Mammalian cystatin and protagonists in brain diseases

Abstract

Cystatins are the thiol Proteinase inhibitors, present ubiquitously in mammalian body. They prevent unwanted proteolysis and play important role in several diseases. Regulation of cysteine Proteinase and their inhibitors is of utmost importance in neurodegenerative diseases like Alzheimer, amyloid angiopathy and in many other diseases. The action of these cysteine proteases is biologically controlled by proteinase inhibitors namely cystatins(cys) they constitute a superfamily of homologous proteins. The major role of cystatins is to protect the organism against endogenous proteases released from lysosomes, invading microorganisms and parasites that use cysteine proteases to enter the body. An enormous progress has been made in understanding of protein degradation process under normal and pathological conditions; in fact proteases are now clearly viewed as important drug targets. Some studies have suggested that cystatin C is a target for intervention in neurological disorders because its expression increases in response to human neurological disorders and in animal models of neurodegenerative states. Although, these studies did not clarify whether CysC up-regulation is a pathogenic factor in neurodegenerative disorders or whether it represents a neuroprotective compensatory response of the organisms aimed to prevent progression of the disease. However, for other diseases in some cases cystatins other than cys C are up regulated and in some it is down regulated.

Cystatins have been implicated in the processes of neuronal degeneration and repair of the nervous system. Both CysC and CysB are potent, reversible inhibitors of most of the currently known cathepsins. The extent of proteolytic activity at any given time and location is the result of a balance between active proteases and physiological inhibitors. Uncontrolled proteolysis as a result of imbalance between active proteases and their endogenous inhibitors has been associated with neuronal cell death in different neuronal diseases, including brain tumors, stroke, some forms of epilepsy, Alzheimer’s disease, and neurological autoimmune diseases.

An antidepressant is a psychiatric medication used to alleviate mood disorders, major depression and other brain diseases. Drugs including the monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), and serotonin-norepinephrine reuptake inhibitors (SNRIs) are most commonly used antidepressant. They are also used to treat other conditions, such as anxiety disorders, obsessive compulsive disorder, eating disorders, and chronic pain. Although the mechanisms of the action of these antidepressants are not precisely understood, their principal target of action is at the monoamine transporter proteins located at nerve endings. Monoamine neurotransmitter transporters act to terminate synaptic neurotransmission. Selective serotonin reuptake inhibitors or SSRIs are also most widely used class of antidepressants. They work by increasing the level of serotonin in the brain. SSRIs have fewer and milder side effects, fewer drug interactions, and are much less likely to be associated with suicide than TCAs.

These antidepressants shows binding when incubated with cystatin, presenting the involvement of these antidepressant in cascade of disease, as leaving no cystatin to inhibit the cathepsin showing the myriad side effect after the administration of antidepressant. This might be one of the reason in the mechanism of action of antidepressant.

So this review expound about the role of cystatins in neurological diseases which is considered to be highly significant as it pave the way for commanding tool in the drug design.

Communicated by Ramaswamy H. Sarma     

The rate of interleukin-1beta secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering

Human myeloid cells activate the NLRP3 inflammasome and secrete interleukin (IL)-1β in response to various Toll-like receptor (TLR) ligands, but the rate of secretion is much higher in primary human monocytes than in cultured macrophages or THP-1 cells. The different myeloid cells also display different redox status under resting conditions and redox response to TLR activation. Resting monocytes display a balanced redox state, with low production of reactive oxygen species (ROS) and antioxidants. TLR engagement induces an effective redox response with increased ROS generation followed by a sustained antioxidant response, parallelled by efficient IL-1β secretion. Drugs blocking ROS production or the antioxidant response prevent the secretion of mature IL-1β but not the biosynthesis of pro-IL-1β, indicating that redox remodeling is responsible for IL-1β processing and release. Unlike monocytes, THP-1 cells and cultured macrophages have up-regulated antioxidant systems that buffer the oxidative hit provided by TLR triggering and suppress the consequent redox response. This aborted redox remodeling is paralleled by low efficiency IL-1β processing and secretion. High doses (5 mM) of H(2)O(2) overcome the high antioxidant capacity of THP-1 cells, restore an efficient redox response, and increase the rate of IL-1β secretion. Together these data indicate that a tightly controlled redox homeostasis in resting cells is a prerequisite for a robust redox response to TLR ligands, in turn necessary for the efficient inflammasome activation. Inflammasome activation by bacterial DNA is not modulated by redox responses, suggesting that redox-dependent regulation of IL-1β secretion is restricted to some inflammasomes including NLRP3 but excluding AIM-2.     

