engleski

Neuropathy target esterase-related enzyme and its kinetic characterization

The object of our study was the kinetic characterization of the neuropathy target esterase-related enzyme (NRE, PNPLA7) in different cell lines. NRE enzyme is a member of the PNPLA (patatin-like phospholipase domain containing proteins) family and a membrane protein associated with endoplasmic reticulum or lipids droplets and is highly expressed in insulin-targeted tissues. Its crystal structure has not been resolved, yet, but according to the sequence analysis and homology modelling it consists of an N-terminal domain with a transmembrane segment and three cyclic nucleotide binding sites, and a C-terminal domain where the active site with catalytic dyad Ser-Asp is located. NRE has been identified as a lysophospholipase that hydrolyses sn-1 esters in lysophosphatidylcholine and lysophosphatidic acid, but its physiological roles are not fully understood. NRE enzyme shares high homology with neuropathy target esterase (NTE, PNPLA6), which suggests that NRE might also be a target of highly toxic organophosphorus compounds (OP) implying involvement of NRE in OP caused pathological conditions such as poorly defined intermediate myopathy syndrome. In our research, we used two commercially available donors of human skeletal muscle cells and the liver hepatocellular carcinoma cell line HepG2 to kinetically characterize NRE activity using known substrate p-nitrophenyl valerate (p-NPV). Firstly, we confirmed presence of NRE in myotubes, differentiated human skeletal muscle cells, and HepG2 by Western blot. Afterwards, esterase activity measurement was optimised for cell lysates. In order to confirm esterase activity, we incubated cell lysates with different OP compounds and measured esterase activity with p-NPV. Our results showed a difference in the NRE activity in different donors of human skeletal muscle cells, probably due to difference in the protein expression. Also, we have concluded that optimal p-NPV concentration for activity measurement is 1 mM with the Km value of 0.4827 mM. Since little is known about this enzyme's physiological role and biological relevance, any findings would most certainly contribute to the understanding of the importance of NRE, which still calls for a detailed clarification. 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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