Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10
ID 15966 Poster Board 88Phospholipase Ce enzymes are required for normal cardiovascular function, and their dysregulation can lead to cardiac hypertrophy and heart failure. PLCe cleaves phosphatidylinositol phosphates into inositol phosphates and diacylglycerol, increasing intracellular Ca 2+ and activating protein kinase C. PLCe activity is increased by direct binding of the Rap1A GTPase, following stimulation of b-adrenergic receptors. This pathway is required for maximum cardiac contractility, but sustained activation causes cardiac hypertrophy. Rap1A binds to the C-terminal Ras Association (RA) domain of PLCe, and we previously showed that activation requires long-range conformational changes in the lipase. However, the residues in PLCe involved in the intramolecular rearrangements are not known. As a first step, I used cryo-electron microscopy single particle analysis (cryo-EM SPA) to determine the 4 Å reconstruction of PLCe PH-C in complex with an antigen binding fragment (Fab). This is the largest fragment of PLCe biochemically characterized to date and is robustly activated by Rap1A. The structure defines the basal state of the enzyme and reveals a potential membrane binding surface on the PH domain. These studies set the stage for investigating the structure of the Rap1A-PLCe PH-C complex and its mechanism of activation. Ultimately, this work supports long-term efforts to develop small molecule modulators of the lipase and its activated complex for treating cardiac hypertrophy.
Nocardia Cholesterolicum NRRL 5767 (NC NRRL5767) is well‐known for its ability to transform ~95% of added oleic acid, an abundant agricultural commodity, to value‐added product of 10‐hydroxystearic acid (10‐HSA). A small amount of unwanted 10‐ketostearic acid (10‐KSA) was also produced. The conversion of oleic acid to 10‐HSA and then to 10‐KSA is catalyzed by oleate hydratase and secondary alcohol dehydrogenase (2°‐ADH), respectively. The objective of this project was to knockout the 2°‐ADH gene in NC NRRL5767 so that the sole biotransformation product from oleic acid would be 10‐HSA. Here, we report construction of CRISPR/Cas9/sgRNA chimeric plasmids that specifically target two different loci in the coding region of the 2°‐ADH gene by golden gate assembly. The constructs were confirmed by DNA sequencing and transformed into NC NRRL 5767 via electroporation. The transformants were selected by apramycin resistance and screened for the presence of the target insert by PCR. The ability of the selected transformants to transform oleic acid to 10‐HSA was examined by TLC. Our results showed that both 10‐HSA and 10‐KSA were produced by the transformants from oleic acid. Apparently, either Cas9 was not expressed or it was expressed but did not target the 2°‐ADH gene. The expression of Cas9 in the cell‐free extracts of selected transformants was examined by Western blotting using Cas9 antibody. However, the Cas9 protein was undetectable in these transformants.Support or Funding InformationWestern Illinois University FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PLC (phospholipase C) enzymes hydrolyze phosphatidylinositol lipids to generate inositol phosphates (IPx) and diacylglycerol (DAG). These second messengers stimulate intracellular calcium release and protein kinase C activation. PLCε is the most recently identified member of the PLC family, and is activated downstream of G protein‐coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with G proteins. In the cardiovascular system, PLCε is necessary for normal cardiac contractility, and its dysregulation contributes to cardiac hypertrophy and heart failure. PLCε shares a highly conserved core that is observed in other PLC subfamilies, and contains a unique N‐terminal CDC25 domain and two C‐terminal Ras association (RA) domains. However, structural insights into the full‐length protein, or catalytically active fragments that contain two or more regulatory domains, have been lacking. Using a combination of structural and functional studies, we previously showed that the PH domain and first two EF hands are dynamic in solution, providing an explanation as to why high‐resolution structures have been elusive. We are now pursuing high‐resolution structural studies of larger PLCε variants and the full‐length enzyme using single particle cryo‐electron microscopy (cryo‐EM). To facilitate this process, we are utilizing domain‐specific fab antibodies as fiduciary markers and to stabilize this conformationally heterogeneous protein. Understanding the structure of PLCε will be critical in identifying sites that can be targeted by small molecule chemical probes, ultimately validating this enzyme as a therapeutic target in cardiovascular disease.
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