Mononuclear copper(I) complexes of O-t-butyl-1,1-dithiooxalate and of O-t-butyl-1-perthio-1-thiooxalate

Described are the syntheses and structures of a phosphonium salt of the anionic ligand O-t-butyl-1,1-dithiooxalate, [PPh₃Bz][i-dto^tBu] ([PPh₃Bz][1]), and of two Cu(I) complexes of this anion, Cu(PPh₃)₂(η²-i-dto^tBu) (2) and Cu(dmp)(PPh₃)(η¹-i-dto^tBu) (3, dmp = 2,9-dimethyl-1,10-phenanthroline). In addition, it was found that the reaction of CuBr₂ with i-dto^tBu⁻ gives a O-t-butyl-1-perthio-1-thiooxalato complex of copper(I), [BzPh₃P][Cu(Br)(S-i-dto^tBu)] ([BzPh₃P][4]), where [S-i-dto^tBu]⁻ is a disulfide-containing anionic ligand. The electronic structure and absorption spectrum of this species were investigated by time dependent DFT methods.     

Effect of glutathione biosynthesis-related modulators on the thiol redox state enzymes and on sclerotial differentiation of filamentous phytopathogenic fungi

In this study, sclerotial differentiation in filamentous phytopathogenic fungi, representing the four main types of sclerotia, was studied in relation to thiol redox state (TRS)-related enzymes and their substrates/products. TRS was altered by the general TRS modulator Ν-acetylcysteine (AcCSH) and by the glutathione (GSH) biosynthesis modulators l-oxo-thiazolidine-4-carboxylate (OTC), and l-buthionine-S,R-sulfoximine (BSO). This study showed that the four studied types of sclerotial differentiation are directly related with the antioxidant –SH groups of GSH and/or CSH, since the decrease of sclerotial differentiation concurred with an increase of these thiols by the GSH biosynthesis modulators AcCSH, OTC, and BSO. Supportive to that conclusion is the fact that, in general, the activities of the TRS-related enzymes GR/GPDH and Ttase decrease in the end of the undifferentiated stage due to the substitution of their antioxidant function by the antioxidant potential of the –SH group providers AcCSH and OTC. Moreover, it was found that BSO expectedly suppressed GSH biosynthesis in the tested fungi, and unexpectedly decreased their sclerotial differentiation by a dose-dependent manner typical for antioxidants. The possible antioxidant role of BSO was supported by the decrease it caused in the antioxidant enzymes GR/GPDH and Ttase. The results of this study are in accordance with our hypothesis that sclerotial differentiation in phytopathogenic fungi is induced by oxidative stress.     

Sulfenic acid in human serum albumin

Summary. Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.     

Detection and quantification of free sulfhydryls in monoclonal antibodies using maleimide labeling and mass spectrometry

The detection of free sulfhydryls in proteins can reveal incomplete disulfide bond formation, indicate cysteine residues available for conjugation, and offer insights into protein stability and structure. Traditional spectroscopic methods of free sulfhydryl detection, such as Ellman’s reagent, generally require a relatively large amount of sample, preventing their use for the analysis of biotherapeutics early in the development cycle. These spectroscopic methods also cannot accurately determine the location of the free sulfhydryl, further limiting their utility. Mass spectrometry was used to detect free sulfhydryl residues in intact proteins after labeling with Maleimide-PEG₂-Biotin. As little as 2% cysteine residues with free sulfhydryls (0.02 mol SH per mol protein) could be detected by this method. Following reduction, the free sulfhydryl abundance on antibody heavy and light chains could be measured. To determine free sulfhydryl location at peptide-level resolution, free sulfhydryls and cysteines involved in disulfide bonds were differentially labeled with N-ethylmaleimide and d₅-N-ethylmaleimide, respectively. Following enzymatic digestion and nanoLC-MS, the abundance of free sulfhydryls at individual cysteine residues was quantified down to 2%. The method was optimized to avoid non-specific labeling, disulfide bond scrambling, and maleimide exchange and hydrolysis. This new workflow for free sulfhydryl analysis was used to measure the abundance and location of free sulfhydryls in 3 commercially available monoclonal antibody standards (NIST Monoclonal Antibody Reference Material (NIST), SILu?Lite SigmaMAb Universal Antibody Standard (Sigma-Aldrich) and Intact mAb Mass Check Standard (Waters)) and 1 small protein standard (β-Lactoglobulin A).     

Naphthoquinone-mediated Inhibition of Lysine Acetyltransferase KAT3B/p300, Basis for Non-toxic Inhibitor Synthesis

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.     

Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H₂O₂), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases.     

The methionine sulfoxide reductases: Catalysis and substrate specificities

Oxidation of Met residues in proteins leads to the formation of methionine sulfoxides (MetSO). Methionine sulfoxide reductases (Msr) are ubiquitous enzymes, which catalyze the reduction of the sulfoxide function of the oxidized methionine residues. In vivo, the role of Msrs is described as essential in protecting cells against oxidative damages and to play a role in infection of cells by pathogenic bacteria. There exist two structurally-unrelated classes of Msrs, called MsrA and MsrB, with opposite stereoselectivity towards the S and R isomers of the sulfoxide function, respectively. Both Msrs present a similar three-step catalytic mechanism. The first step, called the reductase step, leads to the formation of a sulfenic acid on the catalytic Cys with the concomitant release of Met. In recent years, significant efforts have been made to characterize structural and molecular factors involved in the catalysis, in particular of the reductase step, and in structural specificities.     

Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase

Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.