of metabolism are understood, their function in different disease states remains unknown.  To study the role of oxidative stress on PGD2, an isotopically labeled internal standard of each of the metabolites is required for accurate quantification. Though there has been one successful synthesis of PGD-M, there is a need for a more direct and economic route to continue studies on PGD2 metabolism.  We propose PGD-M can be synthetically accessed in 12 convergent steps, utilizing a contrasteric allylation on an a,g-substituted cyclopentenone in the key step.

 

44. "Computational Prediction of Destabilizing Mutations in Membrane Proteins using Rosetta," Amanda Duran

AIMS:
Genetic variations can predispose humans to a number of diseases including cystic fibrosis, long-QT syndrome, and Alzheimer’s disease. Many single nucleotide variations have been linked to loss of function. This is likely because the variations cause a small change in the protein structure which can affect its thermodynamic stability. We propose to use the Rosetta Molecular Modeling Suite to predict whether mutations are destabilizing. Furthermore, the computational models can be used to understand how the mutations affect the structure of the protein. This information can be valuable for determining which therapies are best suited for the particular protein variant.
METHODS: The focus of this study is for membrane proteins, including multi-meric proteins. Few studies have looked at these systems. Rosetta includes both a membrane and symmetry mode which makes it ideal for this study. Existing structures of the wild-type proteins are used to create models with the mutation. The total energy of the wild-type and its respective variants are compared to experimentally reported values for ΔΔG unfolding. Nearly 90 variants of bacteriorhodopsin, rhomboid protease GlpG, and disulfide bond formation protein B were modeled
RESULTS: The AUC for the improved Rosetta Design protocol was 0.718 which indicates that Rosetta can often correctly predict a mutation to be destabilizing.
CONCLUSIONS: The protocol has only tested a specific system with limited experimental data. Additionally, the protocol has not been optimized for minimizing input structures. We plan to continue to test a diverse set of proteins and their variants to fully benchmark Rosetta Membrane and Membrane + Symmetry for a thorough understanding of how accurately Rosetta can predict the stabilizing effects of mutations.

 

45. "Dynamic Exo-metabolomic Response Analysis of 3-Dimensional Liver Bioreactor to Acetaminophen and N-acetylcysteine Exposure," J. Rafael Montenegro-Bruke, Cody Goodwin, Fanny Knöspel, Florian Gerstmann, Katrin Zeilinger, Marc Luebberstedt, Georg Damm, Stacy Sherrod, Srinivas Iyer, Rashi Iyer,  John A. McLean

 

The development of technology that emulates human physiology has gained enormous momentum in recent years. One of the main reasons is the interest in bridging the gap between mouse models and clinical trials in drug development. Herein, we describe the analysis of effluent from three-dimensional liver bioreactors using ultraperformance liquid chromatography-ion mobility-mass spectrometry and multivariate statistical analysis and self-organizing map based approaches for data analysis. Bioreactors with cells from 3 different donors were exposed to different doses of acetaminophen as well as N-acetylcysteine, which is widely used in acetaminophen overdose treatment. These analyses demonstrate an untargeted approach for benchmarking organ-mimic metabolic profiles and uncovering biological ramification of xenobiotic stimuli.

 

46. Identifying protein targets of curcumin and its autoxidative metabolites- The NF-kB pathway," Rebecca L. Edwards and Claus Schneider

 

Curcumin (turmeric) has been used in traditional Asian medicine as an anti-inflammatory agent for centuries. One of the targets for anti-inflammatory activity of curcumin is the NF-kB pathway. Increased levels of NF-kB activity have been associated with over 90 % of cancers. The increased activation keeps the cell in a state of sustained inflammation. We are investigating the hypothesis that inhibition of NF-kB is exerted by oxidative metabolites of curcumin rather than the parent molecule. Oxidative transformation of curcumin is a spontaneous transformation at physiological pH that leads to the formation of a series of electrophilic intermediates (quinone methide, spiroepoxide) in addition to dioxygenated bicyclopentadione (BCP) as the final product. The electrophilic reaction intermediates have the ability to covalently adduct with regulatory cysteines in target proteins. We have prepared novel curcumin synthetic analogs that do or do not undergo oxidative transformation as a tool to investigate if the ability of the molecule to autoxidize is responsible for its ability to inhibit NF-kB activity. The autoxidation rates of curcumin and the analogs were determined using UV/Vis spectrophotometry in ammonium acetate buffer, pH 7.5. RAW267.4 cells were also stably transfected with a pNFkB-TA-MetLuc with TB vector for expression of secreted luciferase. The effect of the addition of curcumin and its analogs on LPS-induced NF-kB activity was monitored using a Ready-to-glow Secreted Luciferase Reporter assay. We observed a correlation between oxidizability and inhibition of NF-kB activation. As a mechanistic explanation it has been shown that curcumin inhibits phosphorylation of Cys-179 of the upstream kinase IKKb. We have developed an LC-MS strategy to identify IKKβ peptide adducts with curcumin, its oxidative intermediates and/or its oxidative end product BCP. Results suggest the oxidative metabolites of curcumin rather than curcumin itself may be a factor in the suppression of NF-kB activity, i.e., its anti-inflammatory properties.

