HG2BG will be a hybrid symposium: in-person and Zoom. Please contact us via our symposium email (hitchhikersguide2biogal@gmail.com) for any questions.

Poster Session Abstracts

Poster Session--Purdue Memorial Union South Ballroom

11:00 AM - 12:00 PM Poster Session Group 1 ---Even numbered posters

1:00 PM - 2:00 PM Poster Session Gropu 2 ---Odd numbered posters

Poster 1--Yu-Chen Yen

Cryo-EM structures of ACKR3 reveal molecular mechanisms for biased signaling and promiscuity 

Atypical chemokine receptor 3 (ACKR3) is a seven-transmembrane chemokine receptor that signals exclusively through beta-arrestin when activated by its endogenous ligand, CXCL12. CXCR4, a canonical receptor that also responds to CXCL12, however activates both G-protein and - arrestin signaling pathways. ACKR3 is also known to display constitutive internalization and recycling, and acts as a decoy receptor that transports CXCL12 to lysosomes for degradation. The scavenging process is important for regulating extracellular CXCL12 concentration and CXCR4 signaling, and hence to cell migration and metastasis. In addition to CXCL12, ACKR3 scavenges a large variety of other ligands, most of which activate ACKR3. To understand the molecular mechanism underlying its biased signaling and this promiscuous activation, we determined seven structures of ACKR3 bound to three different ligands using cryo-electron microscopy (cryo-EM). Our structural models represent the first of atypical chemokine receptor and reveal a unique chemokine binding mode. In the orthosteric binding pocket, several side chains of ACKR3 are rearranged to engage different ligands, underscoring its plasticity for ligand recognition. The cytoplasmic binding pocket of ACKR3 overall adopts an active-like configuration but has a more compact cytoplasmic cleft and a unique conformation of intracellular loop 2 (ICL2), both of which could prevent ACKR3 from binding to heterotrimeric G-proteins and instead promote biased signaling. These features may be more broadly representative of how other receptors can be induced to preclude heterotrimeric G proteins by biased agonists. 

Poster 2--Elias Beretta 

Substrate-based novel dimeric prodrug inhibitors of human P-glycoprotein

The human multidrug resistance transporter P-glycoprotein (P-gp) is highly expressed at blood- tissue barriers, including the blood-brain barrier (BBB), and poses a serious challenge for the delivery of cancer therapeutics to the brain. Additionally, expression of P-gp is also detected in some cancers, and is thought to possibly limit the uptake of therapeutics, leading to a multidrug resistant phenotype (MDR). P-gp has multiple substrate binding sites and uses the energy of ATP hydrolysis to actively transport a variety of hydrophobic compounds out of the cell from the plasma membrane. Our goal is to take advantage of the polyvalency of the substrate binding site to create P-gp inhibitors from substrates through dimerization. Herein, we synthesized and characterized two libraries of dimeric prodrug inhibitors of P-gp based on the substrates temozolomide and dasatinib, a glioblastoma multiforme chemotherapeutic and chronic myelogenous leukemia chemotherapeutic, respectively. In addition to inhibiting P-gp, by containing reversable tethers, these dimers are designed to act as prodrugs and release the therapeutics inside the cell. To improve the efficacy and solubility of our dimers we used the known substrate quinine to generate libraries of temozolomide-quinine and dasatinib-quinine heterodimers with varying tether lengths. Both libraries were shown to be potent inhibitors of P- gp efflux at low micromolar concentrations. Current ongoing experiments include testing the efficacy of the dimers against their respective cellular targets as compared to the therapeutic alone, as well as assessing the kinetics of dimer breakdown and accumulation of therapeutics within the cell. Another area of investigation is using cryo-EM to gain structural insights as to how our dimers bind to and inhibit P-gp efflux.

Poster 3--Weiwei An 

Study of the binding mechanisms with the optimized Inhibitors of Vancomycin-Resistant Enterococcus

Enterococci are the gram-negative cocci that dozens of species commonly exit in human gastrointestinal tract. Two of them are responsible for the majority of human infections including E. faecalisand E. faecium. Vancomycin had been widely used in clinical as a treatment for methicillin-resistant staphylococci and other gram-positive bacteria for decades without marked resistance. However, the Vancomycin-Resistant Enterococcus (VRE) had been widely detected and reported in hospitals in Europe and the United states in 1988.1VRE infection is one of the leading causes for hospital-acquired infections (HAIs) in the United States, which is responsible for 10% mortality rate of 55,000 cases in 2017. To combat VRE, we repurposed the FDA-approved drug Acetazolamide (AZM) and developed series of analogs for structure-activity relationship optimization. One of the leads CAI0019 has significantly improved the potency from AZM MIC = 2 µg/mL to CAI0019 MIC = 0.007 µg/mL for E. faecium strain HM-965.4Other analogs also showed a much lower MICs compared with AZM. To better understanding the mechanism behind the MICs, we further studied the binding affinity towards proteins including Neisseria gonorrhoeaecarbonic anhydrases (NgCA), VRE-carbonic anhydrases 𝛂(VRE-CA𝛂), VRE-carbonic anhydrases y(VRE-CAy), human carbonic anhydrases I (hCAI) and human carbonic anhydrases II (hCAII) together with the binding positions of the analogs with the Schrodinger software. In this poster, we will elaborate the relationships between the MICs of the hits, binding affinity towards the proteins and the binding positions from the computational data.

Poster 4--Arezoo Ardekani 

Monoclonal Antibody Aggregation near Oil−Water Interfaces

In this work, we study the hydrodynamic behavior of monoclonal antibodies in the presence of silicone oil−water interfaces. We model the antibody molecules using a coarse-grained 24-bead model, where two beads are used to represent each antibody domain. We consider the spatial variation of the antibody polarity in our model as each bead represents a set of hydrophilic or hydrophobic amino acids. We use the dissipative particle dynamics scheme to represent the coarse-grained force field which governs the motion of the beads. In addition, interprotein interactions are modeled using an electrostatic force field. The model parameters are determined by comparing the structure factor against experimental structure factor data ranging from a low concentration regime (10 mg/mL) to a high concentration regime (150 mg/mL). Next, we conduct simulations for a suspension of antibody molecules in the presence of silicone oil−water interfaces. Protein loss from the bulk solution is noticed as the molecules adsorb at the interface. We observe dynamic cluster formation in the solution bulk and at the interface, as the antibody molecules self-associate along their trajectories. We quantify the aggregation using a density clustering algorithm and investigate the effect of the antibody concentration on the diffusivity of the antibody solution, aggregation propensity, and protein loss from the bulk. Our study shows that numerical simulations can be an important tool for understanding the molecular mechanisms driving protein aggregation near hydrophobic interfaces. 

Poster 5--Surya Aryal

ER-GCaMP6f: An Endoplasmic Reticulum Targeted Calcium Probe to Study Calcium Signaling in Astrocytic Soma and Processes

Astrocyte calcium signaling is very important to understand normal brain function as well as brain activity during neuropathological conditions and substance use disorder. Despite the importance of understanding calcium activity in astrocytes, one major challenge is currently available sensors target either plasma membrane (PM) or the lumen of the endoplasmic reticulum (ER). We use genetic and molecular biology tool to develop the sensor and utilized fluorescence microscopy-based tools to characterize the sensor. Here we developed an ER targeted calcium sensor which is located at the cytosolic side of the ER. We verified its specific location of the sensor in the ER using super resolution microscopy and showed that it can be well expressed in astrocytic cell soma and processes.  It can detect calcium events in the cell and processes as well as astrocytes in vivo. We also demonstrated that it can be used in combination with TIRF microscopy to detect very small calcium fluctuations. Our pharmacological studies demonstrated this sensor measures calcium activity differently than the currently used plasma membrane sensors. In conclusion our sensor reports calcium signaling in close proximity to the endoplasmic reticulum in astrocytic soma, processes and in vivo.

Poster 6--Abhishek Bandyopadhyay

Structure of EEEV in complex with a neutralizing intact IgG reveals a unique binding mode to a quaternary epitope.

 Alphaviruses are a serious threat to both humans and animals. Currently, there are no approved vaccines or drugs for public use. Among the New World alphaviruses, Eastern equine encephalitis virus (EEEV) poses significant challenge to public health due to a rise in human cases and high mortality. Numerous neutralizing antibodies against several alphaviruses, including EEEV, have been reported in the literature, and the structural basis for their recognition as intact antibodies is lacking. In this report, we present the first description of a 3.8 Å cryo-EM structure of EEEV in complex with a potently neutralizing and therapeutic human IgG isolated from a donor who survived natural EEEV infection. The mechanism of neutralization appears to involve inhibition at the post-attachment step in the virus infection cycle. The structure reveals, that the IgG crosslinks trimeric spikes on the viral surface across the icosahedral two-fold in a unique binding mode to a quaternary epitope. Interestingly, the Fab version of the IgG exhibits negligible binding to the virus, an observation that is explained by the mode of binding and kinetic analysis of the interaction. Given the structural similarity between alphaviruses, the findings described in this work may aid in the development of alphavirus vaccines.

Poster 7--Courtney Bingham

Structural characterization of a pH-dependent photoswitching mechanism in hCRBPII at atomic resolution

Human cellular retinol binding protein II (hCRBPII) is a cytosolic transport protein with a large binding cavity and few evolutionarily conserved residues, making it an ideal engineering scaffold. Rational mutagenesis of the protein binding pocket has been used to design new functionality into hCRBPII. A mutated lysine residue provides a covalent binding site for aldehyde ligands, creating a Schiff base which can exist in either a protonated (PSB) or neutral (SB) state. Previous work hCRBPII was transformed into a fluorescent protein system by mutating the protein binding pocket to bind synthetic fluorophore FR1V. The FR1V-hCRBPII complex displays photoswitching behavior upon exposure to either UV or visible light, turning the system on or off, respectively. Here, x-ray crystallographic methods are employed to characterize the photoswitching behavior of the FR1V-hCRBPII complex at atomic resolution. The pH-dependence of this photoswitching activity is also explored, leading to the discovery of two distinct photoswitching mechanisms driven by microenvironmental pKa changes to the protein binding pocket.

Poster 8--Sherlene Brown 

Discovery and Characterization of a Fic Protein from Bordetella Bronchiseptica with Guanylyltransferase Activity

Fic (filamentation induced by cAMP) proteins regulate diverse cellular processes in bacteria. While Fic proteins predominantly utilize ATP to post-translationally modify target proteins, some utilize other nucleotide derivatives to alter the activity of their target. Bordetella sp. causes respiratory tract infections, including whooping cough in humans. A combination of waning immunity to B. pertussis and the emergence of human-adapted B. bronchiseptica strains have resulted in recent epidemics of whooping cough-like illnesses worldwide – highlighting the presence of novel Bordetella proteins critical for virulence and/or fitness. Such proteins would be key candidates for a more effective vaccine designed for newly circulating Bordetella strains. Interestingly, we discovered a Fic protein, BbFic in Bordetella bronchiseptica, that fits the transcriptional profile of such predicted virulence factors. Unlike most Fic proteins that preferentially bind and utilize ATP as a nucleotide source, BbFic weakly binds ATP and instead shows a 30-fold increased preferential usage for GTP. We thus report the discovery, enzymatic characterization of BbFic as a bona fide guanylyltransferases, and present structural insights into BbFic-GTP interaction. We solved the crystal structure of apo BbFic at 3.1 Å and generated an AlphaFold model of BbFic. Using molecular docking and mutagenesis, we elucidated a mechanism for GTP recognition, which implicates two arginine residues within its nucleotide-binding pocket (Flap). Furthermore, our bioinformatics analyses of the entire Fic protein superfamily (pfam 02661) to identify similarity networks using BbFic as an index protein identified a sub-cluster of proteins that also function as guanylyltransferases. The importance of our work is two-fold: 1) BbFic represents a new category of fitness genes predicted to play a role in new host-adaptations for Bordetella, and 2) BbFic frames the groundwork for understanding Fic-mediated GMPylation (addition of GMP) as a novel post-translational modification in signal transduction.

