RESUMO
The proteasome-activating nucleotidase (PAN) from Methanococcus jannaschii is a complex of relative molecular mass 650,000 that is homologous to the ATPases in the eukaryotic 26S proteasome. When mixed with 20S archaeal proteasomes and ATP, PAN stimulates protein degradation. Here we show that PAN reduces aggregation of denatured proteins and enhances their refolding. These processes do not require ATP hydrolysis, although ATP binding enhances the ability of PAN to prevent aggregation. PAN also catalyses the unfolding of the green fluorescent protein with an 11-residue ssrA extension at its carboxy terminus (GFP11). This unfolding requires ATP hydrolysis, and is linked to GFP11 degradation when 20S proteasomes are also present. This unfolding activity seems to be essential for ATP-dependent proteolysis, although PAN may function by itself as a molecular chaperone.
Assuntos
Adenosina Trifosfatases/metabolismo , Endopeptidases/metabolismo , Mathanococcus/enzimologia , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais , Ativação Enzimática , Magnésio , Chaperonas Moleculares/genética , Desnaturação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , ThermoplasmaRESUMO
Proteasomes are major sites for protein degradation in eukaryotic cells. The recent identification of selective proteasome inhibitors has allowed a definition of the roles of the ubiquitin-proteasome pathway in various cellular processes, such as antigen presentation and the degradation of regulatory or membrane proteins. This review describes the actions of these inhibitors, how they can be used to investigate cellular responses, the functions of the proteasome demonstrated by such studies and their potential applications in the future.
Assuntos
Cisteína Endopeptidases/efeitos dos fármacos , Endopeptidases/metabolismo , Complexos Multienzimáticos/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Apresentação de Antígeno , Compostos de Boro/farmacologia , Cisteína Endopeptidases/fisiologia , Cisteína Endopeptidases/ultraestrutura , Depressão Química , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Humanos , Leupeptinas/farmacologia , Mamíferos/fisiologia , Complexos Multienzimáticos/fisiologia , Complexos Multienzimáticos/ultraestrutura , Oligopeptídeos/farmacologia , Inibidores de Proteases/uso terapêutico , Complexo de Endopeptidases do Proteassoma , Sulfonas/farmacologia , Ubiquitinas/fisiologia , Leveduras/metabolismoRESUMO
Protein degradation in rabbit reticulocytes is a nonlysosomal process requiring ATP. Recently, appreciable evidence has been presented that ATP is required for the covalent binding of the polypeptide ubiquitin to epsilon-amino groups on protein substrates. To test whether linkage of ubiquitin to substrates is required for ATP-dependent proteolysis, the amino groups of 3H-methyl-casein and denatured 125I-bovine serum albumin (BSA) were completely (93-99%) blocked by methylation, acetylation, carbamylation, or succinylation. In each case, the proteins lacking amino groups were still degraded by an ATP-stimulated process, although these various treatments altered absolute rates of proteolysis and reduced the magnitude of the ATP stimulation (two- to fourfold) below that seen measured with the unmodified substrates. When ubiquitin was removed by ion exchange chromatography, ATP still stimulated breakdown of casein and carbamylated casein twofold. The addition of ubiquitin in the presence of ATP caused a further twofold increase in the hydrolysis of unmodified casein but did not affect the degradation of casein lacking amino groups. Thus ubiquitin conjugation to substrates appears important in the breakdown of certain substrates (especially of BSA), but this reaction is not essential for ATP-stimulated proteolysis. The ATP-activated step that is independent of ubiquitin probably is also involved in the degradation of unblocked proteins, since both processes require Mg++ and ATP hydrolysis and are inhibited by hemin but not by protoporphyrin IX. These results suggest that ATP has distinct roles at different steps in the degradative pathway.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas/metabolismo , Reticulócitos/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Caseínas/metabolismo , Hemina/farmacologia , Magnésio/farmacologia , Coelhos , UbiquitinasRESUMO
