title: The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis.
abstract: The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in l-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.
authors.affiliation: Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005, India.
authors.forename: Swapnil S
authors.initials: SS
authors.lastname: Parhad
authors.affiliation: Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005, India; TIFR Centre for Interdisciplinary Sciences, 21 Brundavan Colony, Narsingi, Hyderabad 500075, India.
authors.forename: Deepa
authors.initials: D
authors.lastname: Jaiswal
authors.affiliation: Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005, India. Electronic address: krishanu@tifr.res.in.
authors.forename: Krishanu
authors.initials: K
authors.lastname: Ray
authors.affiliation: Tata Institute of Fundamental Research (TIFR), Homi Bhabha Road, Mumbai 400 005, India. Electronic address: shyamal@tifr.res.in.
authors.forename: Shyamalava
authors.initials: S
authors.lastname: Mazumdar
mesh_terms: D002147:Calmodulin / Q000378:metabolism; D056445:Cytoplasmic Dyneins / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I; C427758:DYNLL1 protein, human; D056445:Cytoplasmic Dyneins
keywords: DLC1; Mechanism of inhibition; Protein-protein interaction; Structural change; nNOS