Acoplamento das unidades catalítica e regulatória do proteassomo e a funcionalidade mitocondrial em leveduras após mutações sítio-específicas na unidade catalítica do proteassomo

Em leveduras da espécie S. cerevisiae, foi descrito uma modificação redox pós-traducional denominada S-glutationilação em resíduos Cys da subunidade α5 da unidade catalítica 20S do proteassomo, especificamente o resíduo α5-C76, posteriormente mutada para α5-C76S. A linhagem carregando essa mutação apresentou como alteração fenotípica um menor tempo de vida cronológico (CLS: chronological life span) e uma maior frequência da conformação fechada da câmara catalítica da unidade 20S. Uma dupla mutação randômica na subunidade α5 (α5-S35P/C221S) criou uma linhagem duplo-mutante (DM), a qual induziu a abertura da câmera catalítica do 20S e também aumentou o CLS da célula. O presente estudo teve como objetivo estudar o grau de acoplamento entre as unidades catalítica e regulatória do proteassomo nas linhagens de levedura (C76S, WT e DM), bem como avaliar a funcionalidade mitocondrial em todas elas. A análise do acoplamento foi feita com eletroforese em gel nativo. Para determinar a funcionalidade mitocondrial, foi medida a atividade da enzima citrato sintase a partir da reação entre DTNB e CoA-SH. Foi observado que na linhagem C76S com CLS reduzido havia um menor grau de acoplamento entre as unidades catalítica 20S e regulatória 19S, além de uma atividade mitocondrial diminuída. Portanto, um menor grau de acoplamento entre as unidades do proteassomo muito provavelmente provocou uma disfunção mitocondrial devido a um importe de proteínas mitocondriais defeituoso, resultando em uma CLS reduzida, fato que pode melhor elucidar a nossa compreensão acerca do processo de envelhecimento e da morte celular prematura vista em doenças degenerativas causadas por acumulação proteica.A post-translational redox modification called S-glutathionylation in S. cerevisiae was described at Cys residues of the α5 subunit of the 20S catalytic unit of the proteasome, specifically α5-C76, posteriorly mutated to α5-C76S. The α5-C76S strain presented, as a phenotypic alteration, a higher frequency of the closed conformation of the catalytic chamber of the 20S unit and a shorter chronological life span (CLS: chronological life span). A double random mutation (DM: double mutated) in the α5 subunit (α5-S35P/C221S) induced the opening of the catalytic chamber and also increased CLS. This project aimed to assess the coupling between the catalytic and the regulatory units of the proteasome in some yeast strains (C76S, WT and DM) and to further evaluate their mitochondrial functionality. The study of the coupling of 20S-19S units was carried out in native gel electrophoresis. To determine mitochondrial functionality, the activity of citrate synthase was measured by the reaction between DTNB with CoA-SH. It was observed that in the C76S strain there was a lower degree of coupling between the 20S catalytic and 19S regulatory units, in addition to a decreased citrate synthase activity. Therefore, less coupling between the proteasomal units triggers mitochondrial dysfunction most likely due to deficient mitochondrial protein import, ultimately leading to decreased CLS, which can better our understanding in regards to the process of aging and premature cell death seen in degenerative disorders caused by protein accumulation.

Aspects of a Distinct Cytotoxicity of Selenium Salts and Organic Selenides in Living Cells with Possible Implications for Drug Design

Selenium is traditionally considered as an antioxidant element and selenium compounds are often discussed in the context of chemoprevention and therapy. Recent studies, however, have revealed a rather more colorful and diverse biological action of selenium-based compounds, including the modulation of the intracellular redox homeostasis and an often selective interference with regulatory cellular pathways. Our basic activity and mode of action studies with simple selenium and tellurium salts in different strains of Staphylococcus aureus (MRSA) and Saccharomyces cerevisiae indicate that such compounds are sometimes not particularly toxic on their own, yet enhance the antibacterial potential of known antibiotics, possibly via the bioreductive formation of insoluble elemental deposits. Whilst the selenium and tellurium compounds tested do not necessarily act via the generation of Reactive Oxygen Species (ROS), they seem to interfere with various cellular pathways, including a possible inhibition of the proteasome and hindrance of DNA repair. Here, organic selenides are considerably more active compared to simple salts. The interference of selenium (and tellurium) compounds with multiple targets could provide new avenues for the development of effective antibiotic and anticancer agents which may go well beyond the traditional notion of selenium as a simple antioxidant

Aspects of a distinct cytotoxicity of selenium salts and organic selenides in living cells with possible implications for drug design

Selenium is traditionally considered as an antioxidant element and selenium compounds are often discussed in the context of chemoprevention and therapy. Recent studies, however, have revealed a rather more colorful and diverse biological action of selenium-based compounds, including the modulation of the intracellular redox homeostasis and an often selective interference with regulatory cellular pathways. Our basic activity and mode of action studies with simple selenium and tellurium salts in different strains of Staphylococcus aureus (MRSA) and Saccharomyces cerevisiae indicate that such compounds are sometimes not particularly toxic on their own, yet enhance the antibacterial potential of known antibiotics, possibly via the bioreductive formation of insoluble elemental deposits. Whilst the selenium and tellurium compounds tested do not necessarily act via the generation of Reactive Oxygen Species (ROS), they seem to interfere with various cellular pathways, including a possible inhibition of the proteasome and hindrance of DNA repair. Here, organic selenides are considerably more active compared to simple salts. The interference of selenium (and tellurium) compounds with multiple targets could provide new avenues for the development of effective antibiotic and anticancer agents which may go well beyond the traditional notion of selenium as a simple antioxidant

A novel proteasome inhibitor acting in mitochondrial dysfunction, ER stress and ROS production