A critical enzyme in protein breakdown in Escherichia coli is protease La (the lon gene product), which hydrolyzes proteins and adenosine triphosphate (ATP) in a coupled process. The mechanism of this process was studied with fluorogenic tripeptides. Although proteins and peptides are degraded at the same active site, protein substrates enhance the ability of the enzyme to degrade these peptides two- to tenfold. Proteins that are not substrates had little or no effect. Thus, protein substrates must bind to protease La at two sites, the active site and an allosteric site whose occupancy enhances proteolytic activity. This effect did not require that the proteins themselves be degraded. Proteins could induce peptide breakdown even in the absence of ATP, and proteins and ATP had additive effects in stimulating peptidase activity. A multistep cyclical mechanism is proposed in which the binding of the substrate and ATP activates the protease. The enzyme can then cleave a peptide bond, but is inactivated through ATP hydrolysis. Such a mechanism may help account for the selectivity of protein breakdown and prevent inappropriate or excessive proteolysis in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , Endopeptidases/metabolismo , Proteínas de Escherichia coli , Proteínas de Choque Térmico , Protease La , Serina Endopeptidases , Proteases Dependentes de ATP , Adenosina Trifosfatases/metabolismo , Caseínas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/enzimologia , Glucagon/farmacologia , Líquido Intracelular/enzimologia , Líquido Intracelular/metabolismo , Cinética , Proteínas/metabolismo , Especificidade por SubstratoRESUMO
The protease inhibitor leupeptin decreases protein degradation in rat skeletal and cardiac muscle incubated in vitro, while protein synthesis remains unaltered. Leupeptin also lowers protein breakdown in denervated rat muscles and affected muscles from mice with hereditary muscular dystrophy. Leupeptin may thus be useful in retarding tissue atrophy. Since homogenates of leupeptin-treated muscles had decreased cathepsin B activity, this lysosomal protease may play a role in protein turnover in normal and diseased muscles.
Assuntos
Leupeptinas/farmacologia , Proteínas Musculares/metabolismo , Músculos/enzimologia , Distrofia Muscular Animal/metabolismo , Oligopeptídeos/farmacologia , Peptídeo Hidrolases/metabolismo , Animais , Catepsinas/antagonistas & inibidores , Técnicas In Vitro , Lisossomos/enzimologia , Denervação Muscular , Miocárdio/enzimologia , Inibidores de Proteases , RatosRESUMO
Heat shock protein (hsp) genes, a group of ubiquitous genes, are activated by various metabolic stresses. The suggestion that denaturation of intracellular proteins may be produced by the metabolic stresses and then signal the activation of the hsp genes was examined by co-injection of purified proteins and hsp genes into frog oocytes. Activation of hsp genes was observed if the proteins were denatured prior to injection but not if they were introduced in their native form. Furthermore, the activation of hsp genes by abnormal proteins and by heat shock appears to occur by a common mechanism. A model for the transcriptional regulation of the genes is based on competition for degradation between abnormal intracellular proteins and a labile regulatory factor.
Assuntos
Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas/fisiologia , Animais , Drosophila melanogaster , Escherichia coli , Regulação da Expressão Gênica/efeitos dos fármacos , Genes , Globinas/farmacologia , Proteínas de Choque Térmico/fisiologia , Temperatura Alta , Oócitos/metabolismo , Proteínas/farmacologia , Xenopus laevisRESUMO
The pattern of organization of the genetic code decreases to a minimum the phenotypic effects of mutation and of base-pairing errors in protein synthesis. Single base changes, especially transitions, usually cause either no amino acid change or the change to a chemically similar amino acid. The degree of degeneracy of the codons for an amino acid is correlated with their guanine-cytosine content. The code gives greater protection (by both degeneracy and guaninecytosine content of codons) to those amino acids that appear more frequently in proteins. Increased reliability of the protein-synthesis system afforded by this pattern of organization nay have determined the fitness of the present code.
Assuntos
Sequência de Aminoácidos , Código Genético , RNARESUMO
The rate of protein degradation in rabbit erythrocytes in normally very low. However, when cells were exposed to agents that oxidize cell proteins (nitrite or phenylhydrazine), the degradation of erythrocyte proteins to amino acids increased 7- to 33-fold. This effect was inhibited by the reducing agent methylene blue. Stimulation of proteolysis also occurred in cell extracts and resulted from the production of substrates (damaged proteins) rather than from activation of proteases. Inhibitors of glycolysis and of the soluble adenosine triphosphate-dependent proteolytic pathway decreased the protein degradation induced by nitrite, whereas inhibitors of lysosomal proteolysis had no effect. Thus, the adenosine triphosphate-dependent proteolytic system is present in mature red cells where it may help protect against the accumulation of proteins damaged by oxidation or other means.
