RESUMO
The intracellular trafficking pathway, macroautophagy, is a recycling and disposal service that can be upregulated during periods of stress to maintain cellular homeostasis. An essential phase is the elongation and closure of the phagophore to seal and isolate unwanted cargo prior to lysosomal degradation. Human ATG2A and ATG2B proteins, through their interaction with WIPI proteins, are thought to be key players during phagophore elongation and closure, but little mechanistic detail is known about their function. We have identified a highly conserved motif driving the interaction between human ATG2 and GABARAP proteins that is in close proximity to the ATG2-WIPI4 interaction site. We show that the ATG2A-GABARAP interaction mutants are unable to form and close phagophores resulting in blocked autophagy, similar to ATG2A/ATG2B double-knockout cells. In contrast, the ATG2A-WIPI4 interaction mutant fully restored phagophore formation and autophagy flux, similar to wild-type ATG2A. Taken together, we provide new mechanistic insights into the requirements for ATG2 function at the phagophore and suggest that an ATG2-GABARAP/GABARAP-L1 interaction is essential for phagophore formation, whereas ATG2-WIPI4 interaction is dispensable.
Assuntos
Autofagossomos , Proteínas de Membrana , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte Proteico , Proteínas de Transporte Vesicular/metabolismoRESUMO
The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 coprecipitates with, and is directly phosphorylated by, mTOR. Using a phosphospecific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Relacionadas à Autofagia/genética , Lisossomos/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Serina-Treonina Quinases TOR/genética , Autofagia/genética , Endossomos/genética , Células HeLa , Humanos , Fosforilação/genética , Ligação Proteica/genéticaRESUMO
Describes part of a wider evaluation exercise undertaken to assess the impact of the introduction of a picture-archiving and communication system (PACS) on the adult intensive care unit (AICU) at the Royal Brompton NHS Trust in London. The objectives of this research were to evaluate the perceptions of PACS of the medical and ancillary staff working within AICU as well as to undertake a preliminary assessment of its impact on the workload of radiographers. Questionnaires, interviews and a process analysis were undertaken. The research findings indicate that the overall perception of staff towards the introduction of the PACS was positive. The impact of the system on the workload of radiographers was significant, reducing the time taken to obtain an image from 90 to 60 minutes. However, lessons to be learned for future PACS implementations include the need to ensure compatibility with existing IT systems, adequate IT support and initiatives to ensure that the benefits of PACS are communicated to the hospital at large.