Poster 9--Patrick Carmody

Modulation of Ribosomal Frameshifting by Cotranslational Folding and Misfolding of the Nascent Polypeptide Chain

Membrane protein folding is an error-prone process that is subject to numerous forms of biochemical regulation. The ribosome and its ensemble of accessory proteins employ a variety of mechanisms to monitor membrane protein folding and misfolding during translation that are essential for the maintenance of protein homeostasis. Nevertheless, it’s unclear how this biosynthetic machinery detects and responds to folding and misfolding in real time. We recently found that the mechanical forces generated by the translocon-mediated membrane integration of transmembrane domains can stimulate a translational recoding event known as -1 programmed ribosomal frameshifting (-1PRF). Alphaviruses modulate the efficiency of -1PRF through crosstalk between an mRNA structure that pauses the ribosome on a slippery-sequence of the transcript during the translocon-mediated cotranslational folding. Based on the features of this motif, we searched the human transcriptome for other potential sites that may promote this cotranslational feedback and identified hundred potential candidates. Preliminary efforts to validate certain hits have identified an active -1PRF motif within the cystic fibrosis transmembrane conductance regulator (CFTR), the misfolding of which causes cystic fibrosis. Ribosomal frameshifting at this site results in the premature termination of translation, and knocking out this motif partially restores the expression of a common CFTR variant (ΔF508) that undergoes cotranslational misfolding. These findings suggest a novel role for ribosomal frameshifting in eukaryotic protein homeostasis.

Poster 10--Laura Chamness

Molecular Basis for the Evolved Instability of a Human G-Protein Coupled Receptor

Many eukaryotic membrane proteins are prone to misfolding, which compromises their function at the plasma membrane. This is particularly true for mammalian gonadotropin-releasing hormone receptors (GnRHRs), which are G-protein coupled receptors involved in reproductive steroidogenesis. There is evidence to suggest that the inefficient folding and cellular trafficking of mammalian GnRHRs is the result of selection for attenuated activity. We show that evolutionary modifications to GnRHR contribute to improper membrane integration of the transmembrane domains (TMDs). In particular, a C-terminal truncation can cause misintegration of the seventh TMD. Additionally, the enhanced polarity of TMDs 2 and 6 compromises their integration into the hydrophobic membrane and induces cotranslational misfolding. A mutation that restores hydrophobicity to TMD 6 causes increased plasma membrane expression in human GnRHR. Evolutionary variations in hydrophobicity at this site also correlate with reproductive phenotypes. Together, these findings suggest that nature has exploited the energetics of membrane protein folding to tune fitness.

Poster 11--Emily Chan

Defining the roles of biophysical membrane tension in breast cancer cell migration

Breast cancer is the most common malignancy and the leading cause of cancer-related death in women worldwide. Triple-negative breast cancer (TNBC), which accounts for 10-20% of all breast cancers, is the most aggressive breast cancer subtype with an overall 5-year relapse of 40% owing to metastatic spread of cells from tumor sites. As metastatic cancer cells detach from a tumor site and acquire a highly migratory phenotype, their successful progression to distant organs relies, in part, on membrane trafficking pathways to uptake and secrete factors that assist them in navigating within their surroundings. Notably, endocytic membrane trafficking, responsible for the cellular uptake of key cell migration-promoting factors, has been shown to be easily disturbed by tension at the plasma membrane. While tension gradients assist in maintaining a front-to-rear polarity of the cell during migration, recently it has been shown that these gradients create an asymmetry in endocytic dynamics across the plasma membrane. However, questions remain about whether this asymmetry is intrinsic to migratory TNBC cells and what its role is in cell migration. We aim to develop experimental and analytical approaches that will allow us to monitor clathrin-mediated endocytosis (CME) and membrane tension in migrating TNBC cells. This involves utilizing a microfluidic platform that simultaneously allows the leading and trailing edge of a cell to be separately treated during migration and enables the application of inputs native to the cellular microenvironment. To establish the role of asymmetric membrane trafficking in cell migration, we will separately apply small molecule CME inhibitors at the leading and trailing edges of migrating TNBC cells. Here, we discuss initial results showing an inverse relationship between CME dynamics and membrane tension in stationary TNBC cells and our optimization of the microfluidic platform.

Poster 12--Hok Wai Chang

Image Analysis and Feature Extraction Pipeline for Spatial Properties of RNA molecules in β-Cells of Type 1 Diabetes

We developed a computational pipeline with the emphasis on analyzing RNA localization and classifying β-cells in human tissue sections that are associated with the type 1 diabetes (T1D). The pipeline first consists of cell segmentation, object detection and localization analysis of RNA, then features are extracted from RNA localization properties using physical and statistical methods with biological implications. Features can also be extracted by measuring the interactions between different types of RNA. Each individual cell can then be represented by a list of RNA localization features and be used as training data in a feature-based classification method. By implementing our pipeline with research projects on T1D, we investigated whether features extracted from RNA localization can be the determining factor for classifying healthy cells and cells with T1D. Results show an 82% accuracy for the constructed classifier when using features extracted solely from RNA localization and RNA interactions properties. The classifier also sheds light on the importance of each feature, allowing further investigations on biological implications of individual features and its connection with T1D. Overall, the pipeline provides a systematic and effective method to analyze and discover the RNA localization properties and functionally of β-cells associated with T1D.

Poster 13--Yueyi Chen

Development of highly potent and selective GRK5 inhibitors

G protein-coupled receptor kinases (GRKs) regulate cell signaling by triggering receptor desensitization via phosphorylation on G protein-coupled receptors (GPCRs). (1)The seven human GRKs (GRK1−GRK7) are classified into three subfamilies: GRK1 (GRK1 and GRK7), GRK2 (GRK2 and GRK3), and GRK4 (GRK4, GRK5, and GRK6). GRK2 and GRK5 are the most abundant in cardiovascular tissue, where they are potential targets for treatment of cardiovascular disease. GRK5 also undergoes Ca2+/calmodulin-dependent nuclear localization, where it phosphorylates histone deacetylase 5 (HDAC5), inducing an increase in transcription of cardiac hypertrophy-related genes. In GRK5-knockdown mice, cardiomyocytes are protected from hypertrophy; however, the specific roles of GRK5 in heart failure and hypertrophic cardiomyopathy are still unclear. Furthermore, GRK5 is required for cancer progression in various cancer types. Depletion of GRK5 has been shown to suppress prostate cancer, breast cancer, and non-small-cell lung cancer. (2)Therefore, targeting GRK5 can also be a chemotherapeutic strategy. Here we are testing a series of inhibitors for GRK5/6 derived from the indolinone scaffold, utilizing Cys474 residue unique in GRK5/6 to enhance selectivity by covalent capture. An immediate goal is to facilitate the development of more selective inhibitors via rational design by determining x-ray crystal structures of inhibitor complexes with GRK5. Recently, we have crystallized a hyper-phosphorylated GRK5, and the active site structure suggested the possibility for compound soaking. Overall, discovery of the inhibitors can significantly facilitate understanding and treatment of cardiovascular diseases and cancer.

Poster 14--Stephanie Clark

One step ahead of a constantly evolving virus: a kinetic comparison of SARS-CoV-2 papain-like protease mutants.

Since emergence of novel coronavirus, SARS-CoV-2, in 2019, there have been over 450 million cases of COVID-19 and over 6 million deaths around the world. This pandemic has brought coronaviruses to the spotlight as a virus family with potential to cause great harm, hinted at by the previous human coronavirus outbreaks of SARS-CoV-1 in 2003 and MERS in 2013. SARS-CoV-2 has proved to be rapidly evolving over time and development of antivirals needs to be studied in the context of emerging mutants in order to understand mechanisms of resistance and how to overcome them. One drug target of SARS-CoV-2 is its papain-like protease (PLpro), encoded in nonstructural protein 3 (nsp3) region of the genome. The PLpro enzyme is a cysteine protease that functions to cleave apart the translated polyprotein sequence, has deubiquinating activity, and has deISGylation activity as an interferon antagonist. As SARS-CoV-2 continues to mutate, some of these mutations, tracked by the database Coronavirus3D, are occurring in the PLpro gene and therefore it is important to understand how they could affect substrate binding compared to the wild type. Kinetic assays were performed on the purified PLpro mutants and were tested against substrates ISG15-AMC, ubiquitin-AMC, and RLRGG-AMC individually to determine comparable kcat and Km values across mutants and compared to the wild type proteases.

Poster 15--Voichita Dadarlat

Best Practices for Responsible Conduct of Research – Life Sciences.

The posterwill be in fact a series of posters that were originally created by DHHS/ORI with minimal modifications by Purdue; the posters are available for review on EVPRPs RCR website at: https://www.purdue.edu/research/oevprp/regulatory-affairs/responsible- conduct.php, in the section RCR Resources, please see RCR Infographics. I would greatly appreciate if the organizing committee would review this proposal for RCR outreach/efforts to promote and enhance a culture of research integrity at Purdue and let me know if is accepted (send an email to voichi@purdue.edu).

Poster 16--Emily David 

Understanding the Lipid-Dependent function of SARS-CoV-2 E Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for COVID-19, a disease which has resulted in the death of millions worldwide over the span of a year. COVID-19 ranges in severity of symptoms which are related to the inflammatory response. This inflammation can impact cardiac health and result in damage. Although the field has progressed significantly, there is still a critical gap in understanding virus-host lipid interactions that contribute to viral assembly and budding of this lipid-enveloped virus. There are four structural proteins encoded in the SARS-CoV-2 genome: membrane, envelope (E), nucleocapsid, and spike. These proteins give shape to the bilayer lipid coat that encapsulate the genomic core necessary for virus infection and replication. Although we know that these proteins are essential for viral reproduction, little is understood on how lipid species regulate their assembly. Inflammation is common during infection and one enzyme that participates in this process is ceramide kinase (CERK), which synthesizes the pro-inflammatory lipid, ceramide-1-phosphate (C1P). CERK is a therapeutic target for a variety of inflammatory disorders. My primary goal is to define the relationship between CERK activity and SARS-CoV-2 assembly. Here we share our preliminary lipid binding interactions between SARS-CoV-2 E and sphingolipids ceramide in vitro and C1P in vivo. With these findings, I am interested in how CERK regulates E protein localization and formation of virus-like particles and the process by which E-lipid interactions contributes to membrane curvature changes necessary for formation of new viral particles. Overall, I anticipate that this study will help define the relationships between a host enzyme and host membranes and the assembly of SARS-CoV-2.