Assuntos
Proteínas Sanguíneas/metabolismo , Endopeptidases/sangue , Eritrócitos/metabolismo , Proteínas de Choque Térmico , Peptídeo Hidrolases/sangue , Serina Endopeptidases , Proteases Dependentes de ATP , Trifosfato de Adenosina/sangue , Eritrócitos/enzimologia , OxirreduçãoRESUMO
As reticulocytes mature into erythrocytes, organelles and many enzymes are lost. Protein degradation during reticulocyte maturation was measured by monitoring the release of tyrosine from cell proteins. Proteolysis in rabbit red blood cells was directly proportional to the number of reticulocytes and was low in erythrocytes. This process was inhibited by blockers of cellular adenosine triphosphate production and by agents, such as o-phenanthroline, N-ethylmaleimide, and hemin, which inhibit the soluble adenosine triphosphate-dependent proteolytic system. The breakdown of endogenous proteins in reticulocyte extracts was also inhibited by these agents and required adenosine triphosphate. Inhibitors of lysosomal function, however, did not affect proteolysis. Thus, the proteolytic system that degrades abnormal proteins also catalyzes the elimination of proteins during red cell development.
Assuntos
Trifosfato de Adenosina/fisiologia , Proteínas Sanguíneas/metabolismo , Reticulócitos/fisiologia , Animais , Diferenciação Celular , Ciclofosfamida/farmacologia , Desoxiglucose/farmacologia , Dinitrofenóis/farmacologia , Lisossomos/enzimologia , Coelhos , Tirosina/análiseRESUMO
The formation of a persistently active cAMP-dependent protein kinase (PKA) is critical for establishing long-term synaptic facilitation (LTF) in Aplysia. The injection of bovine catalytic (C) subunits into sensory neurons is sufficient to produce protein synthesis-dependent LTF. Early in the LTF induced by serotonin (5-HT), an autonomous PKA is generated through the ubiquitin-proteasome-mediated proteolysis of regulatory (R) subunits. The degradation of R occurs during an early time window and appears to be a key function of proteasomes in LTF. Lactacystin, a specific proteasome inhibitor, blocks the facilitation induced by 5-HT, and this block is rescued by injecting C subunits. R is degraded through an allosteric mechanism requiring an elevation of cAMP coincident with the induction of a ubiquitin carboxy-terminal hydrolase.
Assuntos
Aplysia/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Potenciação de Longa Duração/fisiologia , Animais , Aplysia/metabolismo , Bovinos , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Cisteína Endopeptidases/fisiologia , Injeções , Complexos Multienzimáticos/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/fisiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma , Serotonina/farmacologia , Ubiquitinas/metabolismoRESUMO
Tumor necrosis factor (TNF, cachectin) is a macrophage product that has been suggested to signal the loss of body weight, the decrease in adipose tissue and muscle mass, and anorexia during infections or chronic illness. To test this possibility, young growing rats were injected subcutaneously or intraperitoneally with human or murine recombinant TNF. After 3-4 h, these animals developed a 1-2 degrees fever which lasted approximately 4 h. With repeated daily TNF injections for 5 d, the animals developed fevers similarly each day. In contrast, rats injected with endotoxin show a single febrile episode and then are tolerant to subsequent daily injections of endotoxin (but do not develop tolerance to TNF or interleukin-1). On the first day of TNF treatment, the rats did not grow, but on subsequent days, despite their fevers, they grew at similar rates as controls. Although the TNF-treated rats consumed slightly less food than control animals, the ratio of growth per amount of food intake was identical in the two groups. When rats are administered endotoxin, they develop a fever, and their muscles show increased protein degradation and prostaglandin (PG)E2 production. However, when fevers were induced with TNF, there was no change in muscle proteolysis or PGE2 production, and in adipose tissue no increase in basal or catecholamine-induced lipolysis. Also TNF addition in vitro did not enhance lipolysis in epididymal fat pads or proteolysis in soleus muscles. Thus, TNF treatment can induce fever without producing a catabolic state similar to that induced by endotoxin.
