RESUMO
Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.
Assuntos
Autofagia/fisiologia , Desenvolvimento Embrionário/fisiologia , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Animais , Técnicas de Cultura de Células , Citometria de Fluxo , Immunoblotting , Lipólise/fisiologia , Camundongos , Microscopia de Fluorescência , Perilipina-3/metabolismoRESUMO
Basal autophagy plays an essential role as a protein quality control system. Although it has been demonstrated that the loss of autophagy results in the accumulation of ubiquitin-positive aggregates and the development of neurodegenerative diseases, the precise autophagy substrate(s) remain unclear. Here, we determined whether ubiquitinated proteins are direct substrates for basal autophagy using a fluorescent ratiometric probe for ubiquitin. We show that the degradation of polyubiquitinated proteins is not dependent on basal autophagy. Although ubiquitin-positive aggregates are observed in autophagy knockout cultured cells, the aggregates consist of soluble and mobile polyubiquitinated proteins, which are trapped by p62 without an increase in the total amount of ubiquitinated proteins. These results suggest that ubiquitinated proteins are not major targets for basal autophagy.
Assuntos
Autofagia , Poliubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Microscopia de Fluorescência , Proteólise , Interferência de RNA , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Proteínas Ubiquitinadas/genéticaRESUMO
Self-assembled monolayers (SAMs) of pyrene-modified DNA were prepared on gold electrode and their photoelectrochemical properties were investigated. The cathodic photocurrent was generated by photoirradiation of the SAM in the presence of methyl viologen as an electron carrier. The photocurrent efficiency increased with increasing the distance between the pyrene and the gold substrate.