Poster 17--Andrew DeMarco

Coupling auxin-inducible degradation with quantitative proteomics for in-vivo protein functional characterization

Functional characterization of proteins typically involves disrupting expression by genetic deletions, mRNA knockdowns, or by inhibiting function with conditional mutations, followed by observation of concomitant biological effects. These methods, however, are prone to indirect effects resulting from the permanent or long-term changes to the target protein expression/function. In contrast, the auxin-inducible degradation (AID) system allows very rapid depletion of endogenous target protein levels, thereby minimizing deleterious effects from long- term perturbation caused by some traditional methodologies. Combining the AID system with quantitative proteomics in applicable model systems allows detection of proteome changes immediately following target protein degradation. The rapid depletion achieved by AID is well- suited to studying the biological specificity of regulatory enzymes and confidently identifying their direct substrates. Our lab is interested in understanding the role of phosphatases in the regulation of the cell cycle. We demonstrate the power of combining AID and proteomics in the budding yeast, S. cerevisiae, by targeting components of protein phosphatase 2A (PP2A), an abundant heterotrimeric phosphatase with numerous substrates involved in multiple cellular processes, including the cell cycle. The AID system achieves ≥ 85% reduction in the abundance of PP2A components within 10-15 minutes after auxin addition, resulting in detectable phosphoproteome changes by 20 minutes. The rapid response time allows us to selectively probe the proximate phosphoproteome changes enriched with direct PP2A substrates while minimizing long-term, indirect effects resulting from slower transcriptional changes and cellular adaptation responses. Current work is focused on applying this system to studying the contributions of different PP2A complexes at different cell cycle stages, providing novel information on PP2A regulatory contributions to this fundamental biological process. The AID system coupled with proteomics is broadly applicable for studying the role of almost any protein in diverse physiological settings and model organisms.

Poster 18--Shubham Dubey

Structural insight into the transferrin-iron import system from pathogenic Neisseria

N.  gonorrhoeae and N. meningitidis are obligate human pathogens that cause gonorrhea and meningitis, respectively. Vaccines are available against N. meningitidis, however, they are not universally effective and there is currently no vaccine against N. gonorrhoeae. And the rapid emergence of drug resistance in these pathogens has elevated their threat to public health, underpinning an immediate need to find better countermeasures. Essential surface machineries are promising therapeutic targets against Neisseria, one of which is called the Tbp system consisting of a transporter called TbpA and a co-receptor TbpB. Together, these proteins mediate iron scavenging from human transferrin (hTF). Despite knowing the structures of both receptor proteins for more than a decade, exactly how they coordinate with one another and the mechanism for how iron is extracted/imported remain unknown. In our recent studies, we have determined the cryo-EM structures of the double complex between TbpA+holo-hTf and the triple complex between TbpA+TbpB+holo-hTF. Our structural studies have provided new insight into their interactions with each other and provides the framework for deciphering the mechanism for iron piracy.

Poster 19--Nelly Elshafie

miRNome Expression Analysis in Canine DLBCL 

Lymphoma is a prevalent malignancy in dogs. Diffuse large B-cell Lymphoma (DLBCL) is the most common subtype, representing about 50% of the clinically seen lymphoma cases. Thus, the search for additional biomarkers capable of early detection and monitoring of DLBCL is important for improving and sustaining remission rates. The next-generation sequencing technology provides innovative information about biomarkers and can be used to characterize the differential expression of genes. This study broadens the knowledge on the molecular biogenesis of DLBCL by investigating the role of gene expression regulators called microRNAs (miRNA). Noncoding miRNAs negatively regulate gene expression by binding to the 3'-untranslated region of protein-coding mRNA, leading to either targeted RNA degradation or translational repression. MiRNAs' stability and easy accessibility make them promising biomarkers for identifying and sub-classifying lymphoma patients. We isolated and sequenced miRNAs from ten fresh-frozen lymph node tissue samples and compared them to healthy controls (six DLBCL and four healthy). Small RNA sequencing (sRNA-Seq) analysis identified a total of 35 differentially expressed miRNAs (DEMs). RT-qPCR confirmed 23/35 DEMs (14 upregulated and 9 downregulated) in DLBCL. The unpaired parametric Welch's 2-sample t-test and false discovery rate (FDR) were used to determine significant differences in average expression fold-change (2-∆∆Cq) of each miRNA in the DLCBL and healthy groups. The results were normalized using the geometric mean of the expression level of miR-361-5p, miR-101, and miR-29c-3p, the most stably expressed reference candidates. Ultimately, our results demonstrate the potential to harness miRNAs as unique diagnostics and therapeutics targeting DLBCL. 

Poster 20--Isaac Fisher

Single Particle Cryo-EM Reconstruction of an Activated Gβγ-PLCβ Complex

G protein-coupled receptors (GPCRs) regulate diverse physiological processes in health and disease through activation of the heterotrimeric G protein subunits, Gα and Gβγ. G proteins in turn activate effector enzymes, such as phospholipase C β (PLCβ), to produce second messengers. The PLCβ subfamily has modest basal activity which is robustly increased by direct binding of q. PLCβ1-3 are also directly activated by Gβγ. PLCβ hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol-1,4,5 trisphosphate (IP3), activating protein kinase C (PKC) and increasing intracellular Ca2+. Inhibition of G protein-dependent activation of PLCβ has been shown to have therapeutic potential in multiple pathologies, including inflammation, cardiac hypertrophy, opioid analgesia, and cancer. There is no consensus as to the binding site for Gβγ on PLCβ, or its mechanism of activation. Atomic resolution structures of the Gβγ-PLCβ complex are needed to fully understand the mechanism of Gβγ activation of PLCβ. We have isolated a stable, active Gβγ–PLCβ3 complex and are using single particle cryo-electron microscopy (cryo-EM) to investigate its structure. We have so far identified two particle populations using heterogeneous refinement, and generated reconstructions at 4 and 7 Å resolution. The structures reveal strong density for Gβγ binding to PLCβ through multivalent interactions with its pleckstrin homology (PH) domain, EF hands, and C2 domain, dispelling controversies about the Gβγ binding site that have persisted for over three decades. The greatest difference between structures is the position of Gβγ with respect to the PH-EF hand interface. Surprisingly, one conformation of the Gβγ–PLCβ complex is compatible with simultaneous binding of Gαi, which has not been reported in any other Gβγ–effector enzyme structure. In a cellular assay of PLC activity mutants at the Gβγ–PLCβ suggest that the conformation compatible with Gαi binding is less active. This suggests that Gβγ may also function as a scaffold for Gαi and PLCβ, in which a pre-activated complex is maintained at the membrane. Dissociation of activated Gαi would allow rotation of Gβγ, leading to full engagement of PLCβ and maximum activation. Current efforts in the lab are to investigate this scaffolded pre-activated heterotrimer-PLCβ complex using a combination of in vitro and cellular activity assays, as well as further structural characterization of these two conformations of Gβγ-PLCβ.

Poster 21--Hannah Flaherty

Examining Conformational Heterogeneity of PLCβ-Gβγ Complexes 

G protein-coupled receptors (GPCRs) regulate a variety of essential physiological processes in the body. Activation of GPCRs is communicated to the interior of the cell through the activation of heterotrimeric G protein subunits, Gα and Gβγ. These G proteins then bind to activate effector enzymes such as phospholipase C β (PLCβ). PLCβ activity has health and disease implications in many bodily systems, including opioid analgesia as well as cardiovascular diseases such as cardiac hypertrophy. Recently our lab was able to determine structures that show two different conformations of PLCβ in complex with Gβγ. This success has provided us with important structural information for both Gαq and Gβγ in relation to PLCβ. However, mutagenesis in combination with activity assays is needed to validate our structure. One residue on Gβ, W99, appears to be more engaged with PLCβ in one conformation than in the other. Previous studies have demonstrated that this residue is important for Gβγ activation, suggesting that the conformation where W99 is more engaged with PLCβ represents the activated complex. We use mutagenesis, baculovirus production, protein purifications, and in vitro activity assays in order to directly test the importance of W99 in PLCβ binding and activation. Future experiments to learn more about this system include utilizing the same process as previously described with a collection of different mutations to determine exactly which residues and portions of the structure control binding. 

Poster 22--Nicholas Gallina

Hsp60 Receptor-Targeted Bioengineered Probiotic Ameliorates Inflammatory Bowel Disease Pathology in a Colitis Mouse Model

Loss of intestinal barrier function, inflammation, crypt abscesses, ulceration and elevated expression of epithelial heat shock protein 60 (Hsp60) are critical features of inflammatory bowel disease (IBD). Patients often become intolerant or refractory to current immunosuppressive therapies and prone to opportunistic infection and malignancies. Probiotics have been used to alleviate IBD pathologies; however, they are ineffective due to poor adhesion and adaptation to the diseased gut. We hypothesize that enhancing probiotic adhesion to epithelial cells in the inflamed gut may augment immunomodulatory response, mucosal healing, and tight junction restoration. Earlier, we have identified a Listeria adhesion protein (LAP; 94-kDa acetaldehyde alcohol dehydrogenase) that aids in Listeria attachment to the epithelial cells by interacting with the epithelial receptor, Hsp60. Bioengineered Lactobacillus probiotics (BLP) expressing LAP showed strong interaction with epithelial Hsp60, high immunomodulatory response, and sustained epithelial barrier integrity. Here we examined if BLP feeding would ameliorate colitis in dextran sulfate sodium (DSS)-treated mouse model. 

Data show, in DSS (2%,7days)-induced IBD mice, treated with BLP for 10 days, a greater than 50% reduction in FITC-labeled 4 kDa dextran (FD4; epithelial permeability marker) translocation in the BLP-fed group compared to L. casei WT (LbcWT) or water only treatment group. BLP-fed DSS-treated mice gained 3% body weight during 10 days of BLP feeding when compared to the DSS-treated mice that did not receive any probiotics during that period. BLP-feeding conferred a 40% reduction in disease activity index (DAI) when compared to the water or LbcWT-treated mice. All DSS-treated mice showed bloody mucus-covered watery stool after 7 days. BLP treatment restored fecal consistency to Type 3, 4 (Bristol scoring) within 9 days of feeding while the water or LbcWT treatment group failed. Gross examination of the colon showed visible damage (shortening), wall thickening, fragile tissue, and mucus accumulation in water or LbcWT-treated groups, while the cecum and colon of BLP-fed mice appeared healthy. Our BLP offers promising results in ameliorating IBD pathologies thus illuminating a potential treatment plan for the millions of patients suffering from the disease. 

Poster 23--Yichen Han

Structures and Functions of the Human PDGFR-β Promoter G-quadruplexes

The platelet-derived growth factor receptor β (PDGFR-β) is a cell-surface receptor tyrosine kinase. PDGFR-β overexpression is associated with cancers, fibrotic disorders, and vascular diseases. PDGFR-β gene promoter’s activity is controlled by a G-rich nuclease-hypersensitive element (NHE) that forms multiple DNA G-quadruplexes (G4s). The G-quadruplexes formed in the PDGFR-β promoter are found to inhibit transcriptional activity. Unlike the classic G4 which uses four continuous G-runs for its G-core, the major G4 formed in the PDGFR-β promoter adopts a unique broken-strand structure where one G-core-strand uses guanines from two different G-runs. This broken-stranded folding can be considered as an intramolecularly filled-in vacancy-G4 (vG4) in that the terminal tetrad is completed by a guanine from a different G-run. Intriguingly, we found that this vG4 can be filled in and stabilized by exogenous physiologically relevant guanine metabolites and drugs. These results suggest that the equilibrium between intramolecular fill-in and exogenous fill-in vG4s could play an important role in modulating PDGFR-β transcription activity, which may respond to guanine metabolite levels. We determine the high-resolution NMR structures of the intramolecular-fill-in (broken-strand) PDGFR-β vG4 as well as the dGMP-fill-in vG4. Importantly, the NMR structures suggest that the two fill-in vG4s could be differentially targeted by small molecules. Therefore, our study provides a structural basis for rational design of small-molecule drugs that specifically target the novel PDGFR-β G4 to modulate gene expression. Moreover, such small molecules could help delineate potential regulatory functions of the equilibrating vG4s in the PDGFR-β promoter.

