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1.
Cell ; 174(3): 659-671.e14, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30053425

RESUMO

The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.


Assuntos
Fator de Transcrição AP-1/ultraestrutura , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/ultraestrutura , Fator 1 de Ribosilação do ADP/metabolismo , Fator 1 de Ribosilação do ADP/ultraestrutura , Proteínas Adaptadoras de Transporte Vesicular , Antígeno 2 do Estroma da Médula Óssea/metabolismo , Antígeno 2 do Estroma da Médula Óssea/ultraestrutura , Clatrina , Complexo de Golgi , Células HEK293 , HIV-1 , Humanos , Transporte Proteico/fisiologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia
2.
Annu Rev Biochem ; 86: 225-244, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28301741

RESUMO

Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Classe III de Fosfatidilinositol 3-Quinases/química , Classe III de Fosfatidilinositol 3-Quinases/genética , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fagossomos/ultraestrutura , Fosfatidilinositol 3-Quinase/química , Fosfatidilinositol 3-Quinase/genética , Ligação Proteica , Multimerização Proteica , Transdução de Sinais
3.
Cell ; 170(1): 14-16, 2017 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-28666116

RESUMO

A long-standing question in cell biology is how endocytic vesicles and tubules detach from the plasma membrane in the absence of constriction by dynamin. In this issue of Cell, Simunovic et al. describe an elegant biophysical model in which friction between lipids and BAR-domain proteins drives the scission of elongating membrane tubules.


Assuntos
Endocitose , Fricção , Membrana Celular , Dinaminas , Vesículas Transportadoras
4.
Nat Rev Mol Cell Biol ; 18(1): 5-17, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27703243

RESUMO

The narrow membrane necks formed during viral, exosomal and intra-endosomal budding from membranes, as well as during cytokinesis and related processes, have interiors that are contiguous with the cytosol. Severing these necks involves action from the opposite face of the membrane as occurs during the well-characterized formation of coated vesicles. This 'reverse' (or 'inverse')-topology membrane scission is carried out by the endosomal sorting complex required for transport (ESCRT) proteins, which form filaments, flat spirals, tubes and conical funnels that are thought to direct membrane remodelling and scission. Their assembly, and their disassembly by the ATPase vacuolar protein sorting-associated 4 (VPS4) have been intensively studied, but the mechanism of scission has been elusive. New insights from cryo-electron microscopy and various types of spectroscopy may finally be close to rectifying this situation.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Membrana Celular/metabolismo , Endossomos/metabolismo , HIV-1/metabolismo , Humanos , ATPases Vacuolares Próton-Translocadoras/metabolismo
5.
Cell ; 157(2): 300-311, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24725401

RESUMO

Autophagy is directed by numerous distinct autophagy-related (Atg) proteins. These transmit starvation-induced signals to lipids and regulatory proteins and assemble a double-membrane autophagosome sequestering bulk cytoplasm and/or selected cargos destined for degradation upon autophagosome fusion with a vacuole or lysosome. This Review discusses the structural mechanisms by which Atg proteins sense membrane curvature, mediate a PI(3)P-signaling cascade, and utilize autophagy-specific ubiquitin-like protein cascades to tether proteins to autophagosomal membranes. Recent elucidation of molecular interactions enabling vesicle nucleation, elongation, and cargo recruitment provides insights into how dynamic protein-protein and protein-membrane interactions may dictate size, shape, and contents of autophagosomes.


Assuntos
Autofagia , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Fagossomos/metabolismo
6.
Nature ; 614(7948): 572-579, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36697823

RESUMO

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.


Assuntos
Lisossomos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Monoméricas de Ligação ao GTP , Aminoácidos/metabolismo , Domínio Catalítico , Guanosina Difosfato/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação , Multimerização Proteica , Proteína Regulatória Associada a mTOR/metabolismo , Transdução de Sinais
7.
Cell ; 152(4): 755-67, 2013 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-23415225

RESUMO

AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the ß1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1.


Assuntos
Fator 1 de Ribosilação do ADP/química , Fator de Transcrição AP-1/química , Fator 1 de Ribosilação do ADP/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Fator de Transcrição AP-1/metabolismo
8.
Nature ; 610(7933): 761-767, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36261523

RESUMO

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.


