RESUMO
Lysosomes are the degradative endpoints of material delivered by endocytosis and autophagy and are therefore particularly prone to damage. Membrane permeabilization or full rupture of lysosomal or late endosomal compartments is highly deleterious because it threatens cellular homeostasis and can elicit cell death and inflammatory signaling. Cells have developed a complex response to endo-lysosomal damage that largely consists of three branches. Initially, a number of repair pathways are activated to restore the integrity of the lysosomal membrane. If repair fails or if damage is too extensive, lysosomes are isolated and degraded by a form of selective autophagy termed lysophagy. Meanwhile, an mTORC1-governed signaling cascade drives biogenesis and regeneration of new lysosomal components to reestablish the full lysosomal capacity of the cell. This damage response is vital to counteract the effects of various conditions, including neurodegeneration and infection, and can constitute a critical vulnerability in cancer cells.
Assuntos
Autofagia , Endossomos , Lisossomos , Alvo Mecanístico do Complexo 1 de Rapamicina , Transdução de Sinais , Lisossomos/metabolismo , Humanos , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Endossomos/metabolismo , Endocitose , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genéticaRESUMO
Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.
Assuntos
Lisossomos , Macroautofagia , Humanos , Autofagia/fisiologia , Membranas Intracelulares/metabolismo , Lipídeos , Lisossomos/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the extraction of CNN2 and show that other membrane regulators including SNAREs, PICALM, AGFG1, and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.
Assuntos
Macroautofagia , Ubiquitina , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Lisossomos/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.
Assuntos
Proteínas de Ciclo Celular , Ubiquitina , Ubiquitina/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Modelos Moleculares , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
The AAA+-type ATPase p97 governs an ever-expanding number of cellular processes reaching from degradation of damaged proteins and organelles to key signaling events and chromatin regulation with thousands of client proteins. With its relevance for cellular homeostasis and genome stability, it is linked to muscular and neuronal degeneration and, conversely, constitutes an attractive anti-cancer drug target. Its molecular function is ATP-driven protein unfolding, which is directed by ubiquitin and assisted by a host of cofactor proteins. This activity underlies p97's diverse ability to pull proteins out of membranes, unfold proteins for proteasomal degradation, or segregate proteins from partners for downstream activity. Recent advances in structural analysis and biochemical reconstitution have underscored this notion, resolved detailed molecular motions within the p97 hexamer, and suggested substrate threading through the central channel of the p97 hexamer as the driving mechanism. We will discuss the mechanisms and open questions in the context of the diverse cellular activities.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares/metabolismo , Proteína com Valosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Homeostase , Humanos , Desdobramento de Proteína , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa , Holoenzimas/metabolismo , Humanos , Modelos Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Proteína Fosfatase 1/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Ubiquitina/metabolismoRESUMO
During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Anfíbios/genética , Proteínas de Ciclo Celular/genética , Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/genética , Osteoblastos/metabolismo , Reparo de DNA por Recombinação , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Anfíbios/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Regulação da Expressão Gênica , Humanos , Hidrólise , Autoantígeno Ku/metabolismo , Microesferas , Osteoblastos/citologia , Óvulo/química , Óvulo/citologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína com Valosina , Xenopus laevisRESUMO
