engleski

Characterization of lysophospholipase PNPLA7 as a potential target for drug development

Lysophospholipase PNPLA7 is a member of the PNPLA (patatin-like phospholipase domain containing proteins) family of nine enzymes sharing a patatin-like catalytic domain. It has been shown that PNPLA7 is highly expressed in the insulin targeted tissues where it can be associated with endoplasmic reticulum and lipid droplets, however, its physiological and molecular roles are not described well. Crystal structure of PNPLA7 is not known, but according to the sequence analysis and homology modelling, it consists of a transmembrane segment near the N-terminal end, three cyclic nucleotide binding sites, and patatin domain where the active site with catalytic dyad Ser-Asp is located. As the first aim of this study, we investigated the physiological role of this enzyme in the cultured human skeletal muscle cells. Our study confirms that human skeletal muscle cells express PNPLA7 mRNA and protein and that it is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of S6RP and p70, activators of protein translation, as well as the abundance of α1-subunit of Na+, K+- ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important for these cells. As for the second objective of this study, we have tried to express a truncated PNPLA7 enzyme using Escherichia coli as a host. Our results showed that the majority of this enzyme remains in the membrane fractions hence, we are currently working on the optimization of the expression and purification conditions, in order to get the PNPLA7 for possible kinetic characterization. This research was supported by the Croatian Science Foundation, grant number HrZZ-UIP-2017-05- 7260, Slovenian Research Agency (J3-9263 and J3-2523, P3-0043 and J7-8276) and Croatian-Slovenian Bilateral grant 2020-2022 (BI- HR/20-21-041).

NRE ; NTE ; hepatocytes ; kinetics

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