Assuntos
Tecido Adiposo/metabolismo , Febre/induzido quimicamente , Proteínas Musculares/metabolismo , Músculos/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Animais , Temperatura Corporal , Peso Corporal , Dinoprostona , Ingestão de Alimentos , Lipólise , Masculino , Prostaglandinas E/biossíntese , Ratos , Proteínas Recombinantes/toxicidadeRESUMO
Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlysosomal protein breakdown by up to 50% (P < 0.01), and this effect was rapidly reversed upon removal of the inhibitor. The peptide aldehydes did not alter protein synthesis or amino acid pools, but improved overall protein balance in the muscle. Upon treatment with MG132, ubiquitin-conjugated proteins accumulated in the muscle. The inhibition of muscle proteolysis correlated with efficacy against the proteasome, although these agents could also inhibit calpain-dependent proteolysis induced with Ca2+. These inhibitors had much larger effects on proteolysis in atrophying muscles than in controls. In the denervated soleus undergoing atrophy, the increase in ATP-dependent proteolysis was reduced 70% by MG132 (P < 0.001). Similarly, the rise in muscle proteolysis induced by administering thyroid hormones was reduced 40-70% by the inhibitors. Finally, in rats made septic by cecal puncture, the increase in muscle proteolysis was completely blocked by MG132. Thus, the enhanced proteolysis in many catabolic states (including denervation, hyperthyroidism, and sepsis) is due to a proteasome-dependent pathway, and inhibition of proteasome function may be a useful approach to reduce muscle wasting.
Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Leupeptinas/farmacologia , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Proteínas/metabolismo , Animais , Atrofia , Diafragma , Cinética , Masculino , Músculo Esquelético/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos EndogâmicosRESUMO
During sepsis or after injection of endotoxin into rats, there is a large increase in muscle protein breakdown and prostaglandin E2 (PEG2) production. Prior studies showed that partially purified interleukin 1 (IL-1) from human monocytes can stimulate these processes when added to isolated rat muscles. The availability of pure recombinant IL-1 and other monokines has allowed us to investigate the identity of the active agent in this process. Incubation of muscles with recombinant human or murine IL-1 alpha or IL-1 beta or with IL-1 plus a phorbol ester did not stimulate muscle proteolysis or PGE2 production. Homogeneous natural porcine IL-1 ("catabolin") and mouse or human IL-1 beta were also not effective in vitro. In addition, a variety of other human cytokines, including tumor necrosis factor ("cachectin"), epidermal thymocyte-activating factor, eosinophil cytotoxicity-enhancing factor, interferon-alpha, beta, and gamma, platelet-derived growth factor, and transforming growth factor (TGF) beta, which are all released by activated macrophages, TGF-alpha, or mixtures of these polypeptides, also failed to activate proteolysis or PGE2 production. By contrast, a large increase in net protein breakdown could be induced in the rat soleus by polypeptides released from porcine monocytes or by the serum from febrile cattle which had been injected with Pasteurella haemolytica or bovine rhinotracheitis virus. Therefore, a still-unidentified product of activated monocytes appears to be responsible for the negative nitrogen balance that accompanies infectious illness.
Assuntos
Febre/metabolismo , Macrófagos/metabolismo , Proteínas Musculares/metabolismo , Músculos/metabolismo , Prostaglandinas E/biossíntese , Animais , Produtos Biológicos/farmacologia , Bovinos , Citocinas , Dinoprostona , Interleucina-1/farmacologia , Masculino , Monócitos/metabolismo , Ratos , Ratos Endogâmicos , Proteínas Recombinantes/farmacologiaRESUMO
Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas Musculares/metabolismo , Ubiquitinas/metabolismo , Animais , Humanos , Masculino , Complexo de Endopeptidases do Proteassoma , RNA Mensageiro/genética , Coelhos , Ratos , Valores de Referência , Reticulócitos/metabolismo , Transcrição GênicaRESUMO
Induction of NF-kappaB-dependent gene expression plays an important role in a number of biological processes including inflammation and ischemia-reperfusion injury. However, few attempts aimed at selective regulation of this transcription factor have been successful. We report here that a naturally occurring antibacterial peptide PR39 reversibly binds to the alpha 7 subunit of the 26S proteasome and blocks degradation of NF-kappa B inhibitor I kappa B alpha by the ubiquitin-proteasome pathway without affecting overall proteasome activity. I kappa B alpha phosphorylation and ubiquitination occur normally after PR39 treatment, and binding of valosin-containing proteins is not impaired. The inhibition of I kappa B alpha degradation abolishes induction of NF-kappa B-dependent gene expression in cell culture and in mouse models of acute pancreatitis and myocardial infarction, including upregulation of endothelial adhesion proteins VCAM-1 and ICAM-1. In the latter model, sustained infusion of PR39 peptide resulted in significant reduction of myocardial infarct size. PR39 and related peptides may provide novel means to regulate cellular function and to control of NF-kappa B-dependent gene expression for therapeutic purposes.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Complexos Multienzimáticos/metabolismo , Peptídeos/farmacologia , Ubiquitinas/antagonistas & inibidores , Animais , Anti-Infecciosos/metabolismo , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Pancreatite/tratamento farmacológico , Pancreatite/genética , Pancreatite/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Suínos , Ubiquitinas/metabolismoRESUMO
Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NH4Cl-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process. We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5- to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent, proteolytic pathway.