 

47. "Structure-based design of aminopiperidine indoles as modulators of the Ras-SOS interaction," Pratiq A. Patel, R. Nathan Daniels, Michael C. Burns, Jennifer E. Howes, Qi Sun, Taekyu Lee, Jason Phan, Edward T. Olenjniczak, Olivia W. Rossanese, Alex G. Waterson, Stephen W. Fesik

 

K-Ras is an important oncology drug discovery target based on the known functions of Ras, the importance of Ras in oncogenesis, and the presence of Ras mutations in ~30% of all human cancers. Ras exerts its effects through an array of protein-protein interactions, which typically lack binding pockets suitable for small molecule intervention; thus, discovery of potent inhibitors via traditional strategies has proven extremely challenging. An alternate approach for therapeutically intervening in the function of Ras is to target proteins that regulate Ras activity. Of particular interest is the guanidine nucleotide exchange factor (GEF), Son of Sevenless (SOS), which catalyzes the exchange of GDP for GTP, the rate-limiting step in Ras signaling. During the nucleotide exchange process, Ras engages in a protein-protein interaction with SOS to form a Ras:SOS:Ras ternary complex. Recently, our laboratory discovered aminopiperidinyl indole compounds that bind to a unique pocket on the Ras:SOS:Ras complex and increase the rate of nucleotide exchange on Ras. The structure activity relationships of the aminopiperidinyl indole series are described. With the help of X-ray co-crystal structures, a structure-based iterative medicinal chemistry approach was used to determine optimal substitutions upon the indole ring and piperidine moiety. A systematic strategy to combine successful substitutions resulted in compounds that activate nucleotide exchange at low micromolar concentrations and at levels greater than the basal exchange rate. Intriguingly, optimized compounds exhibit a paradoxical inhibition of downstream Ras signaling and produce proliferation defects in cancer cells.

 

48. "Lantibiotics from the anaerobe Ruminococcus flavefaciens FD-1," Xiling Zhao and Wilfred A. van der Donk, University of Illinois at Urbana-Champaign, Howard Hughes Medical Institute

 

Lanthipeptides are a class of ribosomally synthesized and post-translationally modified natural products, a subset of which display antimicrobial activity and are called lantibiotics. Lanthipeptide biosynthetic machinery is conserved, which facilitates discovery efforts. Genome mining revealed a lanthipeptide gene cluster within the anaerobic organism Ruminococcus flavefaciens FD-1 that represents an example of lanthipeptide combinatorial biosynthesis in which a pair of enzymes could be responsible for the modification of twelve substrate peptides. In order to systematically assess the structures and bioactivities of the peptides encoded within the cluster, a heterologous host and in vitro production strategy was employed to access the modified peptides. The presence of characteristic post-translational modifications in the processed peptides was confirmed by chiral gas-chromatography mass spectrometry and tandem mass spectrometry. Furthermore, a preliminary assessment revealed the FlvA peptides to be antibacterial and studies are underway explore this activity.

 

49. "HLTF’s Ancient HIRAN Domain Binds 3′-DNA Ends to Drive Replication Fork Reversal," Diana A. Chavez, Andrew C. Kile, Julien Bacal, Karlene A. Cimprich, Brandt F. Eichman


DNA damage can block the progression of DNA replication, lead to stalled replication forks which may collapse, and lead to genomic instability. In order to evade genomic instability, organisms have developed various DNA damage response pathways that utilize multiple fork-remodeling enzymes to stabilize and repair stalled forks. These fork-remodeling enzymes are DNA-dependent ATPases that are essential in promoting fork restart, but much is still unknown about their mechanism of action and specificity. One such enzyme is the Rad5-related Helicase-like Transcription Factor (HLTF). HLTF is a DNA translocase that is involved in DNA damage response and tumor suppression and has been shown to be involved in transcription and chromatin remodeling. In addition to a RING finger motif located within a SWI/SNF2 helicase domain, HLTF contains a HIP116, Rad5p N-terminal (HIRAN) domain, which has been predicted to function as a DNA-binding domain that recognizes damaged DNA and stalled replication forks. Here, we present structural and biochemical evidence that HIRAN binds the 3′ end of ssDNA and promotes HLTF-dependent fork reversal through its interaction with the 3′ end of ssDNA found at a fork. These studies serve as a basis for understanding the mechanism of HLTF-dependent fork reversal.

