Proteinaceous cysteine protease inhibitors

Studies of proteinaceous cysteine protease inhibitors originated with the discovery of cystatins in the 1960s. Since that time, a rich and fascinating world of proteins that control and regulate a multitude of important physiological processes, ranging from the basics of protein turnover to development and brain function, has been uncovered. Failures in such important and complex systems inevitably lead to pathologies. Many threatening diseases such as cancer or neurological disorders, to mention only some, are attributed to deregulation of proteaseinhibitor balance. Moreover, important aspects of infection pathology and host defense rely on proteolysis and protease inhibition. Recent advances in the field of protease inhibitors have drawn attention to the possible use of this collected knowledge to control related pathological processes. This review attempts to familiarize the reader with proteinaceous cysteine protease inhibitors by providing an overview of current knowledge. The work primarily highlights biological processes in which the inhibitors are involved and focuses on pathologies resulting from aberrant protease-inhibitor balance, pointing out emerging possibilities for their correction.Received 11 October 2004; received after revision 29 November 2004; accepted 6 December 2004     

Cysteine oxidase and cysteine sulfinic acid decarboxylase in developing rat liver

Summary The patterns of development of cysteine oxidase (CO) and cysteine sulfinic acid decarboxylase (CSD) in rat liver are not similar. It was observed that CO is not under sex control as CSD is. The results obtained agree with the idea that, in liver, as well as in brain, CSD is the limiting factor for the regulation of taurine biosynthesis.Acknowledments: The authors thank CEA for financial support for the purchasing of labelled substrates.     

Dynorphin-degrading cysteine protease is highly specific for paired arginine residues

The cleavage of dynorphin and three analogs containing paired basic residues by several proteases was investigated. The cysteine protease of neuroblastoma cells cleaved only the bond between Arg-Arg residues. Submandibular arginylendopeptidase, however, cleaved bonds between both Arg-Arg and Arg-Lys residues, and pancreatic trypsin at the carboxyl sides of both arginine and lysine residues. This shows that the cysteine protease is highly specific for paired arginine residues.     

Dynorphin-degrading cysteine protease is highly specific for paired arginine residues.

The cleavage of dynorphin and three analogs containing paired basic residues by several proteases was investigated. The cysteine protease of neuroblastoma cells cleaved only the bond between Arg-Arg residues. Submandibular arginyl-endopeptidase, however, cleaved bonds between both Arg-Arg and Arg-Lys residues, and pancreatic trypsin at the carboxyl sides of both arginine and lysine residues. This shows that the cysteine protease is highly specific for paired arginine residues.     

Effect of dietary cholic acid and cholesterol on liver and kidney cystathionase and cysteine sulfinate decarboxylase activities and taurine concentrations in the rat

Summary Hepatic cystathionase and cysteine sulfinate decarboxylase activities are drastically affected by cholic acid added to the diet without cholesterol. When cholic acid and cholesterol are given together, only cysteine sulfinate decarboxylase activity is changed. Neither kidney enzyme activity nor taurine concentrations in the liver and kidney are noticeably modified, whatever the diet.This research was supported by INSERM (Contract 78.152.3) and by CEA.     

Reactions of cysteine, its derivatives, glutathione coenzyme A, and dihydrolipoic acid with isothiocyanates.

Isothiocyanates react with deprotonated SH groups of investigated compounds to give the esters of N-monosubstituted dithiocarbamic acid. In the presence of the SH and NH2 groups (Cys, GSH), isothiocyanates react primarily with the SH groups. The reactions are dependent on the pKa SH values.     

Reactions of cysteine,its derivatives,glutathione, coenzyme A,and dihydrolipoic acid with isothiocyanates

Summary Isothiocyanates react with deprotonated SH groups of investigated compounds to give the esters of N-monosubstituted dithiocarbamic acid. In the presence of the SH and NH₂ groups (Cys, GSH), isothiocyanates react primarily with the SH groups. The reactions are dependent on the pK_aSH values.     

