RESUMEN
We screened a series of new epoxysuccinyl peptides for the development of a lysosomal cathepsin L-specific inhibitor. Among the compounds tested, (2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide} (compound CAA0225) was the most potent inhibitor of cathepsin L. CAA0225 inhibited rat liver cathepsin L with IC50 values of 1.9 nM, but not rat liver cathepsin B (IC50, >1000-5000 nM). To assess the contribution of cathepsin L to lysosomal proteolysis, we evaluated autophagy, which is the process of lysosomal self-degradation of cell constituents. In HeLa and Huh-7 cells cultured under nutrient-deprived conditions CAA0225 significantly inhibited degradation of long-lived proteins; however, the magnitude of inhibition was comparable to that in the presence of CA-074-OMe, which is a cathepsin B-specific inhibitor. Thus the contributions of cathepsin L and cathepsin B to autophagic protein degradation of cytoplasmic proteins are nearly equal. During autophagy, microtubule-associated protein IA/IB light chain 3-II (LC3-II) and gamma-aminobutyric acid (A) receptor-associated protein (GABARAP)-II, which are specific markers of autophagosomal membranes that engulf cytoplasmic components, also undergo degradation upon fusion of autophagosomes with lysosomes. CAA0225 effectively inhibited degradation of LC3-II and GABARAP, whereas CA-074-OMe had only a marginal effect on their levels. Therefore we conclude that cathepsin L does not play a general role in the degradation of proteins in the lumen of autophagosomes, but rather is involved specifically in the degradation of autophagosomal membrane markers.
Asunto(s)
Autofagia , Compuestos de Bencilo/síntesis química , Catepsinas/antagonistas & inhibidores , Citoplasma/metabolismo , Óxido de Etileno/análogos & derivados , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Compuestos de Bencilo/química , Compuestos de Bencilo/farmacología , Catepsina B/metabolismo , Catepsina L , Catepsinas/metabolismo , Línea Celular , Cisteína Endopeptidasas/metabolismo , Dipéptidos , Compuestos Epoxi , Óxido de Etileno/síntesis química , Óxido de Etileno/química , Óxido de Etileno/farmacología , Humanos , Hidrólisis , Técnicas In Vitro , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fenilalanina/análogos & derivados , Ratas , Ratas Wistar , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Although conjugation of overexpressed GABARP to phospholipid has been reported during starvation-induced autophagy, it is unclear whether endogenous GABARAP-phospholipid conjugation is also activated under starvation conditions. We observed little accumulation of GABARAP-phospholipid conjugate (GABARAP-PL) in mouse liver and kidney under starvation conditions, whereas endogenous LC3-phospholipid conjugate (LC3-II) accumulated. A small amount of endogenous GABARAP-PL was observed in the heart, independent of starvation. In rapamycin-treated HEK293 cells, there was little accumulation of endogenous GABARAP-PL, even in the presence of lysosomal protease-inhibitors, whereas there was significant accumulation of endogenous LC3-II, together with inactivation of the mTor kinase-signaling pathway. In HeLa and C2C12 cells, GABARAP-PL accumulation in the presence of lysosomal protease inhibitors was independent of starvation-induced autophagy, whereas LC3-II accumulation was significant during starvation-induced autophagy. Interestingly, we observed activation of lysosomal turnover of GABARAP-PL during the differentiation of C2C12 cells to myotubes, along with increased lysosomal turnover of LC3-II. Under these conditions, S6 ribosomal protein was still phosphorylated, suggesting that the mTor kinase-signaling pathway is active during the differentiation of C2C12 cells to myotubes, in contrast to starvation-induced autophagy. These results indicated that lysosomal turnover of GABARAP-PL was activated during the differentiation of C2C12 cells to myotubes without inactivation of the mTor kinase-signaling pathway, whereas little lysosomal turnover of GABARAP-PL was activated during starvation-induced autophagy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fibras Musculares Esqueléticas/fisiología , Fosfolípidos/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antibióticos Antineoplásicos/metabolismo , Proteínas Reguladoras de la Apoptosis , Autofagia , Diferenciación Celular/fisiología , Línea Celular , Proteínas del Citoesqueleto/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sirolimus/metabolismo , Inanición , Serina-Treonina Quinasas TOR , Distribución TisularRESUMEN