 

50. "Compilation and Analysis of Over 10,000 Gas-Phase Collision Cross Section Values," Caleb Morris

 

Gas-phase collision cross section (CCS) is a physical parameter obtained from ion mobility measurements that describe the size and shape of a molecule. To date, no effort has been made to compile the large number of CCS measurements which have been published over the last 50 years. This work presents over ten thousand CCS values obtained from a variety of drift gases and instrument platforms. Most of the CCS values were collected under uniform field conditions using drift tube ion mobility techniques. Here, CCS values were organized by chemical class and plotted as a function of molecular mass to generate “conformational space” maps which describe the scaling of size vs. mass. This projection of the data results in “S” shaped trends for each distinct chemical class (e.g. lipids, proteins, carbohydrates) to which mathematical descriptors, based on best-fit functions, were applied. Preliminary analysis suggests nitrogen drift gas gives better separation between charge states than helium, consistent with gas-polarization effects. Peptides and proteins comprised approximately 69% of the database, revealing the need for expansion of other chemical classes. Further work will involve collating recent CCS data and obtaining new measurements to facilitate more comprehensive comparisons.

 

51. "Characterization of Lysophospholipase A2 – the Major Prostaglandin Glyceryl Ester Hydrolase in Human Cancer Cells," James A. Wepy, Joseph D. Manna, Shu Xu, Lawrence J. Marnett
 
Arachidonic acid and its esterified derivative, 2-arachidonoylglycerol, are oxygenated to form prostaglandins (PG) and prostaglandin glyceryl esters (PGG), respectively. Whereas PGs elicit a wide range of known physiological effects, including vasoregulation, hyperalgesia, and inflammatory responses, very little is known regarding the biological role of PGGs due to rapid hydrolysis to free PGs resulting in low physiological concentrations. Here, we report the major PGG hydrolase in human cancer cells, lysophospholipase A2 (LYPLA2). A recently identified inhibitor of LYPLA2 activity, ML349, was used to significantly increase cellular PGGs in RAW264.7 macrophages. In addition, small interfering RNA was utilized to inhibit LYPLA2 expression in multiple cancer cell lines. These cells were probed for PGE2-G hydrolytic activity to validate LYPLA2 as the major PGG hydrolase. Purified recombinant LYPLA2 was used to evaluate and compare the kinetic rates of hydrolysis of its various substrates, including PGGs, lysophospholipids, and the prostanoid precursors, 2-arachidonoylglycerol and arachidonoylethanolamide. A crystallographic investigation into the structure of LYPLA2 reveals two large channels as well as a mobile loop near its catalytic triad providing critical information regarding the active site of this enzyme. To further investigate active site interactions, a catalytically inactivated serine to alanine mutant was expressed, purified and assessed for hydrolytic activity. Collectively, these data suggest a major role for LYPLA2 in the regulation of cellular PGGs. Furthermore, the perturbation of LYPLA2 in human cancer cells is the first instance of increasing PGG levels without altering endocannabinoid metabolism, providing a suitable target for further study of PGGs and their biological roles.

 

52. "BCL::EvoGen, an evolutionary algorithm for de novo drug design," Alexander Geanes and Jens Meiler

 

In recent years, virtual high-throughput screening (vHTS) techniques have been successfully applied to the drug discovery process.  In many cases, these vHTS techniques are leveraged to prioritize subsets of chemical libraries for acquisition and testing in physical screens.  However, the chemical space that is relevant to a particular pharmacological target may not be reflected in pre-made compound libraries, and as such it is advantageous to have algorithms which are capable of designing new chemical structures, a process known as de novo design.  An evolutionary algorithm was implemented as part of the BCL::ChemInfo suite within the Biochemistry Library (BCL), a C++ library developed in the Meiler laboratory, to iteratively generate sets of compounds for use as focused libraries.  At each step of the algorithm, sets of compounds are evaluated for their biological “fitness” using machine-learning based quantitative structure activity relationship models which correlate two- and three-dimensional molecular properties with biological activities.  The best-scoring molecular structures are then modified or mixed together to generate a subsequent set of candidate compounds.  These steps are repeated until a desired biological activity score is attained.  Here we present the results of this focused library design application, BCL::EvoGen.

