RESUMO
The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation.
Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Multimerização Proteica/fisiologia , Membrana Celular/química , Membrana Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Células HeLa , Humanos , Estrutura Quaternária de ProteínaRESUMO
The cytoplasmic protein Alix/AIP1 (ALG-2 interacting protein X) is involved in cell death through mechanisms which remain unclear but require its binding partner ALG-2 (apoptosis-linked gene-2). The latter was defined as a regulator of calcium-induced apoptosis following endoplasmic reticulum (ER) stress. We show here that Alix is also a critical component of caspase 9 activation and apoptosis triggered by calcium. Indeed, expression of Alix dominant-negative mutants or downregulation of Alix afford significant protection against cytosolic calcium elevation following thapsigargin (Tg) treatment. The function of Alix in this paradigm requires its interaction with ALG-2. In addition, we demonstrate that caspase 9 activation is necessary for apoptosis induced by Tg and that this activation is impaired by knocking down Alix. Altogether, our findings identify, for the first time, Alix as a crucial mediator of Ca(2+) induced caspase 9 activation.
Assuntos
Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Caspase 9/metabolismo , Animais , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular , Cricetinae , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos , Tapsigargina/farmacologiaRESUMO
Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is a ubiquitinous adaptor protein first described for its capacity to bind to the calcium-binding protein, ALG-2. Alix regulates neuronal death in ways involving interactions with ALG-2 and with proteins of the ESCRT (endosomal sorting complex required for transport). Even though all Alix interactors characterized to date are involved in endosomal trafficking, the genuine function of the protein in this process remains unclear. We have demonstrated recently that Alix and ALG-2 form in the presence of calcium, a complex with apical caspases and with the endocytosed death receptor TNFR1 (tumour necrosis factor alpha receptor 1), thus suggesting a molecular coupling between endosomes and the cell death machinery.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Endossomos/metabolismo , Neurônios/citologia , Animais , Caspases/metabolismo , Morte Celular , Endossomos/enzimologia , Ativação Enzimática , Humanos , Neurônios/enzimologiaRESUMO
Alix/AIP1 is a cytosolic protein that regulates cell death through mechanisms that remain unclear. Alix binds to two protein members of the so-called Endosomal Sorting Complex Required for Transport (ESCRT), which facilitates membrane fission events during multivesicular endosome formation, enveloped virus budding and cytokinesis. Alix itself has been suggested to participate in these cellular events and is thus often considered to function in the ESCRT pathway. ESCRT proteins were recently implicated in autophagy, a process involved in bulk degradation of cytoplasmic constituents in lysosomes, which can also participate in cell death. In this study, we shown that, unlike ESCRT proteins, Alix is not involved in autophagy. These results strongly suggest that the capacity of several mutants of Alix to block both caspase-dependent and independent cell death does not relate to their capacity to modulate autophagy. Furthermore, they reinforce the conclusion of other studies demonstrating that the role of Alix is different from that of classical ESCRT proteins.
Assuntos
Autofagia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endossomos/metabolismo , Animais , Autofagia/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cricetinae , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Vacúolos/metabolismoRESUMO
Alix/AIP1 regulates cell death in a way involving interactions with the calcium-binding protein ALG-2 and with proteins of ESCRT (endosomal sorting complex required for transport). Using mass spectrometry we identified caspase-8 among proteins co-immunoprecipitating with Alix in dying neurons. We next demonstrated that Alix and ALG-2 interact with pro-caspase-8 and that Alix forms a complex with the TNFalpha receptor-1 (TNF-R1), depending on its capacity to bind ESCRT proteins. Thus, Alix and ALG-2 may allow the recruitment of pro-caspase-8 onto endosomes containing TNF-R1, a step thought to be necessary for activation of the apical caspase. In line with this, expression of Alix deleted of its ALG-2-binding site (AlixDeltaALG-2) significantly reduced TNF-R1-induced cell death, without affecting endocytosis of the receptor. In a more physiological setting, we found that programmed cell death of motoneurons, which can be inhibited by AlixDeltaALG-2, is regulated by TNF-R1. Taken together, these results highlight Alix and ALG-2 as new actors of the TNF-R1 pathway.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Sítios de Ligação , Caspase 8/metabolismo , Morte Celular , Embrião de Galinha , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Humanos , Crista Neural/embriologia , Neurônios/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
