Protein interactions of NAD(P)H-cytochrome b5 oxidoreductase in the endoplasmic reticulum: its role in prevention of lipotoxicity-induced inflammation
Free-fatty-acid-induced lipotoxicity plays a crucial role in the development of type 2 diabetes mellitus. Saturated fatty acids are remarkably more toxic than their unsaturated analogues; moreover, the latter ones partly counteract the damage caused by the former ones. Therefore, the endogenous cellular fatty acid desaturating activity is considered as a protective feature. Desaturation takes place in the endoplasmic reticulum (ER) membrane, and its rate limiting step is catalyzed by stearoyl-CoA-desaturase (SCD1), which – similarly to cytochrome P450 (CYP) monooxygenases, key enzymes of biotransformation – receives electrons from NAD(P)H via cytochrome b5 (b5) and b5-reductase (b5R). NAD(P)H-cytochrome b5 oxidoreductase (Ncb5or), a particulate cytosolic protein of yet-unresolved function possesses both b5-like and b5R-like domains, and its deficiency enhances lipotoxicity. Ncb5or is implied to be involved in the microsomal electron transfer; nevertheless, its direct interaction with SCD1 and/or certain CYP enzymes remains to be revealed; i.e. its contribution to fatty acid desaturation and drug metabolism needs to be tested.
The present cooperation rests on the synergy of the studies on fatty acid desaturation at Semmelweis University and the studies on CYP enzymes at Research Centre for Natural Sciences in order to learn about Ncb5or. The aim of our work is to elucidate the putative interactions between Ncb5or and SCD1 or CYP isozymes, i.e. the contribution of Ncb5or in fatty acid desaturation and/or biotransformation in cellular and in vitro enzyme activity measurements. We are using the following models: 1) HEK293 (human embryonal kidney) cells overexpressing SCD1 and/or CYP isoenzymes, in which the effect of b5 and/or Ncb5or co-transfection or silencing on enzyme activities is to be measured. 2) Human liver microsome (ER fraction), in which the enzyme activities can be assessed with or without cofactors (NADH, NADPH), cytosolic components and purified Ncb5or protein. 3) Supersomes containing a single CYP isoenzyme combined with certain electron transfer proteins, and so providing a tool to test the role of exogenous Ncb5or. SCD1 and individual CYP isoenzyme activities are measured by using their specific substrates.
The expected results would not only reveal the putative interaction of Ncb5or with ER-resident proteins but also provide a molecular explanation for the observed protective action of Ncb5or in lipotoxicity. This is equally important from the aspect of basic research and of the improvement of anti-diabetic prevention and treatment.
Miklós Csala - Katalin Monostory