In cancer-treatment, potentially therapeutic drugs trigger their effects through apoptotic mechanisms. Generally, cell response is manifested by Bcl-2 family protein regulation, the impairment of mitochondrial functions, and ROS production. Notwithstanding, several drugs operate through proteasome inhibition, which, by inducing the accumulation and aggregation of misfolded or unfolded proteins, can lead to endoplasmic reticulum (ER) stress. Accordingly, it was shown that Amblyomin-X, a Kunitz-type inhibitor identified in the transcriptome of the Amblyomma cajennense tick by ESTs sequence analysis of a cDNA library, obtained in recombinant protein form, induces apoptosis in murine renal adenocarcinoma (RENCA) cells by: inducing imbalance between pro- and anti-apoptotic Bcl-2 family proteins, dysfunction/mitochondrial damage, production of reactive oxygen species (ROS), caspase cascade activation, and proteasome inhibition, all ER-stress inductive. Moreover, there was no manifest action on normal mouse-fibroblast cells (NHI3T3), suggesting an Amblyomin-X tumor-cell selectivity. Taken together, these evidences indicate that Amblyomin-X could be a promising candidate for cancer therapy.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Uniao Quimica Farmaceutica NacionalConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Inst Butantan, Lab Bioquim & Biofis, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilInst Butantan, Programa Posgrad Interunidades Biotecnol, USP, IPT, BR-05503900 São Paulo, BrazilUniv São Paulo, Fac Med, Lab Oncol Expt LIM24, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilFAPESP: FAPESP 2010/52669-3FAPESP: CAT/CEPID - 1998/14307-9Web of Scienc

The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10–50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one

Redox regulation of the proteasome via S-glutathionylation

The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, various signaling processes, antigen presentation, and control of protein synthesis. The central catalytic complex of the proteasome is called the 20S core particle. The majority of these are flanked on one or both sides by regulatory units. Most common among these units is the 19S regulatory unit. When coupled to the 19S unit, the complex is termed the asymmetric or symmetric 26S proteasome depending on whether one or both sides are coupled to the 19S unit, respectively. The 26S proteasome recognizes poly-ubiquitinylated substrates targeted for proteolysis. Targeted proteins interact with the 19S unit where they are deubiquitinylated, unfolded, and translocated to the 20S catalytic chamber for degradation. The 26S proteasome is responsible for the degradation of major proteins involved in the regulation of the cellular cycle, antigen presentation and control of protein synthesis. Alternatively, the proteasome is also active when dissociated from regulatory units. This free pool of 20S proteasome is described in yeast to mammalian cells. The free 20S proteasome degrades proteins by a process independent of poly-ubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20S proteasome comprises two central heptamers (β-rings) where the catalytic sites are located and two external heptamers (α-rings) that are responsible for proteasomal gating. Because the 20S proteasome lacks regulatory units, it is unclear what mechanisms regulate the gating of α-rings between open and closed forms. In the present review, we discuss 20S proteasomal gating modulation through a redox mechanism, namely, S-glutathionylation of cysteine residues located in the α-rings, and the consequence of this post-translational modification on 20S proteasomal function.FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo)Instituto Nacional de Ciência, Tecnologia e Inovação de Processos Redox em BioMedicinaConselho Nacional de Ciência e Tecnologia (CNPq)CAPE

Oxidative modification of proteins: from damage to catalysis, signaling, and beyond

Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteo-toxicity and cellular homeostasis disruption. Recent Advances: Previously, protein oxidation was associated exclusively to damage. However, not all oxidative modifications are necessarily associated with damage, as with Met and Cys protein residue oxidation. In these cases, redox state changes can alter protein structure, catalytic function, and signaling processes in response to metabolic and/or environmental alterations. This review aims to integrate the present knowledge on redox modifications of proteins with their fate and role in redox signaling and human pathological conditions. Critical Issues: It is hypothesized that protein oxidation participates in the development and progression of many pathological conditions. However, no quantitative data have been correlated with specific oxidized proteins or the progression or severity of pathological conditions. Hence, the comprehension of the mechanisms underlying these modifications, their importance in human pathologies, and the fate of the modified proteins is of clinical relevance. Future Directions: We discuss new tools to cope with protein oxidation and suggest new approaches for integrating knowledge about protein oxidation and redox processes with human pathophysiological conditions

Aging and calorie restriction modulate yeast redox state, oxidized protein removal, and the ubiquitin-proteasome system

The ubiquitin-proteasome system governs the half-life of most cellular proteins. Calorie restriction (CR) extends the maximum life span of a variety of species and prevents oxidized protein accumulation. We studied the effects of CR on the ubiquitin-proteasome system and protein turnover in aging Saccharomyces cerevisiae. CR increased chronological life span as well as proteasome activity compared to control cells. The levels of protein carbonyls, a marker of protein oxidation, and those of polyubiquitinated proteins were modulated by CR. Controls, but not CR cells, exhibited a significant increase in oxidized proteins. In keeping with decreased proteasome activity, polyubiquitinated proteins were increased in young control cells compared to time-matched CR cells, but were profoundly decreased in aged control cells despite decreased proteasomal activity. This finding is related to a decreased polyubiquitination ability due to the impairment of the ubiquitin-activating enzyme in aged control cells, probably related to a more oxidative microenvironment. CR preserves the ubiquitin-proteasome system activity. Overall, we found that aging and CR modulate many aspects of protein modification and turnover. (C) 2011 Elsevier Inc. All rights reserved.FAPESP Fundacao de Amparo a Pesquisa do Estado De Sao PauloCNPq Conselho Nacional de Desenvolvimento Cientifico e TecnologicoInstituto Nacional de Ciencia e Tecnologia de Processos Redox em Biomedicina (INCT