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (m) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 M) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05–0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.Received 9 July 2003; received after revision 28 September 2003; accepted 24 October 2003     

Assessment of the scavenging action of reduced glutathione, (+)-cyanidanol-3 and ethanol by the chemiluminescent response of the xanthine oxidase reaction

The free radical scavenging capacity of reduced glutathione (GSH), (+)-cyanidanol-3 and ethanol was assessed by their interference with the maximal chemiluminescent response produced by the xanthine oxidase reaction. GSH and (+)-cyanidanol-3 induce a progressive inhibition of chemiluminescence when increasing amounts are added to the reaction mixture. GSH and (+)-cyanidanol-3 added together at low concentrations (1 and 0.05 mM respectively) exhibit an additive effect. The addition of ethanol presents a biphasic effect. It inhibits chemiluminescence at low concentrations (10-50 mM) while at higher concentrations (75-500 mM) this effect is reversed. Estimation of the concentrations required to produce half of the maximal inhibition of chemiluminescence by these agents revealed that ethanol is less effective than GSH and (+)-cyanidanol-3 as a free radical scavenger in the system used.     

Effect of nonprotein thiols on protein synthesis in isolated rat hepatocytes

The ability of nonprotein thiols to modulate rates of protein synthesis was investigated in isolated rat hepatocytes. Addition of cysteine stimulates protein labelling by [¹⁴C] Leucine. Glutahione depletion, induced by in vivod administration of L-buthionine sulfoximine and diethylmaleate, did not alter the effect of cysteine, although it decreased the rate of protein synthesis by 32%. The effect of cysteine on protein synthesis does not seem to be related to a perturbatin of the redox state of the NAD⁺/NADH system or to changes in the rate of gluconeogenic pathway. The following observations indicate that cysteine may stimulate protein syntheis by increasing intracellular levels of aspartate: 1. Amino-oxyacetate, an inhibitor of pyridoxyal-dependent enzymes, inhibits protein labelling and decreases aspartate cellular content, whereas most amino acids accumulate or remain unchanged; 2. Cysteine, in the absence or in the presence of amino-ocycetate, stimulates protein labelling and induces aspartate accumulation, although mot amino acids diminish or remain unchanged.     

Glutathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALB/c 3T3 cells

The cytotoxicity of arsenic compounds towards BALB/c 3T3 cells in culture was investigated, together with the role of glutathione (GSH) in the induction of the cytotoxic effects. The rank order of cytotoxicity was as follows: arsenite (As³⁺)>arsenate (As⁵⁺)>dimethylarsinic acid (DMAA)>methylarsonic acid (MAA)>trimethylarsine oxide (TMAO). Arsenobetaine, arsenocholine and the tetramethylarsonium ion were less toxic. Depletion of GSH enhanced the cytotoxic effects of As³⁺, As⁵⁺, MAA and TMAO, while the cytotoxicity of DMAA was markedly reduced by depletion of GSH. These results suggest that GSH plays a role in protecting the cells against the toxic effects of As³⁺, As⁵⁺, MAA and TMAO while it is involved in the induction of the cytotoxic effects of DMAA.     

Antiperoxide effect of S-allyl cysteine sulfoxide,an insulin secretagogue,in diabetic rats

Treatment of alloxan diabetic rats with the antioxidant S-allyl cysteine sulfoxide (SACS) isolated from garlic (Allium sativum Linn)., ameliorated the diabetic condition almost to the same extent as did glibenclamide and insulin. In addition, SACS controlled lipid peroxidation better than the other two drugs. Furthermore, SACS significantly stimulated in vitro insulin secretion from B cells isolated from normal rats. Hence it can be surmised that the beneficial effects of SACS could be due to both its antioxidant and its secretagogue actions. The former effect is more predominant and the latter is only secondary. These effects highlight the therapeutic value of garlic, which is a component of many diets.     

Reserpin und γ-Aminobuttersäuregehalt des Gehirns

Summary 1. In mice,reserpine (5 mg/kg) causes a long-lasting depletion of the brain for -aminobutyric acid (GABA), which corresponds temporally to the lowered seizure threshold (electroshock).2.Iproniazid prohibits both the depletion of GABA and the lowering of seizure threshold.3. Relations between amine- and GABA-metabolism of the brain are pointed out.     

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8–10 aa)C signature motif. Structure–function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.     

