RESUMEN
Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Interferón gamma/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Musculares , Péptido Hidrolasas/metabolismo , Proteínas/metabolismo , Animales , Núcleo Celular/enzimología , Cisteína Endopeptidasas/efectos de los fármacos , Citoplasma/enzimología , Retículo Endoplásmico/enzimología , Humanos , Interferón gamma/farmacología , Mamíferos/metabolismo , Complejos Multienzimáticos/efectos de los fármacos , Péptido Hidrolasas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal , Subunidades de Proteína , Proteínas/efectos de los fármacos , Levaduras/metabolismoRESUMEN
In mammalian cells proteasomes can be activated by two different types of regulatory complexes which bind to the ends of the proteasome cylinder. Addition of two 19 S (PA700; ATPase) complexes forms the 26 S proteasome, which is responsible for ATP-dependent non-lysosomal degradation of intracellular proteins, whereas 11 S complexes (PA28; REG) have been implicated in antigen processing. The PA28 complex is upregulated in response to gamma-interferon (gamma-IFN) as are three non-essential subunits of the 20 S proteasome. In the present study we have investigated the effects of gamma-IFN on the level of different proteasome complexes and on the phosphorylation of proteasome subunits. After treatment of cells with gamma-IFN, the level of 26 S proteasomes decreased and there was a concomitant increase in PA28-proteasome complexes. However, no free 19 S regulatory complexes were detected. The majority of the gamma-IFN-inducible proteasome subunits LMP2 and LMP7 were present in PA28-proteasome complexes, but these subunits were also found in 26 S proteasomes. The level of phosphorylation of both 20 S and 26 S proteasome subunits was found to decrease after gamma-IFN treatment of cells. The C8 alpha subunit showed more than a 50% decrease in phosphorylation, and the phosphorylation of C9 was only barely detectable after gamma-IFN treatment. These results suggest that association of regulatory components to 20 S proteasomes is regulated, and that phosphorylation of proteasome alpha subunits may be one mode of regulation.
Asunto(s)
Interferón gamma/farmacología , Proteínas Musculares , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Línea Celular , Cromatografía en Gel , Regulación hacia Abajo , Humanos , Immunoblotting , Modelos Químicos , Péptido Hidrolasas/química , Fosforilación , Proteínas/química , Proteínas/metabolismoRESUMEN
Proteasomes are complex multisubunit proteases which play a critical role in intracellular proteolysis. Immunoproteasomes, which contain three gamma-interferon-inducible subunits, are a subset of proteasomes which have a specialized function in antigen processing for presentation by the MHC class I pathway. Two of the gamma-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7. Immunoproteasomes were strongly enriched around the endoplasmic reticulum as judged by double-immunofluorescence experiments with anti-calreticulin antibodies, but were also present in the nucleus and throughout the cytosol. In contrast, proteasome subunit C2, which is present in all proteasomes, was found to be evenly distributed throughout the cytoplasm and in the nucleus, as was the delta subunit, which is replaced by LMP2 in immunoproteasomes. gamma-Interferon increased the level of immunoproteasomes, but had no effect on their distribution. Our results provide the first direct evidence that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/química , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Anticuerpos Monoclonales , Línea Celular , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Citoplasma/química , Citoplasma/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Interferón gamma/farmacología , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/embriología , Complejo de la Endopetidasa Proteasomal , Subunidades de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas/metabolismoRESUMEN
Proteasomes are multicatalytic proteinase complexes which play a central role in intracellular protein degradation. They catalyse key events in cell cycle regulation and in the activation of the transcription factor NFkappaB. Proteasome inhibitors have been useful for the characterization of proteasome catalytic components and in the elucidation of proteasome functions in animal cells. Potent small peptide inhibitors of proteasomes also represent a novel approach to the treatment of inflammatory diseases (which involve activation of NFkappaB) and cancer. Such compounds have recently been shown to be effective in a variety of animal models, and at least one is currently in use in clinical trials.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Complejos Multienzimáticos/antagonistas & inhibidores , Inhibidores de Proteasas/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Cisteína Endopeptidasas , Interpretación Estadística de Datos , Modelos Animales de Enfermedad , Humanos , Inflamación/patología , Estructura Molecular , Péptido Hidrolasas/metabolismo , Probabilidad , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa ProteasomalRESUMEN
Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.
