Parkin coregulates glutathione metabolism in adult mammalian brain.

We recently discovered that the expression of PRKN, a young-onset Parkinson disease-linked gene, confers redox homeostasis. To further examine the protective effects of parkin in an oxidative stress model, we first combined the loss of prkn with Sod2 haploinsufficiency in mice. Although adult prkn-/-//Sod2± animals did not develop dopamine cell loss in the S. nigra, they had more reactive oxidative species and a higher concentration of carbonylated proteins in the brain; bi-genic mice also showed a trend for more nitrotyrosinated proteins. Because these redox changes were seen in the cytosol rather than mitochondria, we next explored the thiol network in the context of PRKN expression. We detected a parkin deficiency-associated increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in murine brain, PRKN-linked human cortex and several cell models. This shift resulted from enhanced recycling of GSSG back to GSH via upregulated glutathione reductase activity; it also correlated with altered activities of redox-sensitive enzymes in mitochondria isolated from mouse brain (e.g., aconitase-2; creatine kinase). Intriguingly, human parkin itself showed glutathione-recycling activity in vitro and in cells: For each GSSG dipeptide encountered, parkin regenerated one GSH molecule and was S-glutathionylated by the other (GSSG + P-SH [Formula: see text] GSH + P-S-SG), including at cysteines 59, 95 and 377. Moreover, parkin's S-glutathionylation was reversible by glutaredoxin activity. In summary, we found that PRKN gene expression contributes to the network of available thiols in the cell, including by parkin's participation in glutathione recycling, which involves a reversible, posttranslational modification at select cysteines. Further, parkin's impact on redox homeostasis in the cytosol can affect enzyme activities elsewhere, such as in mitochondria. We posit that antioxidant functions of parkin may explain many of its previously described, protective effects in vertebrates and invertebrates that are unrelated to E3 ligase activity.

Glutathione-dependent thioredoxin reduction and lipoamide system support in-vitro mammalian ribonucleotide reductase catalysis: a possible antioxidant redundancy.

BACKGROUND: The thioredoxin system (Trx), comprising of Trx, Thioredoxin reductase (TrxR) and NADPH aids in donating hydrogen group to support Ribonucleotide reductase (RNR) catalysis during de-novo DNA biosynthesis. However, it has been observed that inhibiting TrxR does not affect the viability of cancer cells that are susceptible to pharmacological glutathione (GSH) depletion. This prompted us to study the potential antioxidant redundancies that might prolong RNR activity. METHODS: To study the RNR activity assay, the RNR complex was reconstituted by mixing purified mouse recombinant RNR subunits and the conversion of [3 H] CDP into [3 H] dCDP was monitored. In the assay system, either purified Trx and GSH or Lipoamide system was supplemented as reducing agents to support RNR catalysis. RESULTS: Herein, we have found that GSH-dependent Trx reduction supports mammalian class I RNR catalysis in absence of TrxR in the system. Our data also presents the first report that the LAM system is capable of supporting in-vitro RNR activity in the complete absence of either Trx or Grx systems. CONCLUSIONS: We conclude that GSH-mediated Trx reduction and LAM systems support basal level RNR activity in vitro; in absence of TrxR and complete redoxin systems respectively and hypothesize that potential redundancy between the various antioxidant systems might synergize in sustaining RNR activity.

Ribonucleotide reductase: In-vitro S-glutathionylation of R2 and p53R2 subunits of mammalian class I ribonucleotide reductase protein.

Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.

Stability of S-nitrosothiols and S-nitrosylated proteins: A struggle for cellular existence!

Nitric oxide is a well-known gasotransmitter molecule that covalently docks to sulfhydryl groups of proteins resulting in S-nitrosylation of proteins and nonprotein thiols that serve a variety of cellular processes including cGMP signaling, vasodilatation, neurotransmission, ion-channel modulation, and cardiac signaling. S-nitrosylation is an indispensable modification like phosphorylation that directly regulates the functionality of numerous proteins. However, recently there has been a controversy over the stability of S-nitrosylated proteins (PSNOs) within the cell. It has been argued that PSNOs formed within the cell is a transient intermediate step to more stable disulfide formation and disulfides are the predominant end effector modifications in NO-mediated signaling. The present article accumulates state-of-the-art evidence from numerous research that strongly supports the very existence of PSNOs within the cell and attempts to put an end to the controversy. This review illustrates critical points including comparative bond dissociation energies of S-NO bond, the half-life of S-nitrosothiols and PSNOs, cellular concentrations of PSNOs, X ray crystallographic studies on PSNOs, and stability of PSNOs at physiological concentration of antioxidants. These logical evidence cumulatively support the endogenous stability and inevitable existence of PSNOs/RSNOs within the cell that directly regulate the functionality of proteins and provide valuable insight into understanding stable S-nitrosylation mediated cell signaling.

