2:45 PM Chemistry and Biochemistry Breakout IX: Panel B

Wednesday, August 2 2:45PM – 3:45PM

Location: Odyssey

Cat Avarvarei
UCSD
Chemical Biological Study of Toll-like Receptor (TLR) Structure and Activity in Pharmaceutically Significant Transmembrane Proteins
Transmembrane receptor proteins allow cells to communicate with each other and with the outside world, which is why most pharmaceutically significant drugs are designed to target these proteins. By delivering specific ligands to receptors like TLR4 and mu-opioid, specific signals within a cell can be induced. Therefore, by isolating the polypeptide chains of these natural receptors, their structure and reactivity can be studied. To do this, recombinant protein expression, or total chemical peptide synthesis via solid phase fmoc-synthesis are used. The fragments resulting from these methods are characterized through Liquid Chromatography Mass Spectrometry (LC-MS) and purified through Reverse Phase High Pressure Liquid Chromatography (RP-HPLC). Preliminary SDS-PAGE, LC-MS, and RP-HPLC results show that specific fragments can be isolated and identified. Molecular dynamics (MD) simulations are also used to test and visualize variations in polypeptide design. So new structures predicted to be stable and promising in their activity can be synthesized. Lastly, new fragments can be tested in cell assays to verify their activity.
Regina Jareanpalithapon
University of Minnesota - Twin Cities
Purification of Human Apolipoprotein A1 (ApoA1) from Blood Plasma Using Mixed Mode Chromatography
This research aims to establish a method for purifying human Apolipoprotein A1 (ApoA1) from blood plasma necessary for reconstituting into synthetic high density lipoprotein (sHDL) particles. Purifying ApoA1 is a crucial step in developing sHDL as it makes up approximately 70% of HDL. The function of HDL is believed to promote reverse cholesterol transport. Additionally, HDL has been found to be a potential therapeutic for sepsis since it is a part of regulating the vascular endothelial function and immunity. Given the significance of HDL in these processes, there is a large interest in developing sHDL containing ApoA1 as a potential therapeutic approach and biomarker for various conditions. The method to purify ApoA1 from human blood plasma involves following the procedures from G. et al.’s paper that proposed a seemingly less time-consuming and obtaining a high yield of purified ApoA1 in comparison to conventional methods. For this research project, human blood plasma was precipitated using 60% (NH4)2SO4. Then the supernatant was used onto a HEA HyperCelTM column that eluted the bound proteins with discontinuous pH gradient. The elutions were pooled and the purified ApoA1 was analyzed for homogeneity and yield through SDS-PAGE gel electrophoresis, Western blot analysis, densitometric analysis, and mass spectrometry analysis. The results from this study highlights the challenges faced in purifying ApoA1 and suggest that further purification strategies should be explored to improve the yield and homogeneity of purified ApoA1.
Jennifer Nguyen
University of Colorado Denver
Modeling of CLCF Flouride/Proton Antiporter
Fluoride ions safeguard dental hard tissues and prevent bacterial growth in the oral cavity. However, some bacterial strains, like Streptococcus mutans, which is a significant cause of tooth decay, can survive in environments of high fluoride concentrations. The fluoride resistance of S. mutans is largely due to the CLCF transport proteins that pump fluoride ions out of the cell. The aim of this project is to investigate the mechanistic changes in the fluoride permeation when the gating E318 residue of CLCF is mutated. The crystal structures of a prototypical CLCF protein, CLCF -eca, reveal that two fluoride ions are located at the canonical anion-binding sites. The fluoride ions are coordinated by a number of residues such as Y396 and E318. Of particular interest to us is the glutamate residue E318, which is conserved in CLCF proteins but not found in the closely related homologous CLC proteins that translocate chloride ions. Interestingly, experiments found that, in the E318Q mutant, where E318 is mutated to a charge-neural glutamine residue, the fluoride ion permeation rate increases. Our hypothesis is that fluoride ion interact more strongly with a protonated glutamate through proton sharing than with a glutamine through hydrogen bonding. We will carry out molecular modeling to study the translocation of the fluoride ion through the pore of the E118Q mutant. We will construct an atomistic model of the E318Q mutant through in silica mutation to the experimental structure of E118. The knowledge gained through this study will be valuable in our comprehensive understanding of CLCF operation mechanisms and can help us develop better therapy to deal with these difficult strains.
Maiyia Vang
University of Wisconsin - Madison
Examination of ASBT mRNA and/or Protein Level in the Ileum of MGAT2 KO Mice and its Wild Type Controls
Mice with a deficiency of the intestinal enzyme monoacylglycerol acyltransferase 2 (MGAT2 KO) are protected from metabolic disorders induced by high-fat feeding. Our lab further showed that these protective effects are due to elevated bile acid levels in their plasma, likely resulting from enhanced bile acid reabsorption in the ileum through the apical sodium-dependent bile acid transporter (ASBT). The ASBT transporter, mainly expressed in the ileum, reabsorbed most of secreted bile acid back into the portal vein to the liver. My study this summer aimed to examine whether MGAT2 KO mice have a higher expression of ASBT at the mRNA and/or protein level than their wild-type (WT) controls. I isolated the jejunum (control) and ileum tissues from WT and MGAT2 KO mice to examine ASBT at the protein level utilizing Western Blot with anti-ASBT antibodies and at the mRNA level using qPCR. I found that the relative expression of ASBT proteins in the ileum of MGAT2 KO mice is trending higher. However, ASBT protein expressions were not detected in the jejunum. The ASBT mRNA level in the ileum of MGAT2 KO is trending higher but does not reach statistical significance. The next step is to repeat the experiment with more samples, including MGAT2 KO mice and WT with low and high bile acid levels in plasma. My study may be significant to understanding the factors that protect MGAT2 KO mice from obesity and associated metabolic disorders.