Assuntos
Complexo 1 de Proteínas Adaptadoras , Clatrina , Complexo 1 de Proteínas Adaptadoras/química , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 1 de Proteínas Adaptadoras/ultraestrutura , Clatrina/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo , Imunidade Inata , Proteínas Serina-Treonina Quinases , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Motivos de Aminoácidos , Endossomos/metabolismo , Lisossomos/metabolismo , Fosforilação
9.
Cell ; 151(7): 1501-1512, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23219485

RESUMO

Macroautophagy is a bulk clearance mechanism in which the double-membraned phagophore grows and engulfs cytosolic material. In yeast, the phagophore nucleates from a cluster of 20-30 nm diameter Atg9-containing vesicles located at a multiprotein assembly known as the preautophagosomal structure (PAS). The crystal structure of a 2:2:2 complex of the earliest acting PAS proteins, Atg17, Atg29, and Atg31, was solved at 3.05 Å resolution. Atg17 is crescent shaped with a 10 nm radius of curvature. Dimerization of the Atg17-Atg31-Atg29 complex is critical for both PAS formation and autophagy, and each dimer contains two separate and complete crescents. Upon induction of autophagy, Atg17-Atg31-Atg29 assembles with Atg1 and Atg13, which in turn initiates the formation of the phagophore. The C-terminal EAT domain of Atg1 was shown to sense membrane curvature, dimerize, and tether lipid vesicles. These data suggest a structural mechanism for the organization of Atg9 vesicles into the early phagophore.


Assuntos
Proteínas de Transporte/química , Proteínas Fúngicas/química , Complexos Multiproteicos/química , Fagossomos/metabolismo , Saccharomycetales/química , Saccharomycetales/citologia , Sequência de Aminoácidos , Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomycetales/metabolismo , Alinhamento de Sequência
10.
Mol Cell ; 73(2): 339-353.e6, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30581147

RESUMO

Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ß sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ß sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ß sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.


Assuntos
Autofagia , Proteína Beclina-1/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Relacionadas à Autofagia , Proteína Beclina-1/química , Proteína Beclina-1/genética , Sítios de Ligação , Classe III de Fosfatidilinositol 3-Quinases/química , Classe III de Fosfatidilinositol 3-Quinases/genética , Microscopia Crioeletrônica , Ativação Enzimática , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Simulação de Dinâmica Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Relação Estrutura-Atividade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo
11.
Mol Cell ; 74(2): 330-346.e11, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853400

RESUMO

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.


Assuntos
Autofagossomos/metabolismo , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteína Sequestossoma-1/química , Autofagossomos/química , Autofagia/genética , Proteínas Relacionadas à Autofagia , Cristalografia por Raios X , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mapas de Interação de Proteínas/genética , Proteínas Tirosina Quinases/genética , Proteólise , Proteína Sequestossoma-1/genética , Transdução de Sinais/genética , Ubiquitina/química , Ubiquitina/genética
12.
Proc Natl Acad Sci U S A ; 121(22): e2315690121, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38781206

RESUMO

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.


Assuntos
Citosol , Lisossomos , Proteínas tau , Proteínas tau/metabolismo , Lisossomos/metabolismo , Citosol/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Neurônios/metabolismo , Neurônios/patologia , Humanos , Membranas Intracelulares/metabolismo , Endocitose , Camundongos , Células Cultivadas
13.
Proc Natl Acad Sci U S A ; 121(9): e2318046121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38386713

RESUMO

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.


Assuntos
Fenômenos Fisiológicos Celulares , Membranas Intracelulares , Membranas , Divisão Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética
14.
Cell ; 144(1): 55-66, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21215369

RESUMO

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C ßII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCßII was derived from small-angle X-ray scattering. Together, these results show how PKCßII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.


Assuntos
Proteína Quinase C/química , Regulação Alostérica , Sequência de Aminoácidos , Animais , Catálise , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Ratos , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios X
15.
Nature ; 585(7824): 251-255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848248

RESUMO

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Relacionadas à Autofagia/química , Proteína C9orf72/química , Proteína C9orf72/genética , Proteínas de Transporte/química , Microscopia Crioeletrônica , Demência Frontotemporal/genética , Haploinsuficiência , Complexos Multiproteicos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Demência Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Domínios Proteicos
16.
Cell ; 143(6): 875-87, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21145455

RESUMO

Membrane budding is a key step in vesicular transport, multivesicular body biogenesis, and enveloped virus release. These events range from those that are primarily protein driven, such as the formation of coated vesicles, to those that are primarily lipid driven, such as microdomain-dependent biogenesis of multivesicular bodies. Other types of budding reside in the middle of this spectrum, including caveolae biogenesis, HIV-1 budding, and ESCRT-catalyzed multivesicular body formation. Some of these latter events involve budding away from cytosol, and this unusual topology involves unique mechanisms. This Review discusses progress toward understanding the structural and energetic bases of these different membrane-budding paradigms.