Asunto(s)
Ácidos Borónicos , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Complejos Multienzimáticos/metabolismo , Animales , Células Cultivadas , Quimotripsina/metabolismo , Complejo de la Endopetidasa ProteasomalRESUMEN
Caspase-3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase-3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP-DEVD-YFP). We demonstrate that, once initiated, the activation of caspase-3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Animales , Células COS , Caspasa 3 , Activación Enzimática , Cinética , Potenciales de la Membrana , Microscopía Fluorescente , Mitocondrias/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Fluorescencia , Estaurosporina/farmacología , TransfecciónRESUMEN
Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Quimotripsina/antagonistas & inhibidores , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Aldehídos/farmacología , Péptidos beta-Amiloides/análisis , Precursor de Proteína beta-Amiloide/genética , Ácidos Borónicos/farmacología , Línea Celular , Quimotripsina/metabolismo , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal , TransfecciónRESUMEN
20S proteasomes are large (700 kDa) proteinase complexes which form the central catalytic core of larger complexes (26S proteasomes or PA28-20S complexes) formed by association with regulatory particles. These larger complexes are involved in diverse regulatory processes in the cell including cyclin breakdown, proteolytic control of transcription factors and other short-lived regulatory proteins, and antigen presentation. In order to carry out these diverse functions the proteasome complexes must be held under tight regulatory control. The early recognition of potential phosphorylation sites in a number of core and regulatory subunits suggested that some control of the complexes activities may be via phosphorylation. We have investigated the role of phosphorylation in determining proteasome localization, activities and association with regulatory complexes.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Péptido Hidrolasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Humanos , Fosforilación , Complejo de la Endopetidasa Proteasomal , RatasRESUMEN
Adenovirus early region 1A (Ad E1A) is a multifunctional protein which is essential for adenovirus-mediated transformation and oncogenesis. Whilst E1A is generally considered to exert its influence on recipient cells through regulation of transcription it also increases the level of cellular p53 by increasing the protein half-life. With this in view, we have investigated the relationship of Ad E1A to the proteasome, which is normally responsible for degradation of p53. Here we have shown that both Ad5 and Ad12 E1A 12S and 13S proteins can be co-immunoprecipitated with proteasomes and that the larger Ad12 E1A protein binds strongly to at least three components of the 26S but not 20S proteasome. One of these interacting species has been identified as mammalian SUGI, a proteasome regulatory component which also plays a role in the cell as a mediator of transcription. In vitro assays have demonstrated a direct interaction between Ad12 E1A 13S protein and mouse SUGI. Following infection of human cells with Ad5 wt and Ad5 mutants with lesions in the E1A gene it has been shown that human SUG1 can be co-immunoprecipitated with full-length E1A and with E1A carrying a deletion in conserved region 1 which is the region considered to be responsible for increased expression of p53. We have concluded therefore that Ad EIA binds strongly to SUGI but that this interaction is not responsible for inhibition of proteasome activity. This is consistent with the observation that purified Ad12 E1A inhibits the activity of the purified 20S but not 26S proteasomes. We have also demonstrated that SUGI can be co-immunoprecipitated with SV40 T and therefore we suggest that this may represent a common interaction of transforming proteins of DNA tumour viruses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas E1A de Adenovirus/metabolismo , Proteínas Portadoras/metabolismo , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Factores de Transcripción , ATPasas Asociadas con Actividades Celulares Diversas , Antígenos Transformadores de Poliomavirus/metabolismo , Línea Celular Transformada , Regulación de la Expresión Génica , Genes p53 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas con Dominio LIM , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Células Tumorales CultivadasRESUMEN
The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.
Asunto(s)
Adenosina Trifosfatasas/química , Péptido Hidrolasas/química , Complejo de la Endopetidasa Proteasomal , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Línea Celular , Células Cultivadas , Electroforesis en Gel Bidimensional , Humanos , Hígado/enzimología , Pulmón , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Fosforilación , Pruebas de Precipitina/métodos , RatasRESUMEN
The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially purified from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-fluoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in vitro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Dexametasona/farmacología , Complejos Multienzimáticos/metabolismo , Péptido Hidrolasas/metabolismo , Timo/efectos de los fármacos , Clorometilcetonas de Aminoácidos/farmacología , Animales , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Regulación hacia Abajo/fisiología , Masculino , Complejo de la Endopetidasa Proteasomal , Ratas , Ratas Endogámicas F344 , Timo/enzimologíaRESUMEN
Proteasomes are large multicatalytic proteinase complexes which are responsible for the selective degradation of cellular proteins and the production of peptides for antigen presentation. Proteasomes are localized both in the nucleus and in the cytoplasm, where some are associated with the endoplasmic reticulum membrane. Recent studies have shown differences in the localization of proteasome subpopulations, demonstrated the functional importance of endoplasmic reticulum-associated proteasomes and investigated the role of putative nuclear localization signals and tyrosine phosphorylation on proteasome transport into the nucleus.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Animales , Transporte Biológico , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Complejo de la Endopetidasa ProteasomalRESUMEN
20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-beta-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsin-like activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin-like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyl- and radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly-Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3, 4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by two-dimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Marcadores de Afinidad , Animales , Caseínas/farmacología , Catálisis , Cisteína Endopeptidasas/aislamiento & purificación , Radioisótopos de Yodo , Cinética , Hígado/enzimología , Sustancias Macromoleculares , Complejos Multienzimáticos/aislamiento & purificación , Péptido Hidrolasas/aislamiento & purificación , Complejo de la Endopetidasa Proteasomal , Técnica de Dilución de Radioisótopos , Ratas , Especificidad por Sustrato , TritioRESUMEN
Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder. We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.
