RESUMEN
Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a human CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualized it by cryo-EM. We describe high-resolution structural intermediates, including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimized DCNL1-binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays, help formulate a detailed model for CAND-SCF regulation.
Asunto(s)
Proteínas Cullin , Proteínas Ligasas SKP Cullina F-box , Humanos , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Cullin/metabolismo , Factores de Transcripción/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.
Asunto(s)
Autofagosomas/enzimología , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagosomas/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Fosforilación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de TiempoRESUMEN
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas Portadoras/genética , ADN de Helmintos/genética , Proteínas de Unión al ADN/genética , Recombinasa Rad51/genética , Reparación del ADN por Recombinación , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , ADN de Helmintos/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Unión Proteica , Recombinasa Rad51/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Transducción de Señal , Imagen Individual de MoléculaRESUMEN
NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin-RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here, we re-evaluate studies of non-cullin targets of NEDD8 in light of the current understanding of the neddylation pathway, and suggest criteria for identifying genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights.
Asunto(s)
Proteínas Cullin/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Ubiquitinas/metabolismo , Animales , Proteínas Cullin/genética , Humanos , Proteína NEDD8 , Ubiquitinas/genéticaRESUMEN
DNA repair by homologous recombination