Poster 24--Cassidy Hardin

Developing a broad-spectrum inhibitor against human-infecting Coronaviruses, targeting the papain-like protease (PLP)

Coronaviruses have spread worldwide over the last few decades, some causing mild to moderate respiratory tract infections while others resulting in pandemics with increased mortality rates. The seven human-infected coronaviruses span across the Alpha- and Betacoronavirusgenera within the Coronaviridaefamily, including SARS-CoV-1, SARS-CoV-2, MERS-CoV, NL63, 229E, and others. There have been recent developments of therapeutics and vaccines targeting SARS-CoV-2 (Beta-CoV) in the last year. Despite these developments, these therapeutics only target one CoV whereas the goal of this study is to develop a broad-spectrum inhibitor to target multiple HCoVs. CoVs encode two viral proteases that are essential for virus survival and replication, one specifically being the papain-like protease (PLP). PLP is a cysteine protease, and its function is to process the polyprotein, releasing non-structural protein one through three. Beyond its proteolytic activity, PLP also has deubiquitinating (DUB) and deISGylating activities, assisting the virus in evading the host-innate immune response, making PLP a great drug target for developing small-molecule therapeutics. This study aims to investigate a new covalent, small-molecule scaffold to inhibit PLP activity across multiple HCoVs. Our results identify one specific inhibitor, compound 23224, having lower micromolar IC50 potencies against PLP for both Alpha-and Betacoronaviruses. We hope to determine the X-ray crystal structure of these inhibitors bound to PLP to employ structure-based drug design to develop a potent, broad-spectrum inhibitor.

Poster 25--Kristen Huseman

DDX5 Helicase Unfolds MYC MYC G-Quadruplex to Activate Transcription

DNA G-quadruplexes (G4) are noncanonical secondary structures which form in guanine-rich DNA sequences throughout the human genome. One such structure forms in the proximal promoter region of the MYC oncogene (MycG4) and functions as a transcriptional silencer. MYC is overexpressed in many cancer types, and downregulation of MYC in these cells has been found to induce cancer cell death. Because the MycG4 is highly stable in vitro, MYC transcriptional regulation would require active unfolding of this structure. We have found that DDX5, a founding member of the DEAD-box family of RNA helicases, readily unfolds the MycG4. Interestingly, this unfolding activity is not directly coupled to ATP hydrolysis and does not require a single-stranded overhang. G4-interactive small molecules block the interaction of DDX5 with the MycG4, suppressing MYC activation. DDX5 activates MYC transcription in cancer cells, making it an exciting new target for cancer therapeutic design.

Poster 26--Shreyas Iyer

Efficient Delivery of miR-34a through Ligand Conjugation and Chemical Modifications

MicroRNAs (miRNA) are well-known mediators of gene regulation via their ability to silence or degrade messenger RNAs (mRNAs) or through their ability to promote translational repression. In many cancers, tumor-suppressive miRNAs are downregulated, and we can utilize this knowledge to explore the therapeutic potential of restoration of tumor-suppressive miRNAs. One tumor-suppressive miRNA that is severely downregulated in many cancers is miR-34a. Restoration of miR-34a can lead to the suppression of many tumorigenic pathways; but unfortunately, delivery of miRNAs to cells comes with a unique set of challenges. These challenges range from nuclease degradation of miRNA to endosomal entrapment of the delivered miRNA. The Kasinski lab has previously demonstrated use of a vehicle free, ligand-mediated delivery platform of miRNAs to lung and breast cancer which addresses of a few of these challenges. Here, we expand on that first-generation vehicle, including use of a multivalent ligand that supports delivery to prostate cancer cells and a fully modified miRNA that increases stability and activity of the miRNA.

We show that the tumor suppressor miRNA can be delivered to prostate cancer cells that overexpress the prostate-specific membrane antigen (PSMA) receptor in a targeted, vehicle-free manner through conjugating miR-34a to the PSMA ligand, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA). We also show that a bivalent DUPA (Di-DUPA) molecule conjugated to miR-34a performs comparably to monovalent DUPA-miR-34a using a Renilla luciferase expression system. We explore this direction as multivalent ligands exhibit improved affinity to their receptor often times leading to receptor clustering and enhanced internalization. Finally, we look to improve the stability of our miRNA molecule through various modifications to the ribose and backbone. We compared the ability of partially modified miR-34a to suppress target genes in transfected cancer cells to that of fully modified miR-34a and show that introducing chemical modifications in the miRNA enhances miRNA targeting efficiency.

Poster 27--Utara Jayashankar

Can your dog get infected by the coronavirus? ; Understanding the activity of the novel Canine coronavirus 

After the global pandemic of 2019 caused by SARS-CoV-2 infection, studying the emergence of new coronavirus strains is of significant interest to predict and prevent the onset of future pandemics. A novel canine coronavirus CCoV-HuPn-2018 (CCoV), has recently been isolated from a human patient with pneumonia in Malaysia indicating the ability of the virus to be transmitted from animals to humans. Based on sequence similarity, this novel Canine CoV is classified as an alphacoronavirus, similar to other animal-infecting CoVs like feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV), which infect cats and pigs, respectively. The 3-Chymotrypsin-like (3CL) protease is highly conserved across all coronavirus genera and is essential for viral replication. The absence of human analogs and its highly conserved nature makes it an attractive antiviral drug target. 

In this study, we tested four small molecule inhibitors that are known to inhibit SARS-CoV-2 3CLpro against CCoV 3CLpro. The first compound X77 inhibits CCoV 3CLpro non-covalently with an IC50 value of 1.88 ± 0.17 μM. The 3 additional inhibitors inhibit CCoV 3CLpro by reacting with the active site cysteine through covalent modifications. While the first compound Boceprevir had an IC50 value of 13.17 ± 0.93 μM, the second displayed tight-binding kinetics with a Ki value of 72 ± 8 nM. Unexpectedly, GRL-2420 showed initial activation of the protease followed by inhibition at higher inhibitor concentrations. To understand the structural basis for inhibition of CCoV, we turned to X-ray crystallography. We determined the first X-ray structure of the CCoV 3CLpro enzyme bound to inhibitor X77 to a resolution of 2.2 Å and compared this to the SARS-CoV-2 3CLpro-X77 complex determined previously. The determined structure can further aid us in the rational design of small molecule inhibitors against CCoV 3CLpro. 

Poster 28--Haley Jordan

Determining the Constraints of -1 Programmed Ribosomal Frameshifting in Alphaviruses

Alphaviruses are positive-sense RNA viruses that are transmitted by arthropods to vertebrates. In vertebrates, infection can result in long-term viral polyarthritis and/ or encephalitis that can be fatal. Alphaviruses utilize -1 programmed ribosomal frameshifting (-1PRF) to generate a virulence factor known as the transframe (TF) protein.  Beyond the canonical RNA slip-site and RNA structure element that are known to stimulate -1PRF, we have found that the efficiency of -1PRF is sensitive to the topology of the nascent polypeptide and the pulling forces on the nascent chain that occur during polyprotein synthesis.  Our results reveal that modifications to the hydrophobicity and/ or the position of the second transmembrane domain of the E2 protein (TM2) alter the timing and magnitude of forces generated by its insertion into the ER membrane impact ribosomal frameshifting. To investigate the role of nascent chain pulling forces in alphavirus frameshifting, we characterized a series of chimeric -1PRF reporters bearing combinations of slip sites, RNA secondary structures, and upstream TM domains from different alphaviruses. Consistent with expectations, our results suggest that the slip-site and a correctly positioned RNA secondary structure are essential for -1PRF. However, our results also reveal that nascent chain pulling forces alone cannot overcome the absence of secondary structure. Interestingly, our data also suggest that not all alphaviruses have efficient -1PRF motifs, which implies that many are not reliant on the production of TF. This suggest the role of TF in infection may vary among alphaviruses. These observations merit follow up studies in the context of the viral life cycle. Together our work provides new insights into the -1PRF mechanism within alphaviruses.

Poster 29--Pallavi Joshi

Engineering cpaFx and cpeFx ribozymes to expand the STARzyme tRNA synthetase family

Addition of unnatural amino acids to proteins can be useful to diversify their functions, to aid in their structure-function analysis, and to facilitate peptide drug production. Here, we have engineered a tRNA aminoacylation synthetase ribozyme, called STARzyme which can potentially be used to facilitate incorporation of unnatural amino acids into proteins. We generated this artificial ribozyme by fusing a tRNA binding module with a catalytic ribozyme module. For the tRNA binding module, we used a naturally occurring T-box which can bind and provide specificity towards its cognate tRNA. For the catalytic module of the STARzyme, we used a circular permuted version of an artificial ribozyme named the flexizyme. These are ribozymes capable of aminoacylating any tRNA as they bind to the 3’-CCA tail which is common to all tRNAs. There are three variant flexizymes called dFx, aFx and eFx. These flexizymes recognize the leaving group attached to the activated amino acid and transfer the amino acid to the tRNA. This design allows flexizymes to charge any tRNA with a multitude of different amino acids. In Golden lab, we have demonstrated that circular permutation of the dFx flexizyme module is necessary for simultaneous docking of tRNA to both the flexizyme and T-box modules. The resulting ribozyme, or STARzyme, can be rationally programmed to recognize alternate tRNA sequences, and can selectively charge its cognate tRNAs. Here, we investigate versions of the STARzyme that use alternate flexizymes, aFx and eFx as the catalytic module. We find that like the dFx version of the original STARzymes, circular permutation aFx and eFx endows STARzyme with catalytic activity. These aFx and eFx-containing STARzymes will allow use of alternate substrates, including commercially available activated amino acids and aromatic amino acids, which are not good substrates for the dFx flexizyme.

Poster 30--Samadhi Kulathunga

In-vitrocharacterization and inhibition studies of Sulfotransferase 1A1 as a potential therapeutic target for structure-based design of drugs to overcome drug resistance in breast cancer.

Despite the advancement of early diagnosis and various treatment options, breast cancer is the most commonly diagnosed and the second leading cause of cancer death among women in the US as of 2022. The majority of diagnosed cases correspond to estrogen receptor-positive (ER+) breast cancers, which are typically treated with anti-estrogens such as tamoxifen, the gold standard anti-estrogen for ER+ breast cancers, and fulvestran. However, studies have shown the acquisition of anti-estrogen resistance in patients after several years of anti-estrogen treatments. Thus, it is imperative to identify novel drug targets to broaden the breast cancer treatment efficacy aiming the reduction of prevailing breast cancer death rate.

Human sulfotransferase 1A1 (SULT1A1) is one of the major drug metabolizing enzymes in our body that facilitates the excretion of phenolic drugs by sulfo-conjugating the phenolic groups. This phenomena involves in reducing the bio-availability of phenolic drugs. Significantly, SULT1A1 has been shown overexpressed in ER+ breast cancer cell lines and tamoxifen-resistant tumor tissues. Therefore, we hypothesized that SULT1A1 may be involved in anti-estrogen resistance in ER+ breast cancer patients by increasing the efflux of active drugs from the target cell.

In this study, we focused on the in-vitrosulfonation of fulvestrant, and the two most reactive metabolites of tamoxifen using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) by measuring various kinetic parameters including substrate affinity (Km) and turn over number (kcat). Further, two SULT1A1 inhibitors, were identified and the reduction of SULT1A1 mediated sulfonation of tamoxifen-metabolites and fulvestrant in the presence of these inhibitors was assessed using DESI-MS. Finally, using X-ray crystallographic studies SULT1A1-inhibitor co-crystal structures were determined to 1.27 Å and 1.51 Å resolutions. These high-resolution co-crystal structures will provide a molecular platform for the structure-based design of more potent inhibitors against SULT1A1.