Understanding the role of S-nitrosylation/nitrosative stress in inflammation and the role of cellular denitrosylases in inflammation modulation: Implications in health and diseases.

S-nitrosylation is a very fundamental post-translational modification of protein and non-protein thiols due the involvement of it in a variety of cellular processes including activation/inhibition of several ion channels such as ryanodine receptor in the cardiovascular system; blood vessel dilation; cGMP signaling and neurotransmission. S-nitrosothiol homeostasis in the cell is tightly regulated and perturbations in homeostasis result in an altered redox state leading to a plethora of disease conditions. However, the exact role of S-nitrosylated proteins and nitrosative stress metabolites in inflammation and in inflammation modulation is not well-reviewed. The cell utilizes its intricate defense mechanisms i.e. cellular denitrosylases such as Thioredoxin (Trx) and S-nitrosoglutathione reductase (GSNOR) systems to combat nitric oxide (NO) pathology which has also gained current attraction as novel anti-inflammatory molecules. This review attempts to provide state-of-the-art knowledge from past and present research on the mechanistic role of nitrosative stress intermediates (RNS, OONO-, PSNO) in pulmonary and autoimmune diseases and how cellular denitrosylases particularly GSNOR and Trx via imparting opposing effects can modulate and reduce inflammation in several health and disease conditions. This review would also bring into notice the existing gaps in current research where denitrosylases can be utilized for ameliorating inflammation that would leave avenues for future therapeutic interventions.

Cellular S-denitrosylases: Potential role and interplay of Thioredoxin, TRP14, and Glutaredoxin systems in thiol-dependent protein denitrosylation.

Nitric Oxide is a very well known gaseous second messenger molecule and vasorelaxant agent involved in a variety of signaling in the body such as neurotransmission, ion channel modulation, and inflammation modulation. However, it's reversible covalent attachment to thiol groups of cysteine residues under nitrosative stress leading to aberrant protein S-nitrosylation (PSNO) has been reported in several pathological conditions in the body stemming from neurodegenerative diseases, cancer, cardiovascular system, and immune system disorders. In the cell, PSNOs are partly unstable and transit to a more stable disulfide state serving as an intermediate step towards disulfide formation thus eliciting the biological response. Scientists have identified several cellular thiol-dependent disulfide reductases that have the intrinsic capability to reverse the modification by reducing the stable disulfides formed in PSNOs and thereby rescue S-nitrosylation-induced altered proteins. The physiological roles of these major cellular ubiquitous S-denitrosylases and their probable implementations have not been fully explored. Gaining knowledge from current research and development this review provides a deeper insight into understanding the interplay and role of the major ubiquitous S-denitrosylases in maintaining cellular redox homeostasis. This review umbrellas the mechanism of Thioredoxin, TRP14, and Glutaredoxin systems and highlights their substrates specificities at different cellular conditions, physiological roles, and importance in diseased conditions that would allow researchers to investigate effective therapeutic interventions for nitrosative stress-related diseases and disorders.

Neuron-glia: understanding cellular copper homeostasis, its cross-talk and their contribution towards neurodegenerative diseases.

Over the years, the mechanism of copper homeostasis in various organ systems has gained importance. This is owing to the involvement of copper in a wide range of genetic disorders, most of them involving neurological symptoms. This highlights the importance of copper and its tight regulation in a complex organ system like the brain. It demands understanding the mechanism of copper acquisition and delivery to various cell types overcoming the limitation imposed by the blood brain barrier. The present review aims to investigate the existing work to understand the mechanism and complexity of cellular copper homeostasis in the two major cell types of the CNS - the neurons and the astrocytes. It investigates the mechanism of copper uptake, incorporation and export by these cell types. Furthermore, it brings forth the common as well as the exclusive aspects of neuronal and glial copper homeostasis including the studies from copper-based sensors. Glia act as a mediator of copper supply between the endothelium and the neurons. They possess all the qualifications of acting as a 'copper-sponge' for supply to the neurons. The neurons, on the other hand, require copper for various essential functions like incorporation as a cofactor for enzymes, synaptogenesis, axonal extension, inhibition of postsynaptic excitotoxicity, etc. Lastly, we also aim to understand the neuronal and glial pathology in various copper homeostasis disorders. The etiology of glial pathology and its contribution towards neuronal pathology and vice versa underlies the complexity of the neuropathology associated with the copper metabolism disorders.