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1.
Cell ; 177(3): 766-781.e24, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30955882

RESUMO

During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Sítios de Ligação , Humanos , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
2.
Cell ; 169(4): 722-735.e9, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28475898

RESUMO

The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/isolamento & purificação , Endopeptidases/metabolismo , Modelos Moleculares , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina
3.
Mol Cell ; 84(7): 1290-1303.e7, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38401542

RESUMO

Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.


Assuntos
Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
4.
Mol Cell ; 83(1): 105-120.e5, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36538933

RESUMO

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.


Assuntos
Legionella pneumophila , Ubiquitina , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação , Poliubiquitina/genética , Poliubiquitina/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Enzimas Desubiquitinantes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
5.
Mol Cell ; 83(5): 759-769.e7, 2023 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-36736315

RESUMO

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.


Assuntos
Poliubiquitina , Proteínas de Saccharomyces cerevisiae , Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteína com Valosina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas de Ciclo Celular/metabolismo
6.
Mol Cell ; 83(16): 2976-2990.e9, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37595558

RESUMO

Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.


Assuntos
Mitocôndrias , Proteínas Mitocondriais , Ubiquitina-Proteína Ligases , Humanos , Aclimatação , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Mitocondriais/metabolismo
7.
Mol Cell ; 82(3): 570-584.e8, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34951965

RESUMO

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.


Assuntos
Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Ubiquitinadas/metabolismo , Proteína com Valosina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Desdobramento de Proteína , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Ubiquitinadas/genética , Ubiquitinação , Proteína com Valosina/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
8.
Mol Cell ; 82(14): 2633-2649.e7, 2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35793674

RESUMO

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/metabolismo
9.
Mol Cell ; 82(18): 3453-3467.e14, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35961308

RESUMO

Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.


Assuntos
Retículo Endoplasmático , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
10.
Mol Cell ; 77(5): 1124-1142.e10, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142685

RESUMO

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.


Assuntos
Células-Tronco Embrionárias Humanas/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Mitofagia , Células-Tronco Neurais/enzimologia , Neurogênese , Neurônios/enzimologia , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células HeLa , Células-Tronco Embrionárias Humanas/patologia , Humanos , Cinética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Células-Tronco Neurais/patologia , Neurônios/patologia , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteômica , Transdução de Sinais , Tioléster Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
11.
Mol Cell ; 79(2): 320-331.e9, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645369

RESUMO

Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.


Assuntos
Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismo , Catálise , Citosol/metabolismo , Células HEK293 , Humanos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteólise , Solubilidade , Ubiquitinação
12.
EMBO J ; 42(13): e113609, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37144685

RESUMO

DNA-protein crosslinks (DPCs) pose a serious threat to genome stability. The yeast proteases Wss1, 26S proteasome, and Ddi1 are safeguards of genome integrity by acting on a plethora of DNA-bound proteins in different cellular contexts. The AAA ATPase Cdc48/p97 is known to assist Wss1/SPRTN in clearing DNA-bound complexes; however, its contribution to DPC proteolysis remains unclear. Here, we show that the Cdc48 adaptor Ubx5 is detrimental in yeast mutants defective in DPC processing. Using an inducible site-specific crosslink, we show that Ubx5 accumulates at persistent DPC lesions in the absence of Wss1, which prevents their efficient removal from the DNA. Abolishing Cdc48 binding or complete loss of Ubx5 suppresses sensitivity of wss1∆ cells to DPC-inducing agents by favoring alternate repair pathways. We provide evidence for cooperation of Ubx5-Cdc48 and Wss1 in the genotoxin-induced degradation of RNA polymerase II (RNAPII), a described candidate substrate of Wss1. We propose that Ubx5-Cdc48 assists Wss1 for proteolysis of a subset of DNA-bound proteins. Together, our findings reveal a central role for Ubx5 in DPC clearance and repair.