 

53. "Synthesis and Kinetic Characterization of Mechanism-based Inhibitors of BioA," Carter Eiden, Courtney C. Aldrich, and John D. Lipscomb, University of Minnesota

 

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the second leading cause of mortality among infectious diseases worldwide.  The emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains necessitates the development of new antibiotics, ideally with novel mechanisms of action.  Mechanism-based inhibitor 1 has been shown to kill Mtb in biotin-deprived conditions through inactivation of BioA, a PLP dependent enzyme in the biotin biosynthetic pathway. The rational optimization of mechanism-based inhibitors is challenging since standard equilibrium dissociation constants cannot be used due to the irreversible nature of inhibition.  Rather, one must optimize microscopic rate constants of the individual steps involved in ageing the enzyme.  The inactivation of BioA by 1 is a four step process, beginning with binding of 1 to the PLP and continuing with three more transformations before an irreversible PLP adduct is formed.  Each step shows a unique absorbance signature, allowing us to investigate the kinetics using stopped-flow measurements.  Preliminary analysis suggests the kinetic bottleneck occurs within the second step, suggesting that a new warhead should be designed that can be more easily deprotonated by the lysine of the enzyme.  Using the previous information, two new inhibitors that lower the pKa of the alpha proton were designed (2 and 3).  The synthesis of each of these is described, with the key step in each synthesis the addition of a lithium acetylide to a Weinreb amide followed by cyclization after quench.  Both inhibitors have shown activity against BioA, and experiments are underway to determine their steady-state constants.

 

 

 

54. "Title TBA," Lambrecht, M. J., Brichacek, M.; Barkauskaite, E.; Ariza, A., Ahel, I.; Hergenrother, P. J., University of Illinois

 

Poly(ADP-ribosyl)ation is an important post-translational  modification that mediates cellular processes including DNA damage repair, transcription, ubiquitination, and cell death. While the enzymes that synthesize (poly ADP-ribose polymerase or PARP) and degrade (poly ADP-ribose glycohydrolase or PARG) poly-ADP ribose (PAR) have been known for decades, the actual oligomers and polymers themselves have been challenging to study due to their heterogeneous production by natural sources. The challenges in isolating PAR oligomers of defined length and structure has been a major impediment to studying their underlying function. This heterogeneity has especially been a problem for structural biology studies. To address these issues, we have developed a modular chemical synthesis of a PAR dimer as well as an alkynylated PAR dimer. To demonstrate the power of chemical synthesis to access these compounds homogeneously we have used the synthetic material to obtain the first PAR oligomer-human PARG co-crystal structures. Our crystal structure shows the importance of binding to the ADP-ribose unit of the PAR dimer, consistent with PARG’s binding of PAR through its macrodomain.  We have further used the alkynylated PAR dimer to synthesize a fluorescent PAR dimer and develop a general PAR-protein binding assay using fluorescene polarization (FP). These studies have allowed for the first KD measurements of PAR oligomers with PARG. Using mutant PARGs and this FP assay, we have found amino acids essential for PAR-PARG binding and are using this data to develop better mechanism-based PARG inhibitors.

 

55. "Electrospray Ion Mobility-Mass Spectrometry and Computational Strategies Assist in the Structural Characterization of Isomeric Polyurethane Precursors," Tiffany M. Onifer, Sarah M. Stow, Jay G. Forsythe, Jody C. May, John A. McLean, David M. Hercule

 

Methylenedianiline (MDA) is a common starting material in the production of 4,4-methylene diphenylene diisocyanate, which is a major hard block component in the production of polyurethanes. Industrial grade MDA is composed of multermeric MDA species, formed by the addition of aniline. Determining preferred protonation sites and location of aniline attachments of larger MDA multimers is of great interest to polymer scientists, because of the potential to minimize structural heterogeneity. In our previous studies we structurally characterized 2-ring MDA isomers (4,4-MDA, 2,4-MDA, and 2,2-MDA) using a combination of IM-MS, MS/MS, and computational modeling. Our results showed, similar to aniline, MDA was protonated at either amine positions or ring positions. We found that structural differences between these positional isomers and the sites of protonation contributed to the molecules gas-phase stabilities. In this study, mass spectrometry (MS) techniques were utilized to structurally characterize 3-ring and 4-ring MDA species. Tandem MS (MS/MS) strategies determine fragmentation pathways of these MDA multimers, while ion mobility-MS (IM-MS) provided insight of the gas phase conformations of ionized MDA species. Computational modeling was used to determine additional experimental approaches to gather structural information.