Asunto(s)
Endopeptidasas/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Animales , Proteínas Arqueales , HumanosRESUMEN
Murine IL-2-activated, adherent natural killer (A-NK) cells produce proteolytic activities (including a chymase and a tryptase) which appear to be components of the proteasome/multicatalytic proteinase complex and appear to represent the mouse homologues of the rat A-NK cell A-NKP 2 and A-NKP 1 protease components. The chymase is readily inhibited by Z-Gly-Gly-Phe chloromethylketone (Z-GGF) and to a lesser extent by N-tosyl-L-phenylalanyl-chloromethylketone (TPCK). In addition, this activity is inhibited by 3,4-dichloroisocoumarin (DCI), a suicide inhibitor for both chymotryptic and tryptic proteolytic enzymes. Protease inhibitors reduced A-NK cell-mediated cytotoxicity against P815 target cells, most prominently with inhibitors of chymotryptic and tryptic enzymes, including TPCK, DCI and Z-GGF. A polyclonal rabbit antibody raised against rat liver proteasome immunofluorescently labeled the cytoplasm of 4-day-cultured murine A-NK cells, multiple granules in 5 to 6-day cultures and large intracytoplasmic pools in cells cultured longer. Ultrastructurally this shift in labeling over time corresponded to an immunogold redistribution to non-membrane delineated mucoid masses. A minor nuclear labeling was noted at all time points, whereas the cisternae of the endoplasmic reticulum or Golgi region were negative. It is concluded that murine A-NK cells synthesize and accumulate proteasome in large intracytoplasmic pools, non-delineated by membranes which can occupy up to 80% of the A-NK cellular volume. The potential function of the proteasome produced by A-NK cells including cell-mediated cytotoxicity against tumor target cells remains to be elucidated.
Asunto(s)
Cisteína Endopeptidasas/análisis , Interleucina-2/farmacología , Células Asesinas Naturales/enzimología , Activación de Linfocitos , Complejos Multienzimáticos/análisis , Animales , Línea Celular , Células Cultivadas , Citoplasma/enzimología , Citoplasma/ultraestructura , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/ultraestructura , Citotoxicidad Inmunológica , Inmunohistoquímica , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Microscopía Fluorescente , Orgánulos/enzimología , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa ProteasomalRESUMEN
The proteasome, a multimeric protease, plays an important role in nonlysosomal pathways of intracellular protein degradation. This study was undertaken to determine which subunits of mammalian proteasomes are phosphorylated and to investigate the possible role of phosphorylation in regulating proteasome activity and the association with regulatory components. Rat-1 fibroblasts were grown in the presence of [32P]phosphate and proteasomes were immunoprecipitated from cell lysates with proteasome-specific polyclonal antibodies. Subsequent analysis by two-dimensional polyacrylamide gel electrophoresis showed two radiolabeled proteasome subunits which were identified using monoclonal antibodies as C8 and C9. Treatment of human embryonic lung cells (L-132), under identical conditions, also showed the same two phosphorylated subunits. Phosphoamino acid analysis revealed phosphoserine to be present in both C8 and C9. Examination of the sequence of C9 showed a potential cGMP-dependent phosphorylation site (-Arg3-Arg-Tyr-Asp-Ser-Arg8-), whilst C8 contains several potential casein kinase II phosphorylation sites. Following immunoprecipitation by a monoclonal antibody and dephosphorylation by acid phosphatase, proteasomes were observed to have significantly lower activities when compared to phosphorylated proteasomes, implying that phosphorylation may be an important mechanism of regulating proteasome function. Free proteasomes were separated by gel-filtration from those complexed with regulatory complexes to form the 26S proteinase. The ratio of phosphorylation of C8 and C9 was found to be very similar in the two complexes but the level of phosphorylation was higher in the 26S proteinase than in free proteasomes.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Péptido Hidrolasas/metabolismo , Fosfatasa Ácida/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Línea Celular , Cromatografía en Gel , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/inmunología , Electroforesis en Gel Bidimensional , Endopeptidasas/metabolismo , Humanos , Pulmón/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/inmunología , Péptido Hidrolasas/química , Fosfopéptidos/análisis , Fosforilación , Fosfoserina/análisis , Pruebas de Precipitina , Complejo de la Endopetidasa Proteasomal , RatasRESUMEN