The protease SPRTN degrades DNA-protein crosslinks (DPCs) that threaten genome stability. SPRTN has been connected to the ubiquitin-directed protein unfoldase p97 (also called VCP or Cdc48), but a functional cooperation has not been demonstrated directly. Here, we biochemically reconstituted p97-assisted proteolysis with purified proteins and showed that p97 targets ubiquitin-modified DPCs and unfolds them to prepare them for proteolysis by SPRTN. We demonstrate that purified SPRTN alone was unable to degrade a tightly-folded Eos fluorescent reporter protein even when Eos was crosslinked to DNA (Eos-DPC). However, when present, p97 unfolded poly-ubiquitinated Eos-DPC in a manner requiring its ubiquitin adapter, Ufd1-Npl4. Notably, we show that, in cooperation with p97 and Ufd1-Npl4, SPRTN proteolyzed unfolded Eos-DPC, which relied on recognition of the DNA-crosslink by SPRTN. In a simplified unfolding assay, we further demonstrate that p97, while unfolding a protein substrate, can surmount the obstacle of a DNA crosslink site in the substrate. Thus, our data demonstrate that p97, in conjunction with Ufd1-Npl4, assists SPRTN-mediated proteolysis of tightly-folded proteins crosslinked to DNA, even threading bulky protein-DNA adducts. These findings will be relevant for understanding how cells handle DPCs to ensure genome stability and for designing strategies that target p97 in combination cancer therapy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas , Ubiquitina , Proteína com Valosina , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Instabilidade Genômica , Humanos , Proteínas/metabolismo , Proteólise , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
Rupture of endosomes and lysosomes is a major cellular stress condition leading to cell death and degeneration. Here, we identified an essential role for the ubiquitin-directed AAA-ATPase, p97, in the clearance of damaged lysosomes by autophagy. Upon damage, p97 translocates to lysosomes and there cooperates with a distinct set of cofactors including UBXD1, PLAA, and the deubiquitinating enzyme YOD1, which we term ELDR components for Endo-Lysosomal Damage Response. Together, they act downstream of K63-linked ubiquitination and p62 recruitment, and selectively remove K48-linked ubiquitin conjugates from a subpopulation of damaged lysosomes to promote autophagosome formation. Lysosomal clearance is also compromised in MEFs harboring a p97 mutation that causes inclusion body myopathy and neurodegeneration, and damaged lysosomes accumulate in affected patient tissue carrying the mutation. Moreover, we show that p97 helps clear late endosomes/lysosomes ruptured by endocytosed tau fibrils. Thus, our data reveal an important mechanism of how p97 maintains lysosomal homeostasis, and implicate the pathway as a modulator of degenerative diseases.
Assuntos
Adenosina Trifosfatases/metabolismo , Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endopeptidases/metabolismo , Lisossomos/metabolismo , Proteínas/metabolismo , Tioléster Hidrolases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Proteínas Relacionadas à Autofagia , Células Cultivadas , Humanos , Camundongos , Proteína com ValosinaRESUMO
The autophagic clearance of damaged lysosomes by lysophagy involves extensive modification of the organelle with ubiquitin, but the underlying ubiquitination machinery is still poorly characterized. Here, we use an siRNA screening approach and identify human UBE2QL1 as a major regulator of lysosomal ubiquitination, lysophagy, and cell survival after lysosomal damage. UBE2QL1 translocates to permeabilized lysosomes where it associates with damage sensors, ubiquitination targets, and lysophagy effectors. UBE2QL1 knockdown reduces ubiquitination and accumulation of the critical autophagy receptor p62 and abrogates recruitment of the AAA-ATPase VCP/p97, which is essential for efficient lysophagy. Crucially, it affects association of LC3B with damaged lysosomes indicating that autophagosome formation was impaired. Already in unchallenged cells, depletion of UBE2QL1 leads to increased lysosomal damage, mTOR dissociation from lysosomes, and TFEB activation pointing to a role in lysosomal homeostasis. In line with this, mutation of the homologue ubc-25 in Caenorhabditis elegans exacerbates lysosome permeability in worms lacking the lysosome stabilizing protein SCAV-3/LIMP2. Thus, UBE2QL1 coordinates critical steps in the acute endolysosomal damage response and is essential for maintenance of lysosomal integrity.