 

56. "Probing the conformation of C8-deoxyguanosine adduct of the carcinogenic 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) within the NarI Recognition Sequence," Arnie de Leon

 

Highly mutagenic heterocyclic amines (HCAs) such as 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)  are found in cooked meats. They can form DNA adducts at C8- and N2-positions of deoxyguanosine upon activation by cytochrome P450s. IQ and IQx are structurally similar, but IQ is five times more mutagenic than IQx in the Ames test. The structural similarities but different mutagenic activity of IQ and IQx present a good opportunity to probe the structural basis of genotoxicity of these heterocyclic amines. Our lab has previously reported the synthesis of oligonucleotides containing the C8- and N2-dG adducts of IQ as subsequent structural and bypass studies.  Here, we report the synthesis of C8-2’-deoxyguanosine adduct of IQx (C8-dG-IQx) and its incorporation into oligonucleotides. The C8-IQx-adduct was incorporated into the G1, G2 and G3 positions of the NarI recognition sequence (5’-CTCG1G2CG3CCATC-3’), which is a hotspot for arylamine modification and 2-base frameshift mutations. The modified oligonucleotides were characterized by MALDI-ToF MS, enzyme digestion and LC-MS analysis. Analysis of the UV and CD spectroscopy data are consistent with the IQx adopting a minor groove-bound conformation at G1 and G2 positions while the conformation is intercalated at G3. The sequence-dependent conformation preferences are likely to play an important role in the repair and mutagenicity of the C8-arylamine adducts.

 

57. "Probing Confirmational and Configurational Equilibria of Site-Specific 2´-Deoxyribosylurea DNA Adducts by NMR Spectroscopy," Andrew H. Kellum, and Michael P. Stone


2´-Deoxyribosylurea (urea) lesions within DNA form from the cleavage of thymine with hydroxyl radicals as a consequence of exposure to ionizing radiation.  In addition, urea lesions are formed from oxaluric acid, a lesion results from the reaction of singlet oxygen with 8-oxoguanine, in vitro under salt concentration that are relevant to cells.  Previous NMR studies of urea lesions on the nucleoside level showed the equilibration of alpha (α) and beta (β) anomers on the 2´-deoxyribose ring.  Here, we investigated a urea lesion within the oligodeoxynucleotide 5´-(CTXA)-3´ (X=urea) in single strand DNA.  Reverse phase HPLC revealed the presence of two different species at a ratio of 1:1.  NMR spectroscopy corroborated this finding along with identifying the two different species.  NMR NOESY and TOCSY experiments were used to assign the resonances of the different nucleotides by a process of elimination method.  NOESY experiments confirmed the identity of the two different species as the α and β anomeric configurations of the urea lesion.  In addition, NMR TOCSY and one-dimensional experiments in water showed that urea lesion remained in the trans conformation between the H1´ and NH amino proton regardless of the anomeric configuration. Future experiments will investigate the urea lesion at a primer-template junction as well as in duplex DNA.   

 

58. "Matrix and enzyme pre-coated substrates for high-throughput analysis of biological tissues," Faizan Zubair, Will Swisher, Paul E. Laibinis, Richard M. Caprioli
Introduction
In situ trypsin digestion is critical for direct protein identification from tissue samples while preserving the spatial localization of the analytes. Furthermore, it can enable imaging of high molecular weight proteins that may not be detected directly. Current methodology employs robotic spotters to deposit trypsin in spatially discrete regions followed by matrix deposition. This process requires many hours for a single tissue specimen and the spatial resolution is limited to 200 µm. We have developed alternative approaches that use targets pre-coated with enzymes and matrix enabling high throughput and high resolution imaging. We are looking at both frozen tissues and fixed tissues that are pre-dominant in the clinical environment and pathology.
Methods
We are developing two different approaches. In the post-coating approach, a tissue section is placed on an ITO slide and washed with solvents to remove salts, lipids and paraffin—in the case of FFPE tissues. The sample is spray coated with trypsin, incubated for 2 hrs at 37 °C, and then coated with matrix using a TM SprayerTM. In the pre-coated approach, an ITO slide is first coated with matrix and trypsin, and thin tissue sections are mounted on the top. After a xylene wash, the slide is placed in a hydration chamber saturated with di-isopropylethylamine and water that converts the matrix into an ionic form with neutral pH of 7-8. After incubation, the samples are immersed in cold 10% TFA. We used MALDI TOF/TOF and FT-ICR instrumentation to analyze our samples.
Preliminary Data
We have successfully used spray methods to apply enzymes for protease digestion of fresh frozen tissues, reducing sample preparation time and improving spatial resolution 4-fold down to 100 µm. Thin tissue sections of rat brain were analyzed using the pre-coated approach described above. Several proteins were identified including Myelin Basic Protein, PEP-19, Neurogranin and others. For each parent protein, several peptides were observed in the MALDI FT-ICR spectrum and matched to their theoretical mass to less than 1 ppm. Furthermore, each of the matched peptides for a specific protein was co-localized enabling high confidence in the identifications.
We use similar high-throughput approaches to analyze formalin fixed tissues including mouse brain and heart tissues. Xylene wash was used to remove the paraffin and the samples were antigen retrieved in Tris Buffer at 95°C. Tissue sections were spray-coated with trypsin using a SunChrom Sprayer, incubated, and then coated with CHCA in total spraying time of about 30 minutes. For comparison, a single tissue specimen typically required 4 hours for spotting trypsin and 2 hours for spotting matrix. Using spraying methods, several tissue samples may be prepared simultaneously offering reduced instrument times and enhanced simplicity for the user.
Several hundred peaks were observed in the mass range of 500-3000 m/z that were absent in the control samples that were processed without trypsin. A number of these peaks show discrete localization that can be co-related with the anatomical features of the tissue. For example in the mouse heart tissue, peaks at m/z 855.04 and 1028.10 localize in the left and right ventricles and a peak at 920.90 localizes in the right atrium. Identifications will be confirmed using on-tissue MS/MS analysis and accurate mass measurements.
Novel Aspect
Preparation of trypsin and matrix pre-coated targets for high-through analysis of frozen and fixed tissues using MALDI IMS.