Poster 31--Emma Lendy

Inhibition of SARS-CoV-2 3CLpro by GC376 analogs containing a unique crown-tetrahydrofuran moiety as the P3/P4 ligand

An outbreak of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2), the causative agent of Coronavirus Infectious Disease 2019 (COVID-19), has led to an ongoing, worldwide pandemic. One promising antiviral target in the SARS-CoV-2 genome is the 3-Chymotrypsin-Like Protease (3CLpro, main protease), which is largely responsible for the maturation of the viral polyprotein. GC376, a compound previously designed to inhibit Feline Infectious Peritonitis Virus (FIPV) 3CLpro, was shown to inhibit SARS-CoV-2 3CLpro in the low nanomolar range and served as a starting scaffold for inhibitor design. Here, we report the synthesis of five GC376analogs which were evaluated for their ability to inhibit SARS-CoV-2 3CLpro. These analogs demonstrate the effect of various P1’ (warhead), P1, and P4 substitutions on this scaffold, with all tested compounds displaying low nanomolar potencies. Additionally, the high-resolution crystal structures of all five of these GC376analogs bound to SARS-CoV-2 3CLpro were solved to enable structure-based drug design. These structures illustrate unexpected flexibility of the P4 moiety. This observation prompted the synthesis of a novel GC376analog with a P3 substitution aimed at locking the P4 moiety into a single conformation. Ultimately, these data demonstrate the potential for GC376analogs as potent SARS-CoV-2 3CLpro inhibitors, as well as provide high-resolution structural data that offer insight into the dynamics of this inhibitor scaffold in the 3CLpro active site. These observations also serve as guideposts for future iterations of GC376analog synthesis.

Poster 32--Dongqi Liu 

Secretion and cell surface anchoring of Listeria adhesion protein on Listeria monocytogenes is fastened by internalin B

Listeria adhesion protein (LAP) was identified for its primary role in Listeria monocytogenes (Lm) paracellular transmigration across the intestinal epithelial barrier during the early phase of infection. LAP is a bi-functional acetaldehyde alcohol dehydrogenase which after secretion, re-associates on the bacterial cell surface for interaction with the host epithelial cells, a critical step in Lm pathogenesis. However, the bacterial molecule involved in LAP secretion and surface association is unknown. We demonstrate that LAP anchors to internalin B (InlB) and promotes Lm epithelial adhesion and transmigration. Mutation in inlB or treatment with anti-InlB antibody severely interrupts LAP interaction on the cell surface and consequent LAP-mediated Lm translocation across the epithelial barrier. LAP also interacts with InlB expressed on L. innocua, a non-pathogenic Listeria cloned with Lm inlB. MgCl2, a known ionic bond competitor for InlB also interferes with InlB-LAP bonding, affirming involvement of InlB to fasten LAP on the cell surface for optimal function. Furthermore, molecular dynamics simulation indicates that one or more negative charged amino acid(s) in the crevice of alcohol dehydrogenase (ADH) region of LAP is crucial for interacting with InlB. 

Poster 33--Cody Loy

Discovery of a Destabilizing Ligand of Proteasome Ubiquitin Receptor Rpn-13, a Therapeutic Target for Hematological Cancers

The proteasome, a large multi-catalytic complex of ~2.5 MDa, serves as the major protein degradation pathway in eukaryotic cells. The 20S core particle (20S CP) cleaves proteins into short peptide fragments, and when complexed with the 19S regulatory particle (19S RP), the newly synthesized 26S proteasome is capable of degrading proteins that have been tagged with ubiquitin. This has made it a desirable target for treatment of hematological cancers due to their dependency on high 26S proteasomal activity. Targeting the 19S RP rather than the 20S CP with covalent inhibitors has shown promise in hematological cancer treatment. Rpn-13, a ubiquitin receptor of the 19S RP, is a therapeutic target of interest as it is non-essential in healthy cells but is important for the survival of blood cancers such as multiple myeloma (MM). Our goal is to develop non-covalent scaffolds that bind to the Pru (Pleckstrin-like receptor for ubiquitin) domain of Rpn-13. This work was accomplished in collaboration with Atomwise, a biotechnology company that uses artificial intelligence for drug discovery. Based on their computational analysis, we tested their library of compounds through various biophysical and chemical biology studies such as biochemical & cellular thermal shift, fluorescence polarization, and cell viability assays, in addition to structural biology analysis by 2D protein NMR. From the screen, we have validated a new binder, TCL-11, of the Pru domain on Rpn-13. TCL-11 has demonstrated selectivity for Rpn-13 by eliciting mild toxicity in hematological cancer cells (Ramos & MM.1R), and no toxicity to non-malignant cells (HEK-293T) up to 100µM. TCL-11 also demonstrated dose-dependent destabilization of Pru biochemically.  Confirmation of the binding site by 2D NMR will allow us to further optimize TCL-11’s scaffold through structure-based drug design, with the aim of developing more potent and selective non-covalent Rpn-13 inhibitors. 

Poster 34--Sarah McGovern

Altered Circadian Rhythmicity in the Aging Eye

The circadian rhythm controls 24-hour periodic behaviors and physiology, and is observed across animal kingdoms including animals, plants, and fungi. The circadian clock is synchronized with the solar day, maintaining a robust 24-hour periodicity in consistent environmental conditions. Despite this robustness, behaviors that desynchronize the circadian clock with the solar day (e.g., shift working, sleep deprivation) are detrimental to human health. Additionally, the circadian rhythm in humans shows both an altered phase and a decrease in amplitude with aging. The circadian clock is synchronized by light at the highest level via stimulation of photoreceptors in the eye. During aging, visual function decreases, which can result in decreased circadian photoreception, linking loss in visual function with decreased circadian rhythmicity. Despite these links between aging, alterations in circadian rhythm, and age-related decline in visual function, much remains unknown about the complex interplay between these processes.

Using the fruit fly Drosophila melanogaster as a model for studying the aging eye, we observe that the core circadian clock transcriptional activators Clock (CLK) and Cycle (CYC) have altered transcription factor activity with aging. Additionally, disrupting the CLK:CYC heterodimer complex results in widespread gene dysregulation in the eye. Rhythmic transcriptome profiling of the aging eye using RNA-seq reveals altered rhythmic gene expression of core clock genes and clock output genes. Transcriptome-wide analysis identifies classes of genes that gain rhythmic expression with aging, including genes important for stress and immune response, suggesting a role for circadian transcription in the maintenance of aging photoreceptor cells.

Poster 35--Kedric Milholland

Identification of a Pseudosubstrate Stimulatory Motif Uncovers Novel Roles of Cdc14 Phosphatase in Fungal Cell Wall Integrity

Fungal pathogens are a growing threat to human health and global food security as they are becoming increasingly resistant to the few existing antifungal treatments. Cdc14 is a protein phosphatase primarily known for its essential role in controlling mitotic exit in the model yeast Saccharomyces cerevisiae. However, in other fungal species Cdc14 is non-essential and the reasons for its strict conservation across Dikarya remain unclear. Recently, Cdc14 was shown to be required for host infection by several plant pathogenic fungi. Here, we show that Cdc14 is also required for virulence in the human pathogen, Candida albicans. These observations, coupled with our structural and biochemical knowledge of Cdc14 phosphatases, suggest it could be a useful target for antifungal development. In our search for differences between fungal and animal Cdc14 enzymes that could be exploited for antifungal design, we discovered an invariant motif in the otherwise disordered C-terminal tail of fungal Cdc14 orthologs. This motif resembles the recognition sequence of optimal Cdc14 substrates. We provide evidence from structural prediction and enzyme kinetic studies that this motif is a “pseudosubstrate” that binds the Cdc14 active site and functions to accelerate the rate-limiting catalytic step, providing a plausible mechanism for dynamic control of intracellular Cdc14 activity. Both S. cerevisiae and C. albicans cells expressing Cdc14 variants with point mutations in this motif exhibit a specific and pronounced sensitivity to cell wall stresses, including treatment with echinocandin antifungal drugs that inhibit cell wall synthesis. Moreover, signaling through the cell wall integrity pathway is chronically elevated in these cells, indicative of a cell wall structural defect. Furthermore, infection assays show that C. albicans cdc14Δ/Δ cells are avirulent in two different models of systemic candidiasis. Our findings reveal 1) a novel contribution to catalysis of a structural element outside the conserved catalytic core of Cdc14 enzymes and 2) novel roles of Cdc14 in promoting cell wall integrity and pathogenesis. More detailed characterization of the mechanisms by which Cdc14 contributes to cell wall integrity and host infection in human pathogenic fungi should help assess its future value for antifungal development.

Poster 36--Ramizah Mohd Sabri

CDK2 in neutrophil migration and inflammation

Neutrophils are the first responders upon infection or injury and perform a wide array of functions including phagocytosis, releasing granular contents, cytokines, reactive oxygen species (ROS), and neutrophil extracellular traps. During inflammation, their numbers increase significantly, contributing to massive local cytokines amount that can damage tissues. Neutrophil-dominant inflammation acts as the main driver behind immunopathology underlying a broad range of human diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Due to the major roles neutrophils play in inflammation pathology, they are suitable targets for therapy. Cyclin-dependent-kinases (CDKs) are serine/threonine kinases that bind to co-factors and work by phosphorylating substrates to activate downstream pathways. They are mostly known for progressing the cell cycle. In neutrophils, CDKs are generally downregulated as they no longer proliferate. Due to this, the role of CDKs in terminally differentiated cells like neutrophils is usually overlooked. Our lab has shown that CDK2 is present in neutrophils after differentiation and plays an essential role in neutrophil migration. Abrogation of CDK2 in human neutrophils has been shown to reduce cell migration. Mass spectrometry showed cyclin D3 (CCND3) as a novel co-factor of CDK2 in neutrophils. Knockdown of CCND3 also reduced neutrophil migration and prevents proper cell polarization. In mice with the mutated form, CDK2 DN, neutrophils are less recruited to the infection site. Tumor progression and induced colitis were also dampened in CDK2 DN mice. Continued investigations on CDK2 substrates in neutrophils will provide novel therapeutic targets in regulating neutrophil behavior and alleviate neutrophil-dominant diseases.

Poster 37--Balindile Motsa

Probing the protein-protein and protein-lipid interaction interfaces of Ebola virus matrix protein VP40 with mutagenesis.

Ebola virus (EBOV) is a filamentous RNA virus which causes severe hemorrhagic fever. It is one of the most dangerous known pathogens with a high fatality rate. There is a great need for the development of therapeutics that target EBOV, so it is important to get a detailed understanding of the virus life cycle. EBOV encode for the matrix protein, VP40, which is one of the most conserved viral proteins in the genome. VP40 regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane. The trafficking and assembly of the VP40 dimer to the plasma membrane requires a network of protein-protein and protein-lipid interactions (PPIs and PLIs). In this work we study the effects of VP40 mutations that occur at these interfaces on VP40 plasma membrane binding dynamics and function. We observed that mutations at the membrane binding interface (PLI) affect assembly and budding by either creating more favorable or less favorable interactions with anionic lipids in the plasma membrane inner leaflet. Glycine to arginine mutations at this interface increased VP40 assembly and budding and have increased affinity for PS containing vesicles. In contrast, a glycine to aspartic acid mutation significantly diminished assembly and budding. Mutations at the dimer interface (PPI) affect VP40 oligomerization capabilities. The L117A mutation at the dimer interface prevents dimerization as a result has abolished assembly as well as viral budding. Inhibition of VP40 dimerization leads to a trafficking defect of VP40 to the plasma membrane, the site of VP40 assembly and budding. Understanding the effects of single amino-acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains and for long-term drug discovery aimed at EBOV assembly and budding.

