RESUMEN
How receptors juggle their interactions with multiple downstream effectors remains poorly understood. Here we show that the outcome of death receptor p75NTR signaling is determined through competition of effectors for interaction with its intracellular domain, in turn dictated by the nature of the ligand. While NGF induces release of RhoGDI through recruitment of RIP2, thus decreasing RhoA activity in favor of NFkB signaling, MAG induces PKC-mediated phosphorylation of the RhoGDI N-terminus, promoting its interaction with the juxtamembrane domain of p75NTR, disengaging RIP2, and enhancing RhoA activity in detriment of NF-kB. This results in stunted neurite outgrowth and apoptosis in cerebellar granule neurons. If presented simultaneously, MAG prevails over NGF. The NMR solution structure of the complex between the RhoGDI N-terminus and p75NTR juxtamembrane domain reveals previously unknown structures of these proteins and clarifies the mechanism of p75NTR activation. These results show how ligand-directed competition between RIP2 and RhoGDI for p75NTR engagement determine axon growth and neuron survival. Similar principles are likely at work in other receptors engaging multiple effectors and signaling pathways.
Asunto(s)
FN-kappa B , Neuronas , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/metabolismo , Ligandos , Fosforilación , FN-kappa B/metabolismo , Neuronas/metabolismo , Receptores de Muerte Celular/metabolismo , Axones/metabolismo , Receptor de Factor de Crecimiento Nervioso/metabolismoRESUMEN
The p75 neurotrophin receptor (p75NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.
Asunto(s)
FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de Factor de Crecimiento Nervioso/química , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteína de Dominio de Muerte Asociada a Receptor de TNF/metabolismo , Animales , Dominio de Muerte , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptor de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal , Proteína de Dominio de Muerte Asociada a Receptor de TNF/químicaRESUMEN
Retinoblastoma protein (pRB) regulates cell cycle by utilizing different regions of its pocket domain for interacting with E2F family of transcription factors and with cellular and viral proteins containing an LxCxE motif. An LxCxE-like motif, LxCxD, is present in FZR1, an adaptor protein of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C). The APC/CFZR1 complex regulates the timely degradation of multiple cell cycle proteins for mitotic exit and maintains G1 state. We report that FZR1 interacts with pRB via its LxCxD motif. By using point mutations, we found that the cysteine residue in the FZR1 LxCxD motif is critical for direct interaction with pRb. The direct binding of the LxCxD motif of FZR1 to the pRB LxCxE binding pocket is confirmed by using human papillomavirus protein E7 as a competitor, both in vitro and in vivo. While mutation of the cysteine residue significantly disrupts FZR1 interaction with pRB, this motif does not affect FZR1 and core APC/C association. Expression of the FZR1 point mutant results in accumulation of S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1 (PLK1), while p27Kip1 and p21Cip1 proteins are downregulated, indicating a G1 cell cycle defect. Consistently, cells containing point mutant FZR1 enter the S phase prematurely. Together our results suggest that the LxCxD motif of FZR1 is a critical determinant for the interaction between FZR1 and pRB and is important for G1 restriction.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdh1/metabolismo , Ciclo Celular/fisiología , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos/fisiología , Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas de Ciclo Celular/genética , División Celular/fisiología , Humanos , Proteína de Retinoblastoma/genética , Factores de Transcripción/metabolismoRESUMEN
A prevalent model of Alzheimer's disease (AD) pathogenesis postulates the generation of neurotoxic fragments derived from the amyloid precursor protein (APP) after its internalization to endocytic compartments. The molecular pathways that regulate APP internalization and intracellular trafficking in neurons are incompletely understood. Here, we report that 5xFAD mice, an animal model of AD, expressing signaling-deficient variants of the p75 neurotrophin receptor (p75NTR ) show greater neuroprotection from AD neuropathology than animals lacking this receptor. p75NTR knock-in mice lacking the death domain or transmembrane Cys259 showed lower levels of Aß species, amyloid plaque burden, gliosis, mitochondrial stress, and neurite dystrophy than global knock-outs. Strikingly, long-term synaptic plasticity and memory, which are completely disrupted in 5xFAD mice, were fully recovered in the knock-in mice. Mechanistically, we found that p75NTR interacts with APP at the plasma membrane and regulates its internalization and intracellular trafficking in hippocampal neurons. Inactive p75NTR variants internalized considerably slower than wild-type p75NTR and showed increased association with the recycling pathway, thereby reducing APP internalization and co-localization with BACE1, the critical protease for generation of neurotoxic APP fragments, favoring non-amyloidogenic APP cleavage. These results reveal a novel pathway that directly and specifically regulates APP internalization, amyloidogenic processing, and disease progression, and suggest that inhibitors targeting the p75NTR transmembrane domain may be an effective therapeutic strategy in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas/fisiología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Línea Celular , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Hipocampo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/metabolismo , Neuronas/metabolismo , Placa Amiloide/metabolismo , Receptores de Muerte Celular/metabolismoRESUMEN
The ubiquitin (Ub) ligase anaphase promoting complex/cyclosome (APC/C) and the tumour suppressor retinoblastoma protein (pRB) play key roles in cell cycle regulation. APC/C is a critical regulator of mitosis and G1-phase of the cell cycle whereas pRB keeps a check on proliferation by inhibiting transition to the S-phase. APC/C and pRB interact with each other via the co-activator of APC/C, FZR1, providing an alternative pathway of regulation of G1 to S transition by pRB using a post-translational mechanism. Both pRB and FZR1 have complex roles and are implicated not only in regulation of cell proliferation but also in differentiation, quiescence, apoptosis, maintenance of chromosomal integrity and metabolism. Both are also targeted by transforming viruses. We discuss recent advances in our understanding of the involvement of APC/C and pRB in cell cycle based decisions and how these insights will be useful for development of anti-cancer and anti-viral drugs.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas Cdh1/genética , Complejo de la Endopetidasa Proteasomal/genética , Proteína de Retinoblastoma/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Apoptosis/genética , Proteínas Cdh1/metabolismo , Diferenciación Celular/genética , Proliferación Celular/genética , Fase G1/genética , Humanos , Mitosis/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína de Retinoblastoma/metabolismo , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.