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2017 Research Showcase
BABP Abstracts
Abstract Title : Expression and Purification of Dimers of Recombinant Human Acetylcholinesterase and Computational Analysis of Dimerization Interface in Alpha/Beta-Hydrolase Fold Proteins
Abstract : Human acetylcholinesterase (AChE; EC is one most important proteins in human cholinergic neurotransmission. This serine hydrolase has evolved to catalyze hydrolysis of a single neurotransmitter acetylcholine in a matter of microseconds, under optimal conditions. Catalytic subunit of AChE is ~ 70 kDa large, it has three N-glycosylation sites and forms oligomers in human tissue. We are expressing monomeric form of human AChE in HEK293 cells in milligram quantity for structural and biophysical studies. We have observed that at micromolar concentrations AChE monomers associate reversibly to form homodimers. A hydrophobic dimerization domain is at the enzyme C-terminus and forms a four-helix bundle in a dimer. We have analyzed PDB deposited X-ray structures of dimeric human AChE for geometry of their four helix bundle domain and for relative position of two monomers that form homodimer. Dimerization domains of structurally related proteins of alpha/beta-hydrolase fold, human butyrylcholinesterase, human neuroligin and human carboxylesterase were obtained from PDB, their geometry analyzed and compared to the one of human AChE. This analysis is helping us understand conformational diversity and flexibility of closely related proteins involved in dimerization, that may be of interest for their structural domains directly involved in their physiological function located 20 – 30 A far from the dimer interface.
Abstract Title : Molecular mechanisms regulating scrunching in Dugesia japonica
Abstract : The ability to sense noxious stimuli and trigger an escape mechanism to avoid further damage is key to animal survival. It is known that scrunching, a locomotion gait in the flatworm Dugesia japonica, is one such escape mechanism that helps planarians evade noxious stimuli, including low pH, electric shock, ethanol, and high heat. Transient receptor potential (TRP) channels are known to be molecular detectors of these stimuli and are therefore natural candidates to understand nociception in planarians. In order to test this, we started by showing that capsaicin, a selective TRP agonist, does induce scrunching. Then, through RNA interference-mediated knockdown, we tested the role of the known TRP genes in D. japonica in regulating capsaicin-induced scrunching. The resulting phenotype was assessed through behavioral experiments using automated image analysis that quantified the mean speeds and path lengths. Our results so far are inconclusive and neither confirm nor negate our hypothesis.
Abstract Title : Improving the Binding Affinity of the APY peptide inhibitor for future treatment of ALS
Abstract : ALS is a highly devastating neurodegenerative disease that is characterized by gradual loss of motor neurons in the body. With 5,000 new cases every year in the US and a life expectancy of 3-5 years after disease onset, the disease puts huge financial and psychological strain on patients and family members. Adding to the severity of the disease is the lack of a cure or effective treatment options. The only FDA approved therapeutic, riluzole, only provides a marginal 2-3 month increase in survival of some patients. A recent study, however, identified the EphA4 receptor as a key inducer of ALS disease progression. The EphA4 receptor is part of the Eph family of receptors found generally with the adult nervous system-mainly in the hippocampus, cortex, and motor neurons. Due to its proximity in high neural plasticity regions in the brain, it has been linked with ALS as it regulates downstream activity of other neural molecules and brain structures and studies have shown correlation between increased EphA4 expression in motor neurons affected by ALS. As a receptor tyrosine kinase, it binds to the ephrin ligand, a group of highly promiscuous ligands that can activate multiple different types of Eph receptors. The large size and low selectivity of this ephrin ligand makes it difficult to inhibit the activity of the EphA4 receptor and thus, poses a problem for developing therapeutics. Previous research have identified the APY-βAla8 peptide as the most effective version of the peptide to date. The research presented here continues the development of the APY peptide into an effective therapeutic by investigating different alterations to the peptide sequence to improve binding affinity of the peptide to the EphA4 receptor. Early research dealt with substitution of the N-terminus Ala-1 with the betaine, trimethylglycine. Coupling trimethylglycine, however, proved difficult to achieve and research shifted over to substitution of the aromatic amino acids in the sequence with different analogs. In this range of experiments, Trp10 residue was substituted with 1-Nal and 2-Nal; Tyr6 was substituted with 4-Fluoro-Phe, 4-Bromo-Phe, 4-Chloro-Phe, and DOPA; and Tyr3 was substituted with DOPA. Yields of these APY variants were low and binding affinity are currently being evaluated, with the APY-(1-Nal)10 and APY-(2-Nal)10 variants proving to having comparable IC50 values compared to more effective APY variants, suggesting that the Trp10 position of the peptide sequence is open for additional modifications.
Abstract Title : Conformational Changes in Tertiary and Quaternary Structure of Human Acetylcholinesterase Inhibited by Paraoxon, Analyzed Computationally
Abstract : Human acetylcholinesterase, AChE (EC catalytic activity is essential for maintaining homeostasis and basic life functions for humans. Specifically, the serine hydrolase catalyzes the hydrolysis of more than 10,000 molecules of neurotransmitter acetylcholine every second under optimal conditions. Catalytic serine, which is located in the center of this enzyme, is especially reactive and important for this process. Organophosphate inhibitors form covalent bonds with catalytic Ser203 and block the AChE neurotransmitter. Paraoxon is the active form of organophosphate pesticide parathion that covalently inhibits AchE. Although the metabolic conversion process of parathion to paroxon is much faster in insects than humans, prolonged human exposure to parathion can generate sufficient paraoxon quantities to lead to toxicity and lethal effects in humans as well. In order to create better antidotes of paraoxon poisoning, several PDB (protein data bank) deposited x-ray structures of human AChE were analyzed. Native AChE and AChE inhibited by paraoxon was compared. Changes in AChE backbone conformation on the tertiary level were analyzed by creating overlays through an unbiased structural overlay approach. Furthermore, it was also investigated whether covalent inhibition on the tertiary level affected geometry of hAChE dimers that help create the quaternary structure.