Sulfhydryl oxidation induces calcium release from fragmented sarcoplasmic reticulum even in the presence of glutathione

Alcian blue and plumbagin induced transient Ca²⁺ release from fragmented sarcoplasmic reticulum. Dithiothreitol (DTT) and glutathione (GSH) partially blocked Ca²⁺ release induced by these oxidizing compounds. Pretreatment of alcian blue and plumbagin with DTT or GSH for more than 1 min was required to abolish the ability of the oxidizing compounds to release Ca²⁺. Mg²⁺ and ruthenium red completely blocked alcian blue-and plumbagin-induced Ca²⁺ release. These results suggest that oxidation of sulfhydryls on Ca²⁺ release channels induces Ca²⁺ release even in the presence of GSH in situ.     

Targeting Nrf-2 is a promising intervention approach for the prevention of ethanol-induced liver disease

Alcoholic liver disease (ALD) remains to be a worldwide health problem. It is generally accepted that oxidative stress plays critical roles in the pathogenesis of ALD, and antioxidant therapy represents a logical strategy for the prevention and treatment of ALD. Nuclear factor erythroid-derived 2-like 2 (NFE2L2 or Nrf-2) is essential for the antioxidant responsive element (ARE)-mediated induction of endogenous antioxidant enzymes such as heme oxygenase 1 (HO-1) and glutamate–cysteine ligase [GCL, the rate-limiting enzyme in the synthesis of glutathione (GSH)]. Activation of Nrf-2 pathway by genetic manipulation or pharmacological agents has been demonstrated to provide protection against ALD, which suggests that targeting Nrf-2 may be a promising approach for the prevention and treatment of ALD. Herein, we review the relevant literature about the potential hepatoprotective roles of Nrf-2 activation against ALD.     

Mitochondrial activity: a possible determinant of anoxic injury in renal medulla

In brain, heart and kidney, cell work in the absence of oxygen has been thought to precipitate anoxic damage by increasing the rate of depletion of cellular energy stores. In the medullary thick ascending limb of isolated perfused rat kidneys, however, reduction of ATP synthesis by a variety of mitochondrial or metabolic inhibitors caused ATP depletion comparable to that produced by oxygen deprivation but did not reproduce the lesions of anoxia. In these cells, unrestrained mitochondrial activity may be an important source of anoxic injury.     

S-Nitrosylation of proteins

The transfer of a nitric oxide group to cysteine sulfhydryls on proteins, known as S-nitrosylation, is increasingly becoming recognized as a ubiquitous regulatory reaction comparable to phosphorylation. It represents a form of redox modulation in diverse tissues, including the brain. An increasing number of proteins have been found to undergo S-nitrosylation in vivo. These proteins are called S-nitrosothiols, and may play an important role in many processes ranging from signal transduction, DNA repair, host defense, and blood pressure control to ion channel regulation and neurotransmission. This review focuses on the importance of the S-nitrosylation reaction and describes some recently identified S-nitrosothiols in various fields of research.     

Cysteine-induced effect on amino acids in neonatal rat brain

Summary In neonatal rat brain 6 h after s.c. administration of L-cysteine, an increase was observed in most of the amino acids with the exception of glutamic acid, aspartic acid, phenylalanine, sarcosine, glutamine, hydroxyproline and phosphoethanolamine compared to age-matched saline controls. Cysteine was not present at detectable levels in control brain but was found to be 0.38 to 0.52 mole/g of fresh brain tissue in 2- and 4-day-old rats respectively after cysteine treatment.     

Effect of various stressors on the levels of lipid peroxide,antioxidants and Na+, K+-ATPase actrivity in rat brain

The level of malondialdehyde (MDA), an index of lipid peroxidation, and the antioxidants superoxide dismutase (SOD) and glutathione (GSH), as well as the activity of Na⁺, K⁺-ATPase, were assessed in whole rat brain after immobilization, anemic hypoxia (NaNO₂) and 72 h starvation. The effect of these stressors on plasma glucose and corticosterone levels was also observed. Hypoxia and starvation stimulated the lipidj peroxide formation in braini as indicated by an increase in the level of MDA, being higher after starvation than hypoxia. Brain SOD activity was also increased in response to hypoxia and starvation while GSH content was only diminished ini hypoxia. However, neither MDA nor antioxidants were affected by immobilization. On the other hand, the activity of brain Na⁺, K⁺-ATPase was significantly increased by immobilization and hypoxia but decreased in starvation. A similar pattern of change was also observed in plasma glucose and corticosterone levels in response to these stressors. These results elucidate differences in the biochemical response of animals towards various types of stress, with increased lipid peroxide formation in hypoxia and starvation.