Assuntos
Autofagia , Endossomos/metabolismo , Lisossomos/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Adenosina Trifosfatases , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Sobrevivência Celular , Endossomos/ultraestrutura , Galectinas/metabolismo , Células HeLa , Humanos , Lisina/metabolismo , Lisossomos/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares , Permeabilidade , RNA Interferente Pequeno/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismoRESUMO
Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Omura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Produtos Biológicos/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Produtos Biológicos/química , Estrutura MolecularRESUMO
The ubiquitin-directed AAA-ATPase VCP/p97 facilitates degradation of damaged or misfolded proteins in diverse cellular stress response pathways. Resolving the complexity of its interactions with partner and substrate proteins and understanding its links to stress signaling is therefore a major challenge. Here, we used affinity-purification SWATH mass spectrometry (AP-SWATH) to identify proteins that specifically interact with the substrate-trapping mutant, p97-E578Q. AP-SWATH identified differential interactions over a large detection range from abundant p97 cofactors to pathway-specific partners and individual ligases such as RNF185 and MUL1 that were trapped in p97-E578Q complexes. In addition, we identified various substrate proteins and candidates including the PP1 regulator CReP/PPP1R15B that dephosphorylates eIF2α and thus counteracts attenuation of translation by stress-kinases. We provide evidence that p97 with its Ufd1-Npl4 adapter ensures rapid constitutive turnover and balanced levels of CReP in unperturbed cells. Moreover, we show that p97-mediated degradation, together with a reduction in CReP synthesis, is essential for timely stress-induced reduction of CReP levels and, consequently, for robust eIF2α phosphorylation to enforce the stress response. Thus, our results demonstrate that p97 not only facilitates bulk degradation of misfolded proteins upon stress, but also directly modulates the integrated stress response at the level of signaling.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Proteólise , Transdução de Sinais , Estresse Fisiológico , Arsenitos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Reprodutibilidade dos Testes , Estresse Fisiológico/efeitos dos fármacos , Especificidade por Substrato , Ubiquitina-Proteína Ligases/metabolismo , Raios UltravioletaRESUMO
Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 (PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3). Depletion of I3 does not affect the amount of PP1 at kinetochores but causes quantitative association of SDS22 with PP1 on KNL1 at the kinetochore. Such accumulation of SDS22 at kinetochores interferes with PP1 activity and inhibits Aurora B threonine-232 dephosphorylation, which leads to increased Aurora B activity in metaphase and persistence in anaphase accompanied with segregation defects. We propose a model in which I3 regulates an SDS22-mediated PP1 activation step in solution that precedes SDS22 dissociation and transfer of PP1 to kinetochores, and which is required for PP1 to efficiently antagonize Aurora B.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinetocoros/metabolismo , Modelos Biológicos , Proteína Fosfatase 1/metabolismo , Fuso Acromático/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , Fuso Acromático/genética , Ubiquitina-Proteína LigasesRESUMO
AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Inibidores Enzimáticos/metabolismo , Proteína com Valosina/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Mutagênese Sítio-Dirigida , Ligação Proteica , Relação Estrutura-Atividade , Proteína com Valosina/antagonistas & inibidoresRESUMO
Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Motivos de Aminoácidos , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Transporte/química , Caveolina 1/metabolismo , Linhagem Celular , Dicroísmo Circular , Endossomos/metabolismo , Epitopos/química , Polarização de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Humanos , Lisossomos/metabolismo , Espectroscopia de Ressonância Magnética , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquitina/química , Proteína com ValosinaRESUMO
The ATPase valosin-containing protein (VCP)/p97 has emerged as a central and important element of the ubiquitin system. Together with a network of cofactors, it regulates an ever-expanding range of processes that stretch into almost every aspect of cellular physiology. Its main role in proteostasis and key functions in signaling pathways are of relevance to degenerative diseases and genomic stability. In this Cell Science at a Glance and the accompanying poster, we give a brief overview of this complex system. In addition, we discuss the pathogenic basis for VCP/p97-associated diseases and then highlight in more detail new exciting links to the translational stress response and RNA biology that further underscore the significance of the VCP/p97 system.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adenosina Trifosfatases/genética , Animais , Proteínas de Ciclo Celular/genética , Doença/genética , Regulação da Expressão Gênica , Humanos , Patologia , Estresse Fisiológico , Proteína com ValosinaRESUMO
p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.