Poster 38--Kaushik Muralidharan

Understanding the molecular mechanisms of PLCε regulation

Cardiovascular disease is the leading cause of death in the world. The phospholipase C (PLC) family of enzymes, in particular the PLCε subfamily, are essential for normal cardiovascular function. PLCε hydrolyzes phosphatidylinositol phosphates at cellular membranes, producing inositol phosphates (IPx) and diacylglycerol (DAG). These crucial secondary messengers regulate multiple downstream pathways, including contractility and the expression of hypertrophic genes. In the cardiovascular system, PLCε is regulated through direct interactions with the RhoA and Rap1A GTPases, which in turn are activated downstream of G protein-coupled receptors (GPCRs). RhoA is reported to activate PLCε at the plasma membrane, whereas Rap1A translocates and activates PLCε at the perinuclear membrane. However, the elements within PLCε that regulate basal activity and membrane association have not been fully identified. Similarly, the domains required for Rap1A versus RhoA binding, activation, and translocation have not been mapped. In this work, we use a structure-guided approach, together with cell-based activity assays, epifluorescence, and TIRF microscopy to identify the roles of PLCε regulatory elements and domains in basal activity, subcellular localization, and activation by RhoA and Rap1A GTPases. Functional studies show that the N-terminal region and/or the CDC25 guanine nucleotide exchange factor (GEF) domain dictate its location within the cell, and contribute differently to basal and G protein-dependent activity. We also show that regulatory insertions within the catalytic TIM barrel, including the X–Y linker and Y-box, aid in interfacial activation and membrane association. These studies provide much needed insights into the molecular determinants of PLCε that regulate its localization and activity in cells, which are critical for elucidating its roles in cardiovascular function. 

Poster 39--Tanmay Nallan Chakravarthula

Plasmin Inhibition by Multivalent Benzamidine Derivatives of Varying Valency and Linker Length

Plasmin is a serine protease that cleaves fibrin to achieve clot lysis. Plasmin inhibitors are useful for treating hyperfibrinolysis-associated bleeding, cancer, and inflammatory disorders. There is emerging interest in applying the principles of multivalency to enzymes to achieve highly controlled and strong inhibition. This study aims to determine the effect of valency and linker length of multivalent benzamidine inhibitors on plasmin activity. Reversible monovalent, bivalent and trivalent inhibitors of lengths ranging from 0-10 nm were synthesized using 4-aminomethyl benzamidine (AMB) and monodisperse polyethylene glycol (PEG) molecules. These inhibitors were purified using RP-HPLC and confirmed by mass spectrometry. Inhibition assays were performed for the synthesized inhibitors along with AMB and Pentamidine (FDA approved bivalent benzamidine). A range of inhibitor (0-1200 µM) and chromogenic substrate S-2251 (100-500 µM) concentrations at a fixed human plasmin concentration (42.5 nM) were utilized to determine inhibition constants (Ki values) via Dixon plots. Multivalency parameters such as relative potency (rp) and relative potency per unit benzamidine (rp/n) were computed. Ki values ranged between 259.4 - 521.1 µM, 44.3 - 290.4 µM, 3.9 - 241.9 µM for synthesized monovalent, bivalent, and trivalent inhibitors, respectively (lower the Ki, stronger the inhibition). Ki of free AMB was 1,395 µM, and pentamidine, the shortest bivalent inhibitor was the strongest with a Ki value of 2.1 µM, a rp value of 820.5 and an rp/n much greater than 1 (410.2) indicating a strong multivalent effect. All synthesized inhibitors demonstrated rp/n > 1. The shortest trivalent inhibitor was comparable to pentamidine with a Ki of 3.9 µM. For similar lengths, inhibition increased with valency owing to enhancement in statistical rebinding. For a fixed valency, inhibition linearly decreased (R2>0.9) with increasing length. Since PEG is flexible, increasing its length increases conformational entropic penalties decreasing inhibition. Therefore, higher valency and short linker length enhance inhibition.


Poster 40--Thu Nguyen

Analysis of Epithelial Ovarian Cancer Related-Genetic Markers In a Cancer-free Patient Population Diagnosed with Hypodontia

Early-stage epithelial ovarian cancer (EOC) can be difficult to diagnose, as many of the symptoms are mistakenly assumed to be part of the “normal” changes that occur with the onset of menopause. Based on previous literature, it has been shown that approximately 1/5thof women diagnosed with EOC late in life never formed a complete dentition earlier in life. Although a genetic mutation within the AXIN2gene has been shown to give rise to both oligodontia and colon cancer, little is known regarding the potential molecular mechanism(s) that connect EOC and hypodontia. In this study, we selected five genetic markers associated with EOC from published GWAS data to test for associations to hypodontia in a young orthodontic patient population with strong diagnostic data for hypodontia.

This case-control study enrolled forty-seven subjects diagnosed with hypodontia (i.e., 1-to-6 non-third-molar teeth failed to form within the adult dentition) and eighty-six controls who developed a complete non-third-molar dentition. We tabulated the occurrence of small or peg-shaped teeth and the status of third-molar development. Saliva was collected as a source of genomic DNA for Taqman-based Endpoint genotyping of the following markers: SKAP1rs7207826, WNT4rs3820282, BABAM1rs8170, BNC2rs3814113, and ANKLE1/BABAM1rs4808075.The Chi square test was used to identify any associations between each SNP and hypodontia. A nominal logistic regression analysis was used to model the SNPs together for association to hypodontia. A Fisher’s exact 2-tailed test was used to determine if third-molar agenesis occurred more frequently in the cases versus the controls.

We failed to reject our null hypothesis, which stated there would be no association of the markers with hypodontia. Patients diagnosed with hypodontia were more likely to also have agenesis of third-molar-teeth (p=1.33e-6).

There was not association between subjects with hypodontia and controls among the tested SNPs


Poster 41--Vaani Ohri

Investigation of RhoA-Dependent Regulation of Phospholipase C ε in Cardiovascular Disease 

Cardiovascular diseases are the leading cause of death in the United States. Phospholipase Cε (PLCε) is required for normal cardiovascular function, as it regulates the intracellular Ca2+ concentration and activates protein kinase C (PKC) signaling pathways. PLCε itself is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with small G proteins, including Rap1A and RhoA. Regulation of PLCε by Rap1A at the perinuclear membrane has been well-characterized, as this pathway contributes to cardiac hypertrophy and heart failure. In contrast, RhoA-dependent activation of PLCε is cardioprotective against ischemia and reperfusion injuries. This pathway is initiated by activation of G12/13-coupled receptors, particularly the sphingosine-1-phosphate (S1P) receptors, and leads to the exchange of GDP for GTP on RhoA, activating the small GTPase. RhoA•GTP binds and translocates PLCε to the plasma membrane, where it hydrolyzes membrane phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). The increased DAG activates PKC, which ultimately inhibits mitochondrial apoptosis and prevents cardiomyocyte death. Despite the critical role of RhoA and PLCε in driving the cardioprotective response, little is known about how these proteins interact to increase lipase activity. RhoA was initially thought to bind to PLCε through one of its C-terminal Ras association (RA) domains, which are essential for its regulation by other GTPases. However, the RA domains are dispensable for both RhoA binding and activation, and further truncations of PLCε narrowed its binding site to the highly conserved catalytic core. Functional studies implicated an insertion within the catalytic TIM barrel domain, known as the Y-box, as a requirement for RhoA-dependent activation of PLCε. However, the Y-box does not bind the GTPase. Our goal is to identify the molecular mechanism by which RhoA binds to PLCε and increases its activity using structural and functional studies. The successful completion of these studies will map the interaction between these two critical signaling proteins, as well as identify elements in PLCε required for activation at the membrane. This knowledge can be ultimately exploited to develop lead therapeutic compounds that modulate this interaction to improve cardiovascular health. 

Poster 42--Claire Overly

Characterization of the β-barrel assembly machinery in Fusobacterium nucleatum

The CDC reports more than 2.8 million antibiotic-resistant infections yearly in the U.S, many of which are caused by Gram-negative bacteria. Lack of therapies to treat these infections presents a critical need for new antimicrobial strategies. The β-barrel Assembly Machinery (BAM) complex is a protein complex on the outer membranes of Gram-negative bacteria which functions in the biogenesis of β-barrel outer membrane proteins (OMPs). Inhibiting this complex is lethal for Gram-negative bacteria; thus, it is an important, novel drug target. 

My project studies the BAM complex in Fusobacterium nucleatum, an oral biofilm pathogen. F. nucleatum causes oral infections and is also implicated in colorectal cancer, and it achieves its multifaceted pathogenicity through OMPs which rely on the BAM complex. To study the BAM complex, I am using biophysical techniques like small angle X-ray scattering, X-ray crystallography, and electron microscopy to examine the protein structure of BamA, the OMP anchor of the BAM complex. I use proteomics to probe BamA-interacting proteins. Traditional BAM complex accessory proteins are missing from this organism; thus, I am seeking to understand the novel composition of the complex towards future structural and mechanistic studies and drug design.

Poster 43--Rishi Patel

Structure-guided photoaffinity ubiquitin probes give insight into deubiquitinase binding sites

Ubiquitin (Ub) is a post-translational modifier crucial to eukaryotic biology. The Ub system relays an intricate control over cellular processes through its attachment and detachment on substrate protein lysine residue, giving rise to a “ubiquitin code”. Deregulation in this code has severe consequences, primarily in disease pathogenesis. Proteolytic deubiquitinating enzymes (Dubs) render this PTM reversible by removing or “trimming” Ub off substrate protein. In turn, this interplay between the attachment and detachment machineries establishes a well-defined niche of ubiquitinated protein to relay critical cellular information. Ub modification, unlike other PTMs, is multivalent. Subsequent additions of one Ub moiety to 1 of 8 amines (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys68) on another confers the assembly of polyUb chains with well-defined linkages, each relaying a vastly different function. Dubs must recognize this multivalency by accommodating several topologies of Ub chains. Here we show that genetically encoded photophore Bpa in the Thr-9 position serves as a pan-Dub probe that gives insight into Ub binding sites. We demonstrate that genetic code expansion (GCE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) gives structural insight into the Ub-Dub interaction mode. 

Poster 44--Himani Patel


3-hydroxyl-3-methylglutaryl CoA reductase (HMGR) catalyzes the rate-determining step in the isoprenoid pathway using two equivalents of NADH. Despite significant biomedical interest in the bacterial HMGR and its human homolog, many atomistic details of the mechanism are still unknown. We use a combination of computational studies and time- resolved crystallography. The catalysis of HMGR can be broken down into three major chemical transformations including two hydride transfers separated by a base-catalyzed thiohemiacetal decomposition. Additionally, two cofactor molecules needed for the mechanism necessitate a cofactor exchange process between the hydride transfers. 