Retroviruses use endosomal machinery to bud out of infected cells, and various Gag proteins recruit this machinery by interacting with either of three cellular factors as follows: ubiquitin ligases of the Nedd4 family, Tsg101, or Alix/Aip1. Here we show that the murine leukemia virus Gag has the unique ability to interact with all three factors. Small interfering RNAs against Tsg101 or Alix and dominant-negative forms of Nedd4 can all reduce production of virus-like particles. However, inactivating the Nedd4-binding site abolishes budding, whereas disrupting Tsg101 or Alix binding has milder effects. Nedd4 ubiquitin ligases are therefore essential, and Tsg101 and Alix play auxiliary roles. Most interestingly, overexpression of Alix can stimulate the release of Gag, and this occurs independently of most Alix partners Tsg101, Cin85, Alg-2, and endophilins. In addition, Gag mutants that do not bind Tsg101 or Alix concentrate on late endosomes and become very sensitive to dominant-negative forms of Nedd4 that do not conjugate ubiquitin. This suggests that the direct interaction of Gag with Tsg101 and Alix favors budding from the plasma membrane and relieves a requirement for ubiquitination by Nedd4.1. Other Nedd4-dependent Gag proteins also contain binding sites for Tsg101 or Alix, suggesting that this could be a common feature of retroviruses.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Produtos do Gene gag/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral , Animais , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Produtos do Gene gag/genética , Produtos do Gene gag/fisiologia , Humanos , Vírus da Leucemia Murina/química , Vírus da Leucemia Murina/fisiologia , Camundongos , Mutagênese Sítio-Dirigida , Ubiquitina-Proteína Ligases Nedd4 , Ratos , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transfecção , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
ALG-2-interacting protein X (Alix), also known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes. Alix may regulate apoptosis since it binds apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein necessary for cell death, and also overexpression of its C-terminal half (Alix-CT) blocks death induced by several stimuli. This part of Alix contains a long proline-rich domain containing several potential SH3-binding sites. Using Alix as bait in a yeast two-hybrid system to screen a mouse brain library, we have found that SH3p4, SH3p8, and SH3p13, collectively known as endophilins, bind to Alix. Co-immunoprecipitations and overlay experiments allowed us to demonstrate that endophilins bind to Alix-CT through an SH3/proline-rich domain interaction. We have narrowed the region of Alix interacting with endophilins down to 14 amino acids containing a PXRPPPP consensus sequence, also present in synaptojanin and germinal center kinase-like kinase, allowing their interaction to endophilins. We further show that overexpression of Alix-CT, which blocks cell death, leads to cytoplasmic vacuolization into tubulo-vesicular structures delineated by Alix-CT. This vacuolization phenomenon is greatly enhanced upon co-expression with endophilins and may be part of the protecting mechanism afforded by Alix-CT.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Sítios de Ligação , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Morte Celular , Linhagem Celular , DNA Complementar/metabolismo , Eletroforese em Gel Bidimensional , Complexos Endossomais de Distribuição Requeridos para Transporte , Deleção de Genes , Humanos , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fenótipo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de srcRESUMO
Alix/AIP1 is a cytoplasmic protein, which was first characterized as an interactor of ALG-2, a calcium-binding protein necessary for cell death. Alix has also recently been defined as a regulator of the endo-lysosomal system. Here we have used post-mitotic cerebellar neurons to test Alix function in caspase-dependent and -independent cell death. Indeed, these neurons survived when cultured in 25 mm potassium-containing medium but underwent apoptosis soon after the extracellular potassium was lowered to 5 mm. In agreement with other studies, we show that caspases are activated after K+ deprivation, but that inhibition of these proteases, using the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone, has no effect on cell survival. Transfection experiments demonstrated that Alix overexpression is sufficient to induce caspase activation, whereas overexpression of its C-terminal half, Alix-CT, blocks caspase activation and cell death after K+ deprivation. We also define a 12-amino acid PXY repeat of the C-terminal proline-rich domain necessary for binding ALG-2. Deletion of this domain in Alix or in Alix-CT abolished the effects of the overexpressed proteins on neuronal survival, demonstrating that the ALG-2-binding region is crucial for the death-modulating function of Alix. Overall, these findings define the Alix/ALG-2 complex as a regulator of cell death controlling both caspase-dependent and -independent pathways. They also suggest a molecular link between the endo-lysosomal system and the effectors of the cell death machinery.