Assuntos
Adenosina Trifosfatases/ultraestrutura , Proteínas de Transporte/ultraestrutura , Proteínas de Ciclo Celular/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Proteínas/ultraestrutura , Microscopia Crioeletrônica/métodos , Escherichia coli , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Proteína com ValosinaRESUMO
Caveolin-1 (CAV1) is the defining constituent of caveolae at the plasma membrane of many mammalian cells. For turnover, CAV1 is ubiquitinated and sorted to late endosomes and lysosomes. Sorting of CAV1 requires the AAA+-type ATPase VCP and its cofactor UBXD1. However, it is unclear in which region CAV1 is ubiquitinated and how ubiquitination is linked to sorting of CAV1 by VCP-UBXD1. Here, we show through site-directed mutagenesis that ubiquitination of CAV1 occurs at any of the six lysine residues, 5, 26, 30, 39, 47, and 57, that are clustered in the N-terminal region but not at lysines in the oligomerization, intramembrane, or C-terminal domains. Mutation of Lys-5-57 to arginines prevented binding of the VCP-UBXD1 complex and, importantly, strongly reduced recruitment of VCP-UBXD1 to endocytic compartments. Moreover, the Lys-5-57Arg mutation specifically interfered with trafficking of CAV1 from early to late endosomes. Conversely and consistently, depletion of VCP or UBXD1 led to accumulation of ubiquitinated CAV1, suggesting that VCP acts downstream of ubiquitination and is required for transport of the ubiquitinated form of CAV1 to late endosomes. These results define the N-terminal region of CAV1 as the critical ubiquitin conjugation site and, together with previous data, demonstrate the significance of this ubiquitination for binding to the VCP-UBXD1 complex and for sorting into lysosomes.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Caveolina 1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Adenosina Trifosfatases/genética , Substituição de Aminoácidos , Arginina/genética , Arginina/metabolismo , Proteínas Relacionadas à Autofagia , Western Blotting , Proteínas de Transporte/genética , Caveolina 1/química , Caveolina 1/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HEK293 , Humanos , Lisina/genética , Lisina/metabolismo , Lisossomos/metabolismo , Microscopia Confocal , Mutação , Transporte Proteico , Interferência de RNA , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Proteína com ValosinaRESUMO
The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved α-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.
Assuntos
Proteínas de Ciclo Celular , Ubiquitina , Humanos , Ligação Proteica , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
During exit from mitosis in Xenopus laevis egg extracts, the AAA+ ATPase Cdc48/p97 (also known as VCP in vertebrates) and its adapter Ufd1-Npl4 remove the kinase Aurora B from chromatin to allow nucleus formation. Here, we show that in HeLa cells Ufd1-Npl4 already antagonizes Aurora B on chromosomes during earlier mitotic stages and that this is crucial for proper chromosome segregation. Depletion of Ufd1-Npl4 by small interfering RNA (siRNA) caused chromosome alignment and anaphase defects resulting in missegregated chromosomes and multi-lobed nuclei. Ufd1-Npl4 depletion also led to increased levels of Aurora B on prometaphase and metaphase chromosomes. This increase was associated with higher Aurora B activity, as evidenced by the partial resistance of CENP-A phosphorylation to the Aurora B inhibitor hesperadin. Furthermore, low concentrations of hesperadin partially rescued chromosome alignment in Ufd1-depleted cells, whereas, conversely, Ufd1-depletion partially restored congression in the presence of hesperadin. These data establish Cdc48/p97-Ufd1-Npl4 as a crucial negative regulator of Aurora B early in mitosis of human somatic cells and suggest that the activity of Aurora B on chromosomes needs to be restrained to ensure faithful chromosome segregation.