 

59. "Development of novel and brain penetrant mGlu1 positive allosteric modulators for target validation in schizophrenia," Pedro M. Garcia-Barrantes, Hyekyung P. Cho, Corey R. Hopkins, Colleen M. Niswender, Ryan D. Morrison,  Anna L. Blobaum, J. Scott Daniels, P. Jeffrey Conn, Craig W. Lindsley

 

Metabotropic glutamate receptor type 1 (mGlu1) is a G-protein coupled receptor from the group I of mGlus and is encoded by the GRM1 gene. Stimulation of the receptor leads to an increase in activity of phospholipase C, with subsequent mobilization of calcium and increase of intracellular calcium concentrations. mGlu1 is expressed predominantly in post-synaptic neurons from the hippocampus, hypothalamus, thalamus, amygdala, cerebellum, basal ganglia and spinal cord. The function of mGlu1 has been implicated in several neural processes such as nociception, synaptic plasticity, learning and memory. Hypofunction and deregulation of glutamate signaling has been established as a key concept in schizophrenia pathogenesis. Moreover, mutations in GRM1 gene that lead to hypofunction of the receptor have been observed in patients with schizophrenia and bipolar disorders, supporting the modulatory role of the receptor in neuropsychiatric disorders and suggesting that therapies targeting the receptor’s function recovery might be beneficial. In order to test this hypothesis, it is necessary to have suitable chemical tools to achieve the activation of the receptor and perform target validation studies. This work present the application of molecular switches in an mGlu4 PAM chemotype for the discovery of VU0483605, a novel, selective and submicromolar mGlu1 PAM. The efficacy of this probe was also evaluated in constructs of the mGlu1 receptor with mutations that decrease the functionality of the receptor and that have been identified in the schizophrenia population. Finally, we also present our initial efforts for the optimization of this chemical probe to improve its potency and pharmacokinetic profile.

 

60. "Discovery and Optimization of Novel, CNS-penetrant Positive Allosteric Modulators of the Glucagon-like Peptide 1 Receptor," Kellie D. Nance, Lindsey C Morris, Kellie D NancePatrick R GentryEmily L Days, C David WeaverColleen M NiswenderAnalisa D Thompson, Carrie K JonesChuck W LocusonRyan D MorrisonJ Scott DanielsKevin D Niswender, and Craig W Lindsley

 