Assuntos
Acidose/metabolismo , Cloreto de Amônio/farmacologia , Proteínas Musculares/metabolismo , Músculos/metabolismo , Ubiquitinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína Endopeptidases/metabolismo , Lisossomos/metabolismo , Masculino , Complexos Multienzimáticos/metabolismo , Proteínas Musculares/efeitos dos fármacos , Músculos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Ubiquitinas/genética , Regulação para CimaRESUMO
An accumulation in cells of unfolded proteins is believed to be the common signal triggering the induction of heat shock proteins (hsps). Accordingly, in Saccharomyces cerevisiae, inhibition of protein breakdown at 30 degrees C with the proteasome inhibitor MG132 caused a coordinate induction of many heat shock proteins within 1 to 2 h. Concomitantly, MG132, at concentrations that had little or no effect on growth rate, caused a dramatic increase in the cells' resistance to very high temperature. The magnitude of this effect depended on the extent and duration of the inhibition of proteolysis. A similar induction of hsps and thermotolerance was seen with another proteasome inhibitor, clasto-lactacystin beta-lactone, but not with an inhibitor of vacuolar proteases. Surprisingly, when the reversible inhibitor MG132 was removed, thermotolerance decreased rapidly, while synthesis of hsps continued to increase. In addition, exposure to MG132 and 37 degrees C together had synergistic effects in promoting thermotolerance but did not increase hsp expression beyond that seen with either stimulus alone. Although thermotolerance did not correlate with hsp content, another thermoprotectant trehalose accumulated upon exposure of cells to MG132, and the cellular content of this disaccharide, unlike that of hsps, quickly decreased upon removal of MG132. Also, MG132 and 37 degrees C had additive effects in causing trehalose accumulation. Thus, the resistance to heat induced by proteasome inhibitors is not just due to induction of hsps but also requires a short-lived metabolite, probably trehalose, which accumulates when proteolysis is reduced.
Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Choque Térmico/biossíntese , Leupeptinas/farmacologia , Complexos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/metabolismo , Trealose/biossíntese , Transtornos de Estresse por Calor , Complexo de Endopeptidases do ProteassomaRESUMO
In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.
Assuntos
Endopeptidases/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas de Choque Térmico , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/fisiologia , Trifosfato de Adenosina/metabolismo , Citosol/metabolismo , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP40 , Dobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae , Especificidade por Substrato , Vacúolos/enzimologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A 53,000-dalton protein (p53) present in large amounts in several types of tumorigenic cells was rapidly degraded in nontumorigenic BALB/c 3T3 fibroblasts (t 1/2, approximately 0.5 h) but not in tumorigenic methylcholanthrene-induced mouse sarcoma cells (t 1/2, greater than 2 h). In 3T3 cells, dinitrophenol and 2-deoxyglucose, agents which reduce ATP production, inhibited the rapid degradation of p53 and the slower breakdown of total cell protein. After removal of these agents, the degradation of both p53 and total cell proteins resumed at their normal rates. Inhibitors of intralysosomal proteolysis (Ep475 and chloroquine) did not reduce the rate of degradation of p53. Thus, in 3T3 cells, p53 appears to be degraded by a nonlysosomal, ATP-dependent proteolytic system similar to that previously shown to degrade short- and long-lived proteins in growing fibroblasts. The immunoreactive p53 which remained in ATP-depleted cells had the same molecular weight as the p53 in the control cells. No intermediate products of p53 degradation were detected by immunoprecipitation in either ATP-depleted or control cells. Hence, ATP seems to be required for an initial step in the degradation of p53. Although the amount of labeled p53 was increased in simian virus 40-transformed and methylcholanthrene-induced mouse sarcoma cells, the amount of p53 labeled during a 3-h pulse in Moloney virus- and Rous sarcoma virus-transformed cells and untransformed 3T3 cells was similar. Thus, an increased net rate of p53 accumulation is not a common feature of transformed tumorigenic cells.
Assuntos
Transformação Celular Neoplásica , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Sarcoma Experimental/metabolismo , Vírus 40 dos Símios/genética , Animais , Células Cultivadas , Metabolismo Energético , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Proteínas de Neoplasias/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Radioisótopos de Enxofre , Proteína Supressora de Tumor p53RESUMO
The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.