Assuntos
Reparo do DNA , Saccharomyces cerevisiae , DNA/metabolismo , Dano ao DNA , Endopeptidases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas
13.
Mol Cell ; 74(4): 742-757.e8, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30979586

RESUMO

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Doenças por Armazenamento dos Lisossomos/genética , Doenças Musculares/genética , Proteínas Serina-Treonina Quinases/genética , Proteína com Valosina/genética , Adenosina Trifosfatases/genética , Animais , Autofagia/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Camundongos , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Fosforilação/genética , Estresse Fisiológico/genética , Ubiquitina/genética
14.
EMBO J ; 41(9): e110145, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35349166

RESUMO

Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly-ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell-based ubiquitin replacement strategy to enable selective and conditional abrogation of K27-linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27-linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97-proteasome pathway model substrate (Ub(G76V)-GFP) is directly modified by K27-linked ubiquitylation, and that disabling the formation of K27-linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage-specific binder UCHL3 impedes Ub(G76V)-GFP turnover at the level of p97 function. Our findings suggest a critical role of K27-linked ubiquitylation in supporting cell fitness by facilitating p97-dependent processing of ubiquitylated nuclear proteins.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Núcleo Celular/metabolismo , Proliferação de Células , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
15.
EMBO Rep ; 25(2): 672-703, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38177917

RESUMO

ER protein homeostasis (proteostasis) is essential for proper folding and maturation of proteins in the secretory pathway. Loss of ER proteostasis can lead to the accumulation of misfolded or aberrant proteins in the ER and triggers the unfolded protein response (UPR). In this study, we find that the p97 adaptor UBXN1 is an important negative regulator of the UPR. Loss of UBXN1 sensitizes cells to ER stress and activates the UPR. This leads to widespread upregulation of the ER stress transcriptional program. Using comparative, quantitative proteomics we show that deletion of UBXN1 results in a significant enrichment of proteins involved in ER-quality control processes including those involved in protein folding and import. Notably, we find that loss of UBXN1 does not perturb p97-dependent ER-associated degradation (ERAD). Our studies indicate that loss of UBXN1 increases translation in both resting and ER-stressed cells. Surprisingly, this process is independent of p97 function. Taken together, our studies have identified a new role for UBXN1 in repressing translation and maintaining ER proteostasis in a p97 independent manner.


Assuntos
Proteostase , Resposta a Proteínas não Dobradas , Dobramento de Proteína , Proteínas , Estresse do Retículo Endoplasmático
16.
Mol Cell ; 72(4): 766-777.e6, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30344098

RESUMO

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/metabolismo
17.
Mol Cell ; 72(4): 605-607, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30444996

RESUMO

In this issue of Molecular Cell, Weith et al. (2018) demonstrate that p97, together with a SEP adaptor, can catalyze ordered subunit exchange to facilitate the biogenesis of protein phosphatase-1 (PP1) holoenzyme, establishing a novel ubiquitin-independent "segregase" function for this versatile ATPase.


Assuntos
Proteínas de Ciclo Celular , Ubiquitina , Adenosina Trifosfatases , Holoenzimas , ATPases Translocadoras de Prótons , Proteína com Valosina
18.
Proc Natl Acad Sci U S A ; 120(4): e2208941120, 2023 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-36656859