Glucagon-like peptide 1 (GLP-1) and its seven-member transmembrane receptor (GLP-1R) have recently gained interest for the treatment of type-2 diabetes (T2D). GLP-1 is a gut hormone which functions primarily to effect a post-prandial decrease in blood glucose levels by stimulating insulin secretion in pancreatic β-cells. In the brain, GLP-1 acts to suppress appetite, decrease food intake, produce a sense of satiety and can be strongly correlated with improved synaptic plasticity, neuroprotection, and cognition. Deficiencies in these key neurological aspects may lead to Parkinson’s and Alzheimer’s diseases. GLP-1 has a half-life of 2-4 minutes in the body, and as such does not make for a good therapeutic. A few GLP-1 mimetics have been developed that avoid this issue, but they often cause side effects such as nausea and vomiting, sometimes to the point of patient non-compliance.  Seven-transmembrane receptors, sometimes known as G-protein coupled receptors, belong to a superfamily of cell surface signaling receptors that are implicated in many disease states, and as such are heavily investigated as drug targets. These receptors not only possess an orthosteric site at which to bind molecules that will either activate or inactivate the receptor, they also possess an allosteric site. Allosteric sites allow for “tuning” of the receptor’s activity. Molecules that decrease receptor activity are known as negative allosteric modulators, while molecules that bind and increase receptor activity are known as positive allosteric modulators (PAM). Positive allosteric modulation of the GLP-1 receptor would be useful, not only in increasing the activity of endogenous GLP-1 in patients with T2D, but could also be used as a co-therapy with current mimetics to increase the efficacy of these biologics, decrease the dose required, and potentially decrease side effects. Recent efforts have led to the discovery and optimization of the first highly selective and CNS penetrant GLP-1R positive allosteric modulator (PAM), which has shown to modulate the effects of GLP-1 as well as two GLP-1 mimetics, to augment insulin secretion in primary mouse pancreatic islets, and efficacy in potentiating endogenous GLP-1R to reverse haloperidol-induced catalepsy, an in vivo model for Parkinson’s disease.

 

61. "Acinetobacter baumannii combats membrane stress in the lung through maintenance of lipid asymmetry," Lauren D. Palmer and Eric P. Skaar

 

Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is a leading cause of ventilator-associated pneumonia. A. baumannii is increasingly multi-drug resistant, which necessitates the development of new anti-microbial therapeutics. Understanding bacterial factors required for pathogenesis in the lung may identify novel therapeutic targets. An A. baumannii genetic locus encoding a putative ATP-binding cassette (ABC) transporter was reported as important for colonization and persistence in the murine lung in a high throughput mutant screen. This transporter shares significant homology with the maintenance of lipid asymmetry (mla) locus previously described in Escherichia coli. Deletion of the putative mlaF in A. baumannii results in increased sensitivity to outer membrane stress, confirming its assignment as a member of the mla operon. In a murine model of pneumonia, an A. baumannii ΔmlaF mutant was dramatically attenuated in pathogenesis of the lung (four orders of magnitude lower bacterial burdens) and dissemination to the liver. Interestingly, co-infection with wild-type complemented the ΔmlaF mutant in dissemination to the liver but not in survival in the lung, suggesting the ΔmlaF mutant pathogenesis defect is specific to the lung. The lung presents a unique environmental challenge to bacteria, including the presence of high levels of surfactant proteins and lipids. Therefore, the ΔmlaF mutant strain was tested for survival in pulmonary surfactants ex vivo, and was defective compared to wild-type. These data suggest that the Mla system contributes to bacterial pneumonic pathogenesis by conferring resistance to lung-specific membrane stresses.

 

62. "Molecular probes for non-invasive visualization of hormone receptor expression and activity in disease," Michael Caldwell, Taryn Townsend, Preeti Sukerkar, Georgette Heyrman, Joanna Burdette, Thomas J. Meade


Despite an intense research focus, reproductive cancers and diseases are a significant source of hardship and mortality in the U.S.. As the number and efficacy of targeted neoadjuvant therapies continues to grow, the importance of effective screening and personalized treatment has never been higher. Steroid hormone receptors (HRs) are important biomarkers, therapeutic targets, and drivers of disease in these conditions due to their major roles in cell behavior in reproductive tissues. Further, the expression and activity of HRs are dynamic across the development of disease and transition to treatment-resistant phenotypes. For these reasons, non-invasive detection and monitoring of HRs could provide significant advantages in screening, treatment assignment, and evaluation of response to therapy. Our lab is developing molecular probes to image HRs in disease using magnetic resonance imaging (MRI). These probes incorporate a HR targeting domain conjugated to a paramagnetic Gd(III) chelate to produce MRI contrast. MRI provides a number of key advantages for non-invasive imaging. First, MRI provides excellent spatiotemporal resolution and soft tissue contrast with no depth limitation. Second, because MRI contrast agents (CAs) are non-radioactive and shelf stable, their use does not require special facilities or exposure to ionizing radiation. For these reasons, MRI is excellently suited for screening and longitudinal monitoring applications. Our research is currently focused on the development of a progesterone receptor (PR) targeted CA for applications in breast cancer, ovarian cancer, and endometriosis. We have shown probes synthesized in our lab are able to accumulate in PR(+) tissues and xenografts in vivo to produce detectable contrast. In order to extend this success to more advanced pre-clinical models, it is necessary to optimize the physical and biological properties of these probes. In addition, we are pursuing multimeric strategies to significantly increase the contrast enhancement of each probe by incorporating multiple Gd(III) chelates. Recent efforts have shown that direct conjugation of the targeting domain with polyethylene glycol (PEG) linkers results in significantly increased water solubility, but leads to diminished biological activity. New designs aim to capitalize on the significant increase in hydrophilicity of these linkers while maintaining balanced polarity and sterics near the targeting domain of the probe.