Assuntos
Estruturas da Membrana Celular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Animais , Estruturas da Membrana Celular/química , Vesículas Citoplasmáticas/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Humanos
17.
Mol Cell ; 67(3): 528-534.e3, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28757208

RESUMO

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is required for the initiation of essentially all macroautophagic processes. PI3KC3-C1 consists of the lipid kinase catalytic subunit VPS34, the VPS15 scaffold, and the regulatory BECN1 and ATG14 subunits. The VPS34 catalytic domain and BECN1:ATG14 subcomplex do not touch, and it is unclear how allosteric signals are transmitted to VPS34. We used EM and crosslinking mass spectrometry to dissect five conformational substates of the complex, including one in which the VPS34 catalytic domain is dislodged from the complex but remains tethered by an intrinsically disordered linker. A "leashed" construct prevented dislodging without interfering with the other conformations, blocked enzyme activity in vitro, and blocked autophagy induction in yeast cells. This pinpoints the dislodging and tethering of the VPS34 catalytic domain, and its regulation by VPS15, as a master allosteric switch in autophagy induction.


Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Regulação Alostérica , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/química , Classe III de Fosfatidilinositol 3-Quinases/genética , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Mutação , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Relação Estrutura-Atividade , Proteína VPS15 de Distribuição Vacuolar/química , Proteína VPS15 de Distribuição Vacuolar/genética , Proteína VPS15 de Distribuição Vacuolar/metabolismo
18.
Mol Cell ; 68(5): 835-846.e3, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29107538

RESUMO

The lysosomal membrane is the locus for sensing cellular nutrient levels, which are transduced to mTORC1 via the Rag GTPases and the Ragulator complex. The crystal structure of the five-subunit human Ragulator at 1.4 Å resolution was determined. Lamtor1 wraps around the other four subunits to stabilize the assembly. The Lamtor2:Lamtor3 dimer stacks upon Lamtor4:Lamtor5 to create a platform for Rag binding. Hydrogen-deuterium exchange was used to map the Rag binding site to the outer face of the Lamtor2:Lamtor3 dimer and to the N-terminal intrinsically disordered region of Lamtor1. EM was used to reconstruct the assembly of the full-length RagAGTP:RagCGDP dimer bound to Ragulator at 16 Å resolution, revealing that the G-domains of the Rags project away from the Ragulator core. The combined structural model shows how Ragulator functions as a platform for the presentation of active Rags for mTORC1 recruitment, and might suggest an unconventional mechanism for Rag GEF activity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microscopia Eletrônica , Simulação de Acoplamento Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
19.
Proc Natl Acad Sci U S A ; 119(35): e2205590119, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994655

RESUMO

The endosomal sorting complex required for transport (ESCRT) machinery is centrally involved in the repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to the membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, and CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open an avenue for the mechanistic understanding of endolysosomal membrane repair.


Assuntos
Cálcio , Complexos Endossomais de Distribuição Requeridos para Transporte , Membranas Intracelulares , Lisossomos , ATPases Associadas a Diversas Atividades Celulares , Proteínas Reguladoras de Apoptose , Transporte Biológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo
20.
Proc Natl Acad Sci U S A ; 119(29): e2204536119, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35858336

RESUMO

The endosomal sorting complexes required for transport (ESCRT) system is an ancient and ubiquitous membrane scission machinery that catalyzes the budding and scission of membranes. ESCRT-mediated scission events, exemplified by those involved in the budding of HIV-1, are usually directed away from the cytosol ("reverse topology"), but they can also be directed toward the cytosol ("normal topology"). The ESCRT-III subunits CHMP1B and IST1 can coat and constrict positively curved membrane tubes, suggesting that these subunits could catalyze normal topology membrane severing. CHMP1B and IST1 bind and recruit the microtubule-severing AAA+ ATPase spastin, a close relative of VPS4, suggesting that spastin could have a VPS4-like role in normal-topology membrane scission. Here, we reconstituted the process in vitro using membrane nanotubes pulled from giant unilamellar vesicles using an optical trap in order to determine whether CHMP1B and IST1 are capable of membrane severing on their own or in concert with VPS4 or spastin. CHMP1B and IST1 copolymerize on membrane nanotubes, forming stable scaffolds that constrict the tubes, but do not, on their own, lead to scission. However, CHMP1B-IST1 scaffolded tubes were severed when an additional extensional force was applied, consistent with a friction-driven scission mechanism. We found that spastin colocalized with CHMP1B-enriched sites but did not disassemble the CHMP1B-IST1 coat from the membrane. VPS4 resolubilized CHMP1B and IST1 without leading to scission. These observations show that the CHMP1B-IST1 ESCRT-III combination is capable of severing membranes by a friction-driven mechanism that is independent of VPS4 and spastin.


Assuntos
Membrana Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas Oncogênicas , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fricção , Humanos , Proteínas Oncogênicas/metabolismo , Espastina/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
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