Leveraging the redundancy of S-denitrosylases in response to S-nitrosylation of Caspases: experimental strategies and beyond.

Redox-based protein posttranslational modifications, such as S-nitrosylation of critical, active site cysteine thiols have garnered significant clinical attention and research interest, reasoning for one of the crucial biological implications of reactive messenger molecule, nitric oxide in the cellular repertoire. The stringency of the S-(de)nitrosylation-based redox switch governs the activity and contribution of several susceptible enzymes in signal transduction processes and diverse pathophysiological settings, thus establishing it as a transient yet reasonable, and regulated mechanism of NO adduction and release. Notably, endogenous proteases like cytosolic and mitochondrial caspases with a molecular weight ranging from 33-55 kDa are susceptible to performing this biochemistry in the presence of major oxidoreductases, which further unveils the enormous redox-mediated regulational control of caspases in the etiology of diseases. In addition to advancing the progress of the current state of understanding of 'redox biochemistry' in the field of medicine and enriching the existing dynamic S-nitrosoproteome, this review stands as a testament to an unprecedented shift in the underpinnings for redundancy and redox relay between the major redoxin/ antioxidant systems, fine-tuning of which can command the apoptotic control of caspases at the face of nitro-oxidative stress. These intricate functional overlaps and cellular backups, supported rationally by kinetically favorable reaction mechanisms suggest the physiological relevance of identifying and involving such cognate substrates for cellular S-denitrosylases that can shed light on the bigger picture of extensively proposing targeted therapies and redox-based drug designing to potentially alleviate the side effects of NOx/ ROS in disease pathogenesis.

Nitric oxide and thioredoxin modulate the activity of caspase 9 in HepG2 cells.

The interdependent and finely tuned balance between the well-established redox-based modification, S-nitrosylation, and its counteractive mechanism of S-nitrosothiol degradation, i.e., S-denitrosylation of biological protein or non-protein thiols defines the cellular fate in the context of redox homeostasis. S-nitrosylation of cysteine residues by S-nitrosoglutathione, S-nitroso-L-cysteine-like physiological and S-nitroso-L-cysteine ethyl ester-like synthetic NO donors inactivate Caspase-3, 8, and 9, thereby hindering their apoptotic activity. However, spontaneous restoration of their activity upon S-denitrosylation of S-nitrosocaspases into their reduced, free thiol active states, aided by the members of the ubiquitous cellular redoxin (thioredoxin/ thioredoxin reductase/ NADPH) and low molecular weight dithiol (lipoic acid/ lipoamide dehydrogenase/ dihydrolipoic acid/ NADPH) systems imply a direct relevance to their proteolytic activities and further downstream signaling cascades. Additionally, our previous and current findings offer crucial insight into the concept of redundancy between thioredoxin and lipoic acid systems, and the redox-modulated control of the apoptotic and proteolytic activity of caspases, triggering their cyto- and neurotoxic effects in response to nitro-oxidative stress. Thus, this might lay the foundation for the exogenous introduction of precise and efficient NO or related donor drug delivery systems that can directly participate in catering to the S-(de)-nitrosylation-mediated functional outcomes of the cysteinyl proteases in pathophysiological settings.

De-glutathionylases: The resilient underdogs to keep neurodegeneration at bay.

Proteins become S-glutathionylated as a result of the derivatization of their cysteine thiols with the thiolate anion derivative of glutathione; this process is frequently linked to diseases and protein misbehavior. Along with the other well-known oxidative modifications like S-nitrosylation, S-glutathionylation has quickly emerged as a major contributor to a number of diseases, with a focus on neurodegeneration. The immense clinical significance of S-glutathionylation in cell signaling and the genesis of diseases are progressively coming to light with advanced research, which is also creating new opportunities for prompt diagnostics that utilize this phenomenon. In-depth investigation in recent years has revealed other significant deglutathionylases in addition to glutaredoxin, necessitating the hunt for their specific substrates. The precise catalytic mechanisms of these enzymes must also be understood, along with how the intracellular environment affects their impact on protein conformation and function. These insights must then be extrapolated to the understanding of neurodegeneration and the introduction of novel and clever therapeutic approaches to clinics. Clarifying the importance of the functional overlap of glutaredoxin and other deglutathionylases and examining their complementary functions as defense systems in the face of stress are essential prerequisites for predicting and promoting cell survival under high oxidative/nitrosative stress.

S-Denitrosylation: A Crosstalk between Glutathione and Redoxin Systems.