 

63. "Development of a screening assay platform as a novel approach to material fabrication & design," Jenny Nesbitt

 

Biomineralization is a great source of inspiration for the material scientist due to the mild synthetic conditions, control, and functionality of inorganic materials produced in biological systems.  While many studies focus on mimicking biomineralization techniques in vitro, we are adopting a novel approach to material development by utilizing biological methods in vivo.  Diatoms, a subset of algae, are photosynthetic single-cell eukaryotes that create a silica cell wall, or frustule, that is both species-specific and nano-patterned.  We will exploit the diatom’s hierarchical control of silica patterning by adding small molecules to the diatom environment to induce changes in the silica formation.  We are developing a higher-throughput assay to more rapidly examine a large library of diverse small molecules to maximize the possible morphological changes of the frustule. In this way, we hope to fabricate, with a high degree of reproducibility, a large amount of non-orthogonal three-dimensional structures with nanoscale patterning.

 

64. "Incorporating Small Angle X-Ray Scattering Experimental Data into the BCL::Fold Protein Structure Prediction Software, Oanh Vu, Dainel Putnam

Small angle X-ray scattering (SAXS) is a structural characterization method which provides information of protein shapes and sizes. We seek to combine experimental SAXS data with BCL::Fold, - a de Novo protein prediction algorithm that assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs). We previously implemented a method in BCL::Fold to regenerate SAXS profiles from assembled models and compare those simulated SAXS profiles with experimental counterparts by computing SAXS scores. A lower SAXS score suggests higher structural consistency between a BCL::Fold model and its native protein, and vice versa. To prepare positive controls for our benchmarks, side chains and loop regions of either nuclear magnetic resonance (NMR) or crystallographic structures of 17 protein samples were omitted, and then simulated and recovered by BCL::Fold. During a protein folding run with BCL::Fold we will penalize models that deviate from the overall SAXS score by incorporating it as a term in the linear weighted scoring function.  Further, we plan to compare our fold benchmark sets with and without SAXS restraints. We expect that overall lower SAXS scores with SAXS restraint implementation will allow us to filter poorly assembled protein models early in the prediction process.

 

65. "Investigating the role of DksA and ppGpp in regulation of rRNA promoters and photosynthetic growth in Rhodobacter sphaeroides," Kemardo Henry, Kimberly Lemmer, Wilma Ross, Timothy Donohue, Richard Gourse, University of Wisconsin-Madison


Ribosomal RNA (rRNA) synthesis is a rate-limiting step in the production of ribosomes (Paul et. al., Cell. 2004). The small molecule, ppGpp, and protein DksA are regulators of transcription in Escherichia coli and have been studied extensively for their effects on regulation of rRNA and amino acid biosynthesis genes. DksA and ppGpp work synergistically to inhibit open complex formation and thus reduce rRNA synthesis, whereas they increase transcription from certain amino acid promoters. Like all bacteria studied to date, the photosynthetic bacterium Rhodobacter sphaeroides synthesizes ppGpp, but it has been reported that it is made in response to different signals than in E. coli; ppGpp appears to control responses to light rather than to amino acid availability (Campbell and Lueking, J. Bac. 1983). In addition, R. sphaeroides rRNA synthesis has been previously shown to be upregulated during photosynthetic growth (Dryden and Kaplan, J. Bac. 1993). Recently, we showed that there is a DksA homolog in R. sphaeroides (RSP2654) with properties similar to E. coli DksA (DksAEc). Like DksAEc, RSP2654 interacts with E.coli RNA polymerase (RNAPEc) and reduces the lifetime of RNAP complexes with the rrnB P1 promoter. DRSP2654 cells were unable to grow photosynthetically or to utilize exogenous amino acids, inferring it might play roles in photosynthesis and amino acid transport (Lennon et. al., mBio 2014). To further characterize the roles of DksA and ppGpp in transcription regulation, we are developing an in vitro transcription system to determine the mechanism(s) by which these factors exert their effects on R. sphaeroides RNAP (RNAPRs). Using the in vitro system, I will determine if ppGpp and DksARs are involved in the regulation of R. sphaeroides rRNA synthesis. We are utilizing expression microarrays to identify promoters regulated by DksA and ppGpp in R. sphaeroides, and use the developed in vitro transcription system to determine which of these effects are direct.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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