Mammalian proteasomes are composed of 14-17 different types of subunits, some of which, including major-histocompatibility-complex-encoded subunits LMP2 and LMP7, are non-essential and present in variable amounts. We have investigated the distribution of total proteasomes and some individual subunits in rat liver by quantitative immunoblot analysis of purified subcellular fractions (nuclei, mitochondria, microsomes and cytosol). Proteasomes were mainly found in the cytosol but were also present in the purified nuclear and microsomal fractions. In the nuclei, proteasomes were soluble or loosely attached to the chromatin, since they could be easily extracted by treatment with nucleases or high concentrations of salt. In the microsomes, proteasomes were on the outside of the membranes. Further subfractionation of the microsomes showed that the proteasomes in this fraction were associated with the smooth endoplasmic reticulum and with the cis-Golgi but were practically absent from the rough endoplasmic reticulum. Using monospecific antibodies for some proteasomal subunits (C8, C9, LMP2 and Z), the composition of proteasomes in nuclei, microsomes and cytosol was investigated. Although there appear not to be differences in proteasome composition in the alpha subunits (C8 and C9) in the different locations, the relative amounts of some beta subunits varied. Subunit Z was enriched in nuclear proteasomes but low in microsome-associated proteasomes, whereas LMP2, which was relatively low in nuclei, showed a small enrichment in the microsomes. These differences in subunit composition of proteasomes probably reflect differences in the function of proteasomes in distinct cell compartments.
Asunto(s)
Cisteína Endopeptidasas/análisis , Hígado/enzimología , Complejos Multienzimáticos/análisis , Animales , Anticuerpos , Fraccionamiento Celular , Núcleo Celular/enzimología , Cisteína Endopeptidasas/inmunología , Citoplasma/enzimología , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/enzimología , Técnica del Anticuerpo Fluorescente , Aparato de Golgi , Immunoblotting , Técnicas para Inmunoenzimas , Hígado/ultraestructura , Microscopía Electrónica , Microsomas Hepáticos/enzimología , Complejos Multienzimáticos/inmunología , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , Ratas , Tripsina/metabolismoRESUMEN
Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5. This protein has a free N-terminus, having undergone post-translational processing. After immunoprecipitation from [35S]methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3. However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA. Therefore rapid amplification of cDNA ends ("RACE') was carried out to determine the full sequence. The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex. The half-life of the processing was determined to be 31 min in growing L-132 cells. The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies. There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits. Interestingly also, the site of cleavage of N3 (ITR decreases TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G decreases TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cartilla de ADN/genética , ADN Complementario/genética , Escherichia coli/genética , Semivida , Humanos , Técnicas In Vitro , Hígado/enzimología , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. RESULTS: We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. CONCLUSIONS: Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression.
Asunto(s)
Presentación de Antígeno , Cisteína Endopeptidasas , Endopeptidasas/metabolismo , Hemaglutininas Virales/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Secuencia de Bases , Línea Celular , Endopeptidasas/genética , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón gamma/farmacología , Linfoma de Células T/inmunología , Ratones , Datos de Secuencia Molecular , Mutación , Oligodesoxirribonucleótidos , Orthomyxoviridae/inmunología , Células Tumorales CultivadasRESUMEN
Proteasomes are high-molecular-mass multisubunit complexes which are believed, either by themselves or as a part of the 26S proteinase complex, to play a central role in extralysosomal pathways of intracellular protein breakdown. We have addressed the degradation of proteasomes in rat liver, investigating the possible role of lysosomes. Affinity-purified antibodies against rat liver proteasomes were used for immunoblot analysis of isolated lysosomes. Although proteasomes are not found in lysosomes from normally fed rats, they were found to accumulate in lysosomes of rats treated with leupeptin (an inhibitor of lysosomal proteases) and could also be detected in lysosomes isolated from livers of starved (24 h) rats. Proteinase-K treatment of these fractions, as well as immunogold procedures, show that a proportion of the proteasomes are inside lysosomes. Comparison of the amount of proteasomes found in lysosomes by immunoblotting with their experimentally determined half life (8.3 days) is consistent with an important role of these organelles in the degradation of rat liver proteasomes. Nevertheless, these data do not exclude the possibility that some nonlysosomal degradation of proteasome components also occurs. Since proteasomes were localized in autophagic vacuoles, it is likely that they are taken up mainly by nonselective autophagy. However, using an in vitro system, it was found that, under conditions of starvation, proteasomes may also be taken up into lysosomes and degraded via the heat-shock cognate protein of 73 kDa (hsc73)-mediated transport.