S-nitrosylation of proteins occurs as a consequence of the derivatization of cysteine thiols with nitric oxide (NO) and is often associated with diseases and protein malfunction. Aberrant S-nitrosylation, in addition to other genetic and epigenetic factors, has gained rapid importance as a prime cause of various metabolic, respiratory, and cardiac disorders, with a major emphasis on cancer and neurodegeneration. The S-nitrosoproteome, a term used to collectively refer to the diverse and dynamic repertoire of S-nitrosylated proteins, is relatively less explored in the field of redox biochemistry, in contrast to other covalently modified versions of the same set of proteins. Advancing research is gradually unveiling the enormous clinical importance of S-nitrosylation in the etiology of diseases and is opening up new avenues of prompt diagnosis that harness this phenomenon. Ever since the discovery of the two robust and highly conserved S-nitrosoglutathione reductase and thioredoxin systems as candidate denitrosylases, years of rampant speculation centered around the identification of specific substrates and other candidate denitrosylases, subcellular localization of both substrates and denitrosylases, the position of susceptible thiols, mechanisms of S-denitrosylation under basal and stimulus-dependent conditions, impact on protein conformation and function, and extrapolating these findings towards the understanding of diseases, aging and the development of novel therapeutic strategies. However, newer insights in the ever-expanding field of redox biology reveal distinct gaps in exploring the crucial crosstalk between the redoxins/major denitrosylase systems. Clarifying the importance of the functional overlap of the glutaredoxin, glutathione, and thioredoxin systems and examining their complementary functions as denitrosylases and antioxidant enzymatic defense systems are essential prerequisites for devising a rationale that could aid in predicting the extent of cell survival under high oxidative/nitrosative stress while taking into account the existence of the alternative and compensatory regulatory mechanisms. This review thus attempts to highlight major gaps in our understanding of the robust cellular redox regulation system, which is upheld by the concerted efforts of various denitrosylases and antioxidants.

NO news: S-(de)nitrosylation of cathepsins and their relationship with cancer.

Tumor formation and progression have been much of a study over the last two centuries. Recent studies have seen different developments for the early diagnosis and treatment of the disease; some of which even promise survival of the patient. Cysteine proteases, mainly cathepsins have been unequivocally identified as putative worthy players of redox imbalance that contribute to the premonition and further progression of cancer by interfering in the normal extracellular and intracellular proteolysis and initiating a proteolytic cascade. The present review article focuses on the study of cancer so far, while establishing facts on how future studies focused on the cellular interrelation between nitric oxide (NO) and cancer, can direct their focus on cathepsins. For a tumor cell to thrive and synergize in a cancerous environment, different mutations in the proteolytic and signaling pathways and the proto-oncogenes, oncogenes, and the tumor suppressor genes are made possible through cellular biochemistry and some cancer-stimulating environmental factors. The accumulated findings show that S-nitrosylation of cathepsins under the influence of NO-donors can prevent the invasion of cancer and cause cancer cell death by blocking the activity of cathepsins as well as the major denitrosylase systems using a multi-way approach. Faced with a conundrum of how to fill the gap between the dodging of established cancer hallmarks with cathepsin activity and gaining appropriate research/clinical accreditation using our hypothesis, the scope of this review also explores the interplay and crosstalk between S-nitrosylation and S-(de)nitrosylation of this protease and highlights the utility of charging thioredoxin (Trx) reductase inhibitors, low-molecular-weight dithiols, and Trx mimetics using efficient drug delivery system to prevent the denitrosylation or regaining of cathepsin activity in vivo. In foresight, this raises the prospect that drugs or novel compounds that target cathepsins taking all these factors into consideration could be deployed as alternative or even better treatments for cancer, though further research is needed to ascertain the safety, efficiency and effectiveness of this approach.

Ribonucleotide reductase: Implications of thiol S-nitrosylation and tyrosine nitration for different subunits.

Ribonucleotide reductase (RNR) is a multi-subunit enzyme responsible for catalyzing the rate-limiting step in the production of deoxyribonucleotides essential for DNA synthesis and repair. The active RNR complex is composed of multimeric R1 and R2 subunits. The RNR catalysis involves the formation of tyrosyl radicals in R2 subunits and thiyl radicals in R1 subunits. Despite the quaternary structure and cofactor diversity, all the three classes of RNR have a conserved cysteine residue at the active site which is converted into a thiyl radical that initiates the substrate turnover, suggesting that the catalytic mechanism is somewhat similar for all three classes of the RNR enzyme. Increased RNR activity has been associated with malignant transformation, cancer cell growth, and tumorigenesis. Efforts concerning the understanding of RNR inhibition in designing potent RNR inhibitors/drugs as well as developing novel approaches for antibacterial, antiviral treatments, and cancer therapeutics with improved radiosensitization have been made in clinical research. This review highlights the precise and potent roles of NO in RNR inhibition by targeting both the subunits. Under nitrosative stress, the thiols of the R1 subunits have been found to be modified by S-nitrosylation and the tyrosyl radicals of the R2 subunits have been modified by nitration. In view of the recent advances and progresses in the field of nitrosative modifications and its fundamental role in signaling with implications in health and diseases, the present article focuses on the regulations of RNR activity by S-nitrosylation of thiols (R1 subunits) and nitration of tyrosyl residues (R2 subunits) which will further help in designing new drugs and therapies.