RESUMO
Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimer's disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.
Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia , Western Blotting , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteína Huntingtina , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Peptídeos/genética , Fagossomos/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Ligação Proteica , Interferência de RNA , Transfecção , Repetições de Trinucleotídeos/genéticaRESUMO
Ubiquilin proteins are conserved across all eukaryotes and function in the regulation of protein degradation. We found that ubiquilin functions to regulate macroautophagy and that the protein is also a substrate of chaperone-mediated autophagy.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Unwanted proteins in the endoplasmic reticulum (ER) are exported into the cytoplasm and degraded by the proteasome through the ER-associated protein degradation pathway (ERAD). Disturbances in ERAD are linked to ER stress, which has been implicated in the pathogenesis of several human diseases. However, the composition and organization of ERAD complexes in human cells is still poorly understood. In this paper, we describe a trimeric complex that we propose functions in ERAD. Knockdown of erasin, a platform for p97/VCP and ubiquilin binding, or knockdown of ubiquilin in human cells slowed degradation of two classical ERAD substrates. In Caenorhabditis elegans, ubiquilin and erasin are ER stress-response genes that are regulated by the ire-1 branch of the unfolded protein response pathway. Loss of ubiquilin or erasin resulted in activation of ER stress, increased accumulation of polyubiquitinated proteins, and shortened lifespan in worms. Our results strongly support a role for this complex in ERAD and in the regulation of ER stress.