RESUMO

p97 is an essential AAA+ ATPase that extracts and unfolds substrate proteins from membranes and protein complexes. Through its mode of action, p97 contributes to various cellular processes, such as membrane fusion, ER-associated protein degradation, DNA repair, and many others. Diverse p97 functions and protein interactions are regulated by a large number of adaptor proteins. Alveolar soft part sarcoma locus (ASPL) is a unique adaptor protein that regulates p97 by disassembling functional p97 hexamers to smaller entities. An alternative mechanism to regulate the activity and interactions of p97 is by posttranslational modifications (PTMs). Although more than 140 PTMs have been identified in p97, only a handful of those have been described in detail. Here we present structural and biochemical data to explain how the p97-remodeling adaptor protein ASPL enables the metastasis promoting methyltransferase METTL21D to bind and trimethylate p97 at a single lysine side chain, which is deeply buried inside functional p97 hexamers. The crystal structure of a heterotrimeric p97:ASPL:METTL21D complex in the presence of cofactors ATP and S-adenosyl homocysteine reveals how structural remodeling by ASPL exposes the crucial lysine residue of p97 to facilitate its trimethylation by METTL21D. The structure also uncovers a role of the second region of homology (SRH) present in the first ATPase domain of p97 in binding of a modifying enzyme to the AAA+ ATPase. Investigation of this interaction in the human, fish, and plant reveals fine details on the mechanism and significance of p97 trimethylation by METTL21D across different organisms.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases , Metiltransferases , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Lisina/metabolismo , Metilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Proteína com Valosina/metabolismo , Metiltransferases/metabolismo
19.
J Biol Chem ; 300(5): 107230, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38537699

RESUMO

Arsenite-induced stress granule (SG) formation can be cleared by the ubiquitin-proteasome system aided by the ATP-dependent unfoldase p97. ZFAND1 participates in this pathway by recruiting p97 to trigger SG clearance. ZFAND1 contains two An1-type zinc finger domains (ZF1 and ZF2), followed by a ubiquitin-like domain (UBL); but their structures are not experimentally determined. To shed light on the structural basis of the ZFAND1-p97 interaction, we determined the atomic structures of the individual domains of ZFAND1 by solution-state NMR spectroscopy and X-ray crystallography. We further characterized the interaction between ZFAND1 and p97 by methyl NMR spectroscopy and cryo-EM. 15N spin relaxation dynamics analysis indicated independent domain motions for ZF1, ZF2, and UBL. The crystal structure and NMR structure of UBL showed a conserved ß-grasp fold homologous to ubiquitin and other UBLs. Nevertheless, the UBL of ZFAND1 contains an additional N-terminal helix that adopts different conformations in the crystalline and solution states. ZFAND1 uses the C-terminal UBL to bind to p97, evidenced by the pronounced line-broadening of the UBL domain during the p97 titration monitored by methyl NMR spectroscopy. ZFAND1 binding induces pronounced conformational heterogeneity in the N-terminal domain of p97, leading to a partial loss of the cryo-EM density of the N-terminal domain of p97. In conclusion, this work paved the way for a better understanding of the interplay between p97 and ZFAND1 in the context of SG clearance.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Grânulos de Estresse , Proteína com Valosina , Humanos , Arsenitos/metabolismo , Arsenitos/química , Cristalografia por Raios X , Ligação Proteica , Domínios Proteicos , Grânulos de Estresse/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/química , Proteína com Valosina/genética , Dedos de Zinco , Dobramento de Proteína , Imageamento por Ressonância Magnética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
20.
EMBO J ; 40(9): e105853, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33555040

RESUMO

p97ATPase-mediated membrane fusion is required for the biogenesis of the Golgi complex. p97 and its cofactor p47 function in soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for p97/p47-mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47-binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or p97 via its association with their polyglutamate motifs. FTCD functions in p97/p47-mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to p97. We also showed that FTCD, p47, and p97 form a big FTCD-p97/p47-FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous p97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the p97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD-p97/p47-FTCD complex in p97/p47-mediated Golgi membrane fusion.


Assuntos
Amônia-Liases/metabolismo , Glutamato Formimidoiltransferase/metabolismo , Complexo de Golgi/metabolismo , Enzimas Multifuncionais/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteína com Valosina/metabolismo , Amônia-Liases/química , Sítios de Ligação , Glutamato Formimidoiltransferase/química , Células HeLa , Células Hep G2 , Humanos , Fusão de Membrana , Mitocôndrias , Mitose , Enzimas Multifuncionais/química , Complexos Multiproteicos/metabolismo , Ligação Proteica
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