This theoretical study explores the atomic details of the enzyme and ligands for the thiohemiacetal breakdown in PmHMGR whose catalytic site is structurally similar to that of human HMGR. Theozymes models used to calculate the transition state of thiohemiacetal decomposition at the M06/6-31G(d,p) level indicate a higher energy of activation for the theozyme models containing NAD relative to the NADH ones. These models show Nδ of His381 stabilizes the negative charge of the thiolate anion. While QM/MM models show a smaller C-S bond elongation relative to the theozyme models, the former models establish the Ser85 hydrogen-bonding network with substrate amide, a function performed by His381 in the first hydride transfer. Molecular dynamics simulations of different protonation states of His381 reveal a greater stabilization of the active site with a positively charged His381. Transition state force fields (TSFFs) are generated with the theozyme models reference data using the quantum guided molecular mechanics (Q2MM) program for the thiohemiacetal decomposition. The TSFF allows a μsec timescale molecular dynamics simulations to probe further mechanistic questions such as the flap domain movement that facilitates the cofactor exchange. This project focuses on not only developing computational biophysics methods for studying enzyme mechanisms but also using time-resolved x-ray crystallography to obtain snapshots along the HMGR reaction pathway. 

Poster 45--Tyler Pikes

MicroRNA-31 Regulates Neutrophil Migration and Activation

Neutrophils are the most abundant white blood cell in circulation and are involved in fighting infections as well as initiating inflammatory immune responses and contribute to certain autoimmune diseases. Regulation of neutrophil activity is crucial in maintaining homeostasis in the immune system. However, the specific mechanisms or which molecules are involved in regulating neutrophil functions are still not fully understood. Previous studies in our lab discovered that microRNA-31 (miR-31) regulates neutrophil motility in zebrafish. Over expressing miR-31 in zebrafish neutrophils leads to a decrease in neutrophil motility and neutrophil recruitment to sites of infection. Through a small-scale genetic screen using the neutrophil-specific CRISPR/Cas9-based gene inactivation technique to knockout miR-31 target genes, I identified that efnb2b is involved in regulating neutrophil motility. Efnb2b is a ligand for Eph receptors important for heart morphogenesis and angiogenesis via regulation of cell adhesion and cell migration. Its role in the immune system or specifically in neutrophils, is not yet characterized. Future work is to understand the signaling pathway regulated by efnb2 in neutrophils. Completion of the proposed study will advance our understanding of neutrophils and innate immunity, leading to possible therapeutic developments for neutrophil-related diseases. 

 Poster 46--Aaditya Prabhu

Spectrophotometric Analysis of Synechococcus sp. Questions the Concept of the Q-cycle in Oxygenic Photosynthesis.

A current concept in the theoretical presentation of oxygenic photosynthesis suggests that the rate of light-dependent electron transfer depends on the ‘Q-cycle’ in which the reduction of cytochrome heme-b6 contributes to the electrochemical proton gradient required for the various downstream reactions. The ‘Q-cycle’ has been studied extensively in mitochondria; however, there is no definite experimental evidence for its existence in chloroplasts. It was assumed to be conceptually relevant due to the shared structural features between the two organelles. The project aims to obtain spectrophotometric data on the existence of the Q-cycle in intact cyanobacterial cells. Late log-phase Synechococcus sp. cells were exposed to actinic light, and a flash spectrophotometer was used to measure the change in absorbance as a function of wavelength. We observed a clear absorbance change corresponding to the oxidation of cytochrome f; however, there was no evidence for the reduction of heme b6. The latter contradicts the expectation of the ‘Q-cycle’ mechanism and can only be explained if the internal environment of the cells is already reduced in their resting unexcited state. This development would imply that the Q-cycle does not operate in cyanobacterial cells and that the rate-determining step requires an alternate mechanism to explain it.

Poster 47--Jack Reynolds

Linearly Paired Variational Autoencoders: A Contrastive Machine Learning Approach to Modeling Transient Behavior 

We propose a novel implementation of Variational Auto Encoders (VAE) using contrastive learning to maximize the value of complex observations of transient behavior. The system introduced by this work, a Linearly Paired Variational Auto Encoder (LP-VAE), demonstrates the ability of machine learning algorithms to ingest multiple sparce epochs of event-based time series data and both interpolate and extrapolate new data. This is done by “pairing” multiple VAEs and providing a linear loss to the implicit latent space each VAE produces, with only the maintained deep feature of the VAE changing. The linear loss acts to have each transient event follow a linear path through the high dimensional latent space. We argue that the development of systems that augment non-periodic environmentally driven events are a necessary step in the evolution of time series networks in machine learning. Systems such as there maximize the value of possible expensive and irregular observations by acting as valuable data enrichment. This is demonstrated with early success via simulation of supernova spectral evolution. Supernova spectra are complex in nature, require limited and specialized equipment to observe, and often have non-negligible interactions with the surrounding environment. 

Poster 48--Hannah Rondon

Structure-based design of covalent inhibitors for UCHL1, UCHL3, and PfUCHL3

Protein ubiquitination and deubiquitination are important post-translational modifications that affect virtually all cellular processes. Ubiquitination of substrate protein is mediated by an E1 (activating), E2 (conjugating) and E3 (ligase) cascade of enzymes. Together with  deubiquitinating enzymes (Dubs), this system establishes a well-defined niche of ubiquitinated protein.  Deregulation of  Dubs is accompanied by disease development  notably including cancer and neurodegenerative diseases. The ubiquitin C-terminal hydrolase (UCH family of Dubs  are becoming  a great interest for therapeutic intervention. The UCHL1 Dub composes 1-5% of all protein in neurons and is implicated in many neurodegenerative diseases. Another member of the UCH family, UCHL3 is more expressed throughout other tissue. Its deregulation is described in several cancers. These enzymes have increasingly gained interests as a promising therapeutic target. Along with this, UCH enzymes can also be found in parasitic organisms such as the causative agent of malaria, Plasmodium falciparum. According to World Health Organization, this parasite is responsible for nearly 600,000 deaths worldwide. It contains an enzyme with deubiquitinating activity that shares homology with the human UCHL3. P.f.UCHL3 plays a large role in parasite physiology and inhibitors of this Dub can serve as potential drugs target. In collaboration with the Flaherty Lab in the Department of Medicinal Chemistry and Molecular Pharmacology, we use X-ray crystallography to help elucidate the structural basis of inhibition of these enzymes with covalent inhibitors. This data gives insight into structure-based optimization for more potent and selective molecules. Structure solved in this study of UCHL1 in complex with a lead compound has led to further improvement on inhibitors. I have co-crystallized UCHL1 in complex with the most potent inhibitor IMP1710 as verified by mass spectrometry. The devised co-crystallization strategy is applied to both UCHL3 and P.f.UCHL3 with their respective lead compounds. These structures will show interactions critical for inhibitory function.

 Poster 49--Manalee Samaddar

Listeria adhesion protein (LAP) is a 94-kDa oligomer present in Listeria monocytogenes (Lm) that regulates the intestinal epithelial tight junction barrier to allow Lm translocation across the epithelium. The interaction of LAP with epithelial receptor Hsp60 is critical for LAP-mediated adhesion and translocation of Lm across the intestinal epithelial layer. In the human gastrointestinal tract, the tight junction of the epithelial barrier is the major obstacle to the efficient delivery of medicinal drugs. Among the current drug delivery systems, the paracellular flux via different transporter has limited success, whereas existing tight junction modulators have a nonspecific mode of action and toxic side effects. Hence, we investigated LAP as a potential tight junction modulator for oral drug delivery.

Initial experiments were performed to resolve the three-dimensional structure of LAP-Hsp60 interaction using transmission (TEM) and cryo-electron microscopy (Cryo-EM).  To determine LAP-mediated translocation of a model drug-analog (FITC-conjugated 4-kDa Dextran, FD4) and peptide drugs (vancomycin and desmopressin), in vitro cell (Caco-2 and MDCK) culture and in vivo animal models were used. Histology, the epithelial tight junction architecture and levels of inflammatory markers (Lipocalin-2, C-reactive protein & IL-6) in sera or feces were analyzed. 

TEM micrographs revealed LAP to form a distinct spirosome-like structure that is predicted to interact with host Hsp60. Cryo-EM images showed a more detailed tetrameric structure of LAP at a 3.1Å resolution. A complex structure of LAP-Hsp60 revealed interaction of N terminal domain  of LAP is more intended with Hsp60. In the cell culture model, LAP facilitated FD4 translocation across the epithelial barrier in a dose and time-dependent manner with maximum activity was observed after 1h. LAP-mediated FD4 translocation was 10,000-fold more efficient than the established permeability enhancers (EDTA & Na-caproate). In a mouse model, LAP-facilitated translocation of FD4 by 18 fold while vancomycin and desmopressin by 2 fold. Furthermore, LAP neither induced tissue inflammation nor elevated levels of inflammatory markers (lipocalin-2, C-Reactive protein & IL-6) suggesting LAP to be an efficient and safe tight junction modulator for oral delivery of peptide drugs.

Poster 50--Kadidia Samassekou

FABulous Tools for Studying PLCε Structure 

Phospholipase C (PLC) enzymes are critical regulators of cell proliferation, differentiation, and survival. The PLCε subfamily is required for normal cardiovascular function, and its dysregulation can lead to cardiac hypertrophy. This enzyme cleaves the phosphatidylinositol phosphates PIP2 and PI4P into inositol phosphates (IPX) and diacylglycerol (DAG), which in turn stimulate the release of Ca2+ from intracellular stores and activates protein kinase C (PKC). PLCε shares a highly conserved catalytic core that is flanked by unique N- and C-terminal regulatory domains. The N-terminal CDC25 domain is as a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1A. The two C-terminal Ras association domains, RA1 and RA2, are involved in stabilizing the lipase core and binding Rap1A, respectively. However, whether or how these regulatory domains interact with the catalytic core to modulate activity is not known. PLCε is a large, conformationally dynamic protein, which presents numerous technical challenges for determining its structure. Towards this goal, and in collaboration with the Kossiakoff lab, we have developed antigen binding fragments (Fabs) that recognize specific domains of PLCε for use in single-particle cryo-electron microscopy (cryo-EM) experiments. We now determined a ~4 Å reconstruction a Fab-bound PLCε PH-C variant that contains conformationally flexible PH domain, catalytic core, and RA domains. This structure provides new insights into the molecular architecture of the protein, including a unique conformation of the PH domain. Work is ongoing to improve resolution and validate the structure through functional assays.