Advisor : JOHN YATES
Abstract Title : Missing protein node prediction and protein quantitation in bipartite network representations of complex proteomes
Abstract : We analyzed the protein-peptide relationships via bipartite networks in order to quantify at the protein level, the varying proteoforms present in samples. These networks showcase the power of using bottom-up proteomics to show protein-peptide relationships to show that the quantitative measurements of the peptide nodes accurately determine the weights of the protein nodes, and thus can predict the presence of additional species-specific orthologues that were not included in the initial analysis. An initial set of experiments were run using different Drosophila species in order to test the accuracy. The distribution of weights in peptide nodes showed the species specificity of the peptide nodes, and therefore we were able to identify significantly regulated protein variants. The networking algorithm was also tested on Cystic Fibrosis cells CFBE41o- compared to HBE41o-, and identified serine/threonine protein kinase variants PKN1 and PKN4 were less abundant in the Cystic Fibrosis cells. The bipartite network analysis of peptide-protein relationships allows for a quantitation at protein level and prediction of additional proteoform nodes in a given sample.
Abstract Title : Site directed mutagenesis of ACP to determine the sequestration location of a pantetheine analogue probe, 4-DMN
Abstract : The acyl carrier protein (ACP) is an essential protein in fatty acid biosynthesis. ACP acts as a scaffold, tethering the growing acyl chain as it is elongated by various tailoring enzymes during fatty acid biosynthesis. Before entering the fatty acid biosynthetic pathway, ACP is converted from apo, an inactive form, to holo, the activated form, by the addition of a phophopantetheinyl arm (PPant) catalyzed by a phosphopantetheinyl transferase, PPtase. A promiscuous PPtase can accept synthetic analogues of the PPant arm, allowing for various probes to be attached to ACP. Recently, we attached a synthetic pantetheine analogue containing a fluorescent solvatochromic dye, 4-DMN. It was originally thought that 4-DMN is sequestered in the active site of E. coli ACP. However, recent computational analysis has provided evidence for the sequestration of the probe between alpha helices 2 and 3 of ACP. To test the hypothesis, site directed mutagenesis was performed on residues within these alpha helices, after which the synthetic probe was attached, and changes in fluorescence response were recorded.
Abstract Title : Substrate stabilization as a means of identifying ERAD-M retrotranslocation SUSpects
Abstract : ER associated degradation (ERAD) is a quality control pathway that destroys misfolded proteins in the ER lumen or membrane. The HRD pathway is a notable pathway in ERAD. Within the HRD complex, the E3 ligase Hrd1 functions in transferring ubiquitin from the E2 ligase Ubc7 to ERAD substrates. The substrates are then removed to the cytosol to be degraded by the 26S proteasome in a process called “retrotranslocation,” powered by the hexameric AAA-ATPase p97/Cdc48. In order to study the mechanisms of retrotranslocation of ERAD membrane (ERAD-M) substrates in Saccharomyces cerevisiae, we developed a self-ubiquitinating fusion substrate (SUS). SUS consists of the Hrd1 RING domain fused to the transmembrane domain of HMG-CoA reductase Hmg1; allowing SUS to degrade independently of Hrd1, but still relying on Cdc48 and the proteasome for retrotranslocation and degradation. We employed a genetic optical screen with GFP-tagged SUS and found that yeast Derlin, Dfm1, has a direct role in ERAD-M retrotranslocation. Biochemical studies show that both SUS-GFP and Hmg2-GFP depend on Dfm1 for retrotranslocation and degradation. As these findings directly contradicted the previous studies for Dfm1 being uninvolved in ERAD-M retrotranslocation, we reasoned that cells lacking Dfm1 are susceptible to suppressions. In fact, passaging of dfm1Δ strains expressing SUS-GFP over time, caused SUS-GFP to degrade to wild-type levels. In addition, we monitored the growth of dfm1Δ strains expressing galactose-driven Hmg2-GFP. When grown on galactose media, dfm1Δ strains show a substrate-induced slow growth. This growth phenotype of dfm1Δ strains under substrate-induced stress may provide insight on the interplay between Dfm1 and other ERAD components to reveal the requirements of ERAD-M retrotranslocation.
Abstract Title : Finding novel protein structures in Chlamydomonas reinhardtii in order to engineer algae for biofuels
Abstract : Finding the structure of algae acyl carrier protein (ACP) and thioesterase (TE) can facilitate research on engineering algae to create fatty acid profiles of our choice, and in particular, algae biofuels that can compete with dwindling fossil fuel reserves. Analysis of these proteins in Chlamydomonas reinhardtii have initiated via high-throughput protein crystallography methods, and further optimization of crystallizing conditions is needed to proceed with x-ray crystallography.