Regulation of cellular functions by nitric oxide pathway

Abstract

Nitric oxide (NO) is a highly diffusible gas that is synthesized by three distinct isoforms of nitric oxide synthases (NOS). Two isoforms are constitutively expressed and generate NO for cell-signalling purposes: neuronal NOS (nNOS) and endothelial NOS (eNOS), and the third member of the family is an inducible isoform (iNOS) which releases NO in larger quantities during inflammatory or immunological defence reactions and is involved in host tissue damage. NO has been shown to act as a multifunctional gaseous modulator in many cellular events. It is an important messenger molecule synthesized in a variety of tissues and involved in various physiological and pathological functions. The regulatory actions of NO are explained both by the physiological intracellular concentrations of NO and by some well known NO-dependent cell signalling and regulatory pathways. During this research project we focused on two of these regulatory pathways: the NO-dependent protein modifications of cysteine residues and the reversible inhibition of mitochondrial cytochrome c oxidase (CcOX), the terminal complex of the mitochondrial respiratory chain. Modulation of thiol-disulfide status of critical cysteines on enzymes, receptors, transport proteins, and transcription factors is recognized as an important mechanism of signal transduction and an important consequence of oxidative/nitrosative stress associated with aging, cardiovascular and neurodegenerative diseases. Within these contexts, a prevalent form of cysteine modification is reversible formation of protein mixed disulfides with glutathione (GSH), the major non-protein thiol compound in cells. Protein S-glutathionylation increases globally during overt oxidative stress, but selective/local generation of reactive oxygen and nitrogen species mediates physiological redox signaling. Moreover, reversible modifications, as S-glutathionylation, have been suggested to have a dual role: protection from cysteine irreversible oxidation and modulation of protein function. Among the wide list of proteins demonstrated to be susceptible to oxidative cysteine modifications, as S-nitrosylation, we focused on Metallothionein (MT) and the transcription factor Sp1, two zinc-binding metalloproteins that play fundamental roles in cellular functions, and whose activity has been shown to be affected upon ageing. Thus we investigated the susceptibility to S-glutathionylation of these proteins. Analysis of the three-dimensional structure of both MT and Sp1 showed the presence of some Cys residues likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity glutathionylable cysteine residues. Western blot and dot blot assays performed after in vitro exposure to GSNO, diamide and H2O2 (oxidant agents acting through different mechanisms) revealed that both MT and Sp1 can be susceptible to S-glutathionylation upon oxidative/nitrosative stress conditions. Moreover, this effect was completely reversed by treatment with the reducing agent DTT, indicating the involvement of protein-mixed disulphides. Together our findings support a potential functional role for S-glutathionylation in protecting these proteins from irreversible oxidation that could occur during oxidative/nitrosative stress into the cell, and may represents an important antioxidant mechanism in the ageing process. NO has been observed to act as an effective signal molecule that regulates mitochondrial events, including oxygen consumption and reactive oxygen species production. Binding of NO with the terminal electron acceptor of the mitochondrial electron transport chain, CcOX, plays crucial roles in mediating the physiological effects of NO. Interaction between NO and CcOX is bidirectional resulting not only in the modulation of the mitochondrial enzyme activity by NO, but also in the regulation of NO concentrations by CcOX. Recently, we reported that mtNOS is physically associated with CcOX, and that this binding is mediated by the PDZ motif of mtNOS. In order to further analyze the role played by NO in CcOX activity modulation, in the present work we explored whether and how the interaction between the mtNOS PDZ motif and the subunit Va of CcOX can be regulated. Through molecular modeling simulations we individuated a potentially critic specific tyrosine residue (Tyr77) in the PDZ of nNOS (whose alpha isoform is identical to mtNOS). Considering that the importance of phosphorylation/dephosphorylation in regulating NOS activity and, more recently, mitochondrial processes has been recognized and several protein kinases and phosphatases have been identified in mitochondria, we investigated whether nNOS PDZ Tyr77 phosphorylation could be implicated in the mtNOS/CcOX interaction modulation. To this purpose we utilized different experimental approaches such as nNOS alpha cloning and mutagenesis, expression of wild type and mutant nNOS in mammalian cells, and, finally, analysis of nNOS/CcOX interaction in wild type and transfected cells by means of confocal microscopy and co-immunoprecipitation assays. Together our results suggest phosphorylation as a likely mechanism of mtNOS/CcOX interaction regulation. Moreover it seems that this modulation is mediated by the action of some src tyrosine kinases.