Poster 51--Roopashi Saxena

Stapled peptides to probe for biophysical studies of the Ebolavirus VP40 dimer interface

Ebola virus (EBOV) belongs to family of filoviruses, which is an enveloped single stranded RNA virus that causes hemorrhagic fever in humans. Several outbreaks of EBOV have been reported in Western and Sub-Saharan Africa with fatality rates of 50-80%. FDA has approved use of two monoclonal antibodies treatment and a vaccine against Zaire Ebolavirus. With emerging strains of EBOV (at least six known) and limited therapeutics, it is important to study the mechanisms of viral replication and viral assembly and budding.
EBOV encodes for lipid binding matrix protein VP40 which is one of the most abundant and conserved viral protein. VP40 is sufficient to form filamentous virus-like particles from host cell membrane in the absence of other filovirus proteins, eluding towards the importance of this protein-protein and protein-lipid interactions for viral assembly and budding. VP40 undergoes structural changes to perform multiple functions during the viral life cycle. It predominantly exists as a dimer to serve as building block for filament formation and as octamer to bind viral RNA to regulate gene transcription. Equilibrium exists between VP40 oligomers for successful viral infection. We aim to understand the biophysical mechanism involved in dimer stability and oligomerization equilibrium. VP40 dimerizes by helix-helix interactions occurring at the hydrophobic N-terminal domain. Residues 106-120 constitute the dimerization interface and computational analysis has assessed the contribution of each of these residues in helix formation. Combining computational data and mutant studies, alpha-helical peptides mimicking the dimer contact surface were designed and a library of compounds with different kind and position of staple was synthesized. These peptides are expected to bind VP40 monomer or dimer and disrupt the VP40 dimerization interface. Peptides were screened for binding to VP40 using thermal shift assay (TYCHO) and binding kinetics of potential peptides was measured using microscale thermophoresis (MST). FB02, FB05, FB06 and FB09 peptides exhibited binding to VP40 and FB09 showed the highest binding affinity. Mechanism of binding of peptides to VP40 dimer is being investigated and complimented with cellular studies to elucidate the energetics of VP40 oligomerization

Poster 52--Beinan Yang

Characterization of small-molecule inhibitors against human sulfotransferase 1A1 as a new cancer drug target

Human sulfotransferases (SULTs) are enzymes from a supergene family, which catalyze sulfonation reaction that is crucial for metabolism of both endogenous compounds and xenobiotics. Generally, conjugation of a charged sulfonate group to a molecule increases its hydrophilicity, thus facilitates its excretion. Hence, SULTs have been recognized as a major class of detoxification enzyme in adult as well as fetal development. However, recent evidence has shown that the major sulfotransferase isoform 1A1 (SULT1A1) in liver and GI tract is responsible for bioactivation of promutagens and procarcinogens such as N-hydroxy-PhIP. It is implicated that SULT1A1 activity is closely associated carcinogenesis in liver, prostate, colon and breast in rats and human. Moreover, bioavailability of various drugs can be impaired by SULT1A1 catalyzed sulfonation. Therefore, it is important to find and design small molecules that can bind to SULT1A1 and inhibit its activity subsequently. Inhibitory potency against SULT1A1 of nine candidate molecules including known bioactive molecules from HTS screen and FDA approved drugs were tested using a continuous, fluorescent-based activity assay. Here, we report the dose response curves of four candidates with most potent inhibitor that has IC50 = 1.3 ± 0.24 μM. Sulfonation state of each compound was confirmed by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) analysis. We also report the X-ray crystallography structure of SULT1A1 co-crystallized with our most potent inhibitor and cofactor 3-phosphoadenosine 5-phosphate (PAP) to 1.9 Å. The crystal structure of these candidate molecules provide insights for us to understand the mechanism of SULT1A1 and sulfotransferase in general, and might serve as a cornerstone for designing potent, cancer specific, small-molecular inhibitors.

Poster 53--Kevin Scrudders

Bridging the gap: single molecule, single cell reconstitution of engineered T cell activation

The use of Chimeric Antigen Receptor (CAR) T cells is a promising new immunotherapy whose potential to treat a wide range of cancers has been incompletely realized. Clinical efficacy against liquid tumors has not translated to solid tumors which may be due, in part, to the deviation of signal integration from the native T cell receptor context. Here, we apply in vitro reconstitution using supported lipid bilayers functionalized with adhesion and tumor antigen biomolecules to mimic the surface of a solid tumor and measure the minimal, threshold criteria for CAR T cell activation. The flexibility to control the density of tumor markers, spanning orders of magnitude, reflects normal-to-disease conditions as well as expression level changes that occur during disease progression. We focus on a second-generation, anti-FITC CAR that targets tumor cells via a small molecule ligand. This bridging molecule consists of a folic acid (tumor ligand) moiety linked to FITC and includes a bright, photostable fluorescent marker for detection of single CAR triggering events. Binding is captured using high spatiotemporal resolution imaging in a Total Internal Reflection Fluorescence (TIRF) configuration and mapped to both early cellular activation and cytotoxic granule release. A mechanistic understanding of CAR activation is expected to improve therapeutic implementation by tuning bridge and T cell dosing based on tumor stage and type.

Poster 54--Sajad Shiekh

FRET-PAINT for Studying Long Telomeric DNA Overhangs and their Interactions with Shelterin Proteins 

I will be presenting the single molecule FRET-PAINT measurements and computational modeling studies where we investigated the accessibility of range of different human telomeric overhangs. These overhangs can form 1-7 tandem G-quadruplex (GQ) structures, which, to our knowledge, is the most comprehensive range studied to date and covers a significant portion of the physiologically relevant telomeric overhang length scale. Our measurements demonstrate novel accessibility maps where certain regions of telomeric overhang are significantly more accessible than others. We also observe folding frustration patterns with a well-defined periodicity and constructs with a certain number of telomeric repeats demonstrate elevated levels of frustration compared to others. These patterns have significant implications for telomere organization, protection of free 3’-end against exonuclease activity and telomerase-catalyzed extension, and folding cooperativity between neighboring GQ structures. We further investigated the impact of POT1 and a four-protein Shelterin complex on the accessibility of human telomeric DNA constructs. with physiologically relevant overhang lengths (28-150 nt). To quantify telomere accessibility, we monitored transient binding events of a Cy5-labeled, short peptide nucleic acid strand to available sites on the telomere using FRET-PAINT methodology.  When averaged over 11 constructs investigated in this study, we observed approximately 2.5-fold reduced accessibility in the presence of POT1 (compared to DNA-only case) and about 5-fold reduced accessibility in the presence of Shelterin. These results suggest the protection of exposed telomeric segments by POT1 was effective enough to dominate over its mild GQ unfolding activity. The more effective protection in the presence of Shelterin compared to POT1-only case suggests the restructuring of the junction region between single and double stranded telomere, which is otherwise the most accessible part of the overhang, serves an important function in telomere maintenance. 

Poster 55--Katelyn Silva

Mechanistic Evaluation of the Structure and Function of Tautomerases Cg10062 and cis-CaaD

The enzymes cis-CaaD and Cg10062 are members of the tautomerase superfamily defined by a homologous β-α-β structure and a catalytic proline at the N-terminus. While the native function of cis-CaaD was determined in the conversion of cis-3-chloroacrylic acid from nematocides to exclusively malonic semialdehyde, it is reported to utilize other substrates such as acetylenecarboxylic acid (ACA). Additionally, Cg10062 more favorably utilizes acetylenecarboxylic acid, although decarboxylation to acetaldehyde affords low yield of malonic semialdehyde. Since malonic semialdehyde, can be utilized in the production of value-added products, avoidance of the decarboxylation step is essential. Here we present the kinetic characterization of Cg10062 and cis-CaaD variants and atomic resolution crystal structures for apo and substrate-soaked variants of these two tautomerases. In our evaluation of the trapped intermediates of the cis-CaaD reaction with acetylenecarboxylic acid, we see a similar route as that of Cg10062. We find that in the formation of malonic semialdehyde, nucleophilic addition of the terminal proline to acetylenecarboxylic acid substrate to likely form an iminium intermediate. Following addition of water yields a hydroxypropionate covalent intermediate, which cleaves to form malonic semialdehyde. In contrast, the iminium intermediate may decarboxylate and subsequently hydrate to form the lesser yield product, acetaldehyde. This structural evaluation provides insight into the mechanisms of these tautomerases and reveals what structural characteristics contribute to their different products.

Poster 56--Karthik Srinivasan

Towards the Architecture of the TOC protein complex

Protein translocation across the chloroplast outer membrane is essential for photosynthesis in all green plants. This is because most chloroplast proteins (over 90%) are encoded in the nucleus, translated in the cytoplasm, and must be imported into the chloroplasts to perform their functions. The translocon on the outer chloroplast membrane (TOC) complex orchestrates this vital translocation process and consists of three components: Toc75, Toc33/34 and Toc159 with unknown stoichiometries. Our lab seeks to elucidate the structural architecture of the TOC complex to gain mechanistic insights into protein translocation in chloroplasts. Toc75 is a β-barrel membrane protein that forms the channel of the TOC translocon and our lab has previously reported a crystal structure of the N-terminal polypeptide transport-associated (POTRA) domains. In this work, we demonstrate the generation of antigen-binding fragments (Fabs) that specifically recognize the POTRA domains from both Arabidopsis thaliana and Pisum sativum. Further, we characterize this interaction using size exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS), isothermal titration calorimetry (ITC), and X-ray crystallography. We also show that we are able to use these fabs to pull down part of the TOC core complex from pea leaves bought at a local grocery store. Future directions include testing if the Fab fragments can pull down the full TOC complex which may then be used for structural elucidation using cryo electron microscopy.

Poster 57--Trung Thach

CRISPR/Cas engineering for effective and direct delivery-mediated gene suppression

The RNA-based CRISPR interference (CRISPRi) system is a powerful tool for transcriptional repression. However, the intracellular delivery of the CRISPRi system is challenging due to the large size. Here, we identified a newly small CRISPR/Cas9 system in Rhodobacter sp. (termed RsCas9), yet previously described. Furthermore, we engineered a small CRISPRi system (sCRISPRi) derived from the truncated variant of RsCas9 protein, which could be directly delivered into mammalian cells via the 7-Arg tag, while that of origin RsCas9 fails to be delivered. The sCRISPRi system specifically suppressed endogenous eGFP and MyoD gene expression in HEK and skeletal muscle cells with low incidence of off-target, respectively. Collectively, The structure-based sCRISPRi engineering shows direct delivery efficiency, thus providing a versatile platform approach for CRISPRi-based applications. 

 Poster 58--Jacob Verburgt

Computational Modeling of GPCR-Ligand Complexes in GPCR-Dock 2021

G-Protein Coupled Receptors (GPCRs) are a superfamily of transmembrane proteins involved in a wide range of cell signaling pathways and are thus considered extremely viable drug targets. Highly accurate in-silico modeling of GPCR-ligand complexes is invaluable for efficient characterization and design of therapeutics that may target these receptors. Recently, a community assessment of GPCR-ligand modeling (GPCR-Dock 2021) was organized to assess the modeling capabilities of 5 GPCR targets, in complex with both small-molecule and peptide receptors. Our modeling approach, which incorporates AlphaFold, and the AlphaFold-Multimer variant, traditional ligand docking, and Molecular Dynamics (MD) was able to successfully produce one of the best models of all groups and our group consistently produced high-ranking models for all targets. Building from these results, along with recent studies of IDP-Receptor modeling, we propose ways to improve GPCR-Peptide docking.  

Poster 59--Dan Xie

Structural characterization of substrate recognition and array-based functional peptide screening of Src tyrosine kinase 

Protein kinases are a highly targeted class of enzymes for cancer therapeutics. All current FDA approved drugs bind within the ATP site and many demonstrate toxicity due to off-target effects. A new avenue is to design drugs that compete with substrate interactions. This requires the knowledge of how kinases interact with their substrates. Current structure information on kinase-substrate interactions is particularly deficient due to the transient nature of tyrosine kinase-substrate complexes. To overcome this deficiency, we are using NMR approaches and the native solution state to characterize structural patterns for substrate binding of Src tyrosine kinase, a well-known cancer-related drug target. Paramagnetic relaxation enhancement, chemical shift perturbation and exchange-transferred NOE are exploited to define the peptide pose for Src-peptide complexes. Our data suggests alternative binding modes for different peptide sequences. While peptide substrates inform on interactions near the active site, the native protein substrate involves additional contacts. We obtained high-quality NMR spectra of a kinase-protein substrate complex that probe for the first time interaction of the substrate near the kinase catalytic site. Further, with the knowledge of Src- peptide interaction, we developed a new design method to construct a peptide library in order to screen the potential for high-affinity binders with a SPOT peptide array. As a result, we found a large group of peptides that have five- to ten-fold greater activity and binding affinity than current well-known peptides. Given the strong correlation between protein tyrosine kinase dysfunction and cancer, substrate recognition patterns become critical for drug discovery. Finding of tight binders would facilitate development of more specific drugs for future cancer treatments.