Murine Atg8L/Apg8L has significant homology with the other known mammalian Atg8 homologs, LC3, GABARAP and GATE-16. However, it is unclear whether murine Atg8L modification is mediated by human Atg4B, Atg7 and Atg3. Expression of Atg8L in HEK293 cells led to cleavage of its C-terminus. In vitro, the C-terminus of Atg8L was cleaved by human Atg4B, but not human Atg4A or Atg4C. Atg8L-I formed an E1-substrate intermediate with Atg7(C572S), and an E2-substrate intermediate with Atg3(C264S). A modified form of Atg8L was detected in the pelletable fraction in the presence of lysosomal protease inhibitors under nutrient-rich conditions. Cyan fluorescent protein (CFP)-Atg8L colocalized with yellow fluorescent protein (YFP)-LC3 in HeLa cells in the presence of the inhibitors. However, little accumulation of the modified form of Atg8L was observed under conditions of starvation. These results indicate that Atg8L is the fourth modifier of mammalian Atg8 conjugation.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/fisiología , Animales , Proteína 7 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Línea Celular , Células HeLa , Humanos , Riñón , Ratones , Proteínas Asociadas a Microtúbulos , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/farmacología , Plásmidos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.
Asunto(s)
Autofagia/fisiología , Lisosomas/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/fisiología , Transporte Biológico Activo , Biomarcadores/metabolismo , Línea Celular , Humanos , Hidrolasas/antagonistas & inhibidores , Leucina/análogos & derivados , Leucina/farmacología , Lisosomas/ultraestructura , Microscopía Electrónica de Transmisión , Pepstatinas/farmacología , Fagosomas/ultraestructuraRESUMEN
In yeast, Atg4/Apg4 is a unique cysteine protease responsible for the cleavage of the carboxyl terminus of Atg8/Apg8/Aut7, a reaction essential for its lipidation during the formation of autophagosomes. However, it is still unclear whether four human Atg4 homologues cleave the carboxyl termini of the three human Atg8 homologues, microtubule-associated protein light chain 3 (LC3), GABARAP, and GATE-16. Using a cell-free system, we found that HsAtg4B, one of the human Atg4 homologues, cleaves the carboxyl termini of these three Atg8 homologues. In contrast, the mutant HsAtg4B(C74A), in which a predicted active site Cys(74) was changed to Ala, lacked proteolytic activity, indicating that Cys(74) is essential for the cleavage activity of cysteine protease. Using phospholipase D, we showed that the modified forms of endogenous LC3 and GABARAP are lipidated and therefore were designated LC3-PL and GABARAP-PL. When purified glutathione S-transferase-tagged HsAtg4B was incubated in vitro with a membrane fraction enriched with endogenous LC3-PL and GABARAP-PL, the mobility of LC3-PL and GABARAP-PL was changed to those of the unmodified proteins. These mobility shifts were not seen when Cys(74) of HsAtg4B was changed to Ala. Overexpression of wild-type HsAtg4B decreased the amount of LC3-PL and GABARAP-PL and increased the amount of unmodified endogenous LC3 and GABARAP in HeLa cells. Expression of CFP-tagged HsAtg4B (CFP-HsAtg4B) and YFP-tagged LC3 in HeLa cells under starvation conditions resulted in a significant decrease in the punctate pattern of distribution of YFP-tagged LC3 and an increase in its cytoplasmic distribution. RNA interference of HsAtg4B increased the amount of LC3-PL in HEK293 cells. Taken together, these results suggest that HsAtg4B negatively regulates the localization of LC3 to a membrane compartment by delipidation.
Asunto(s)
Cisteína Endopeptidasas/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae , Alanina/química , Secuencia de Aminoácidos , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Sistema Libre de Células , Medios de Cultivo , Cisteína/química , Cisteína Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Lípidos/química , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/química , Microtúbulos/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fagocitosis , Fosfolipasa D/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Interferencia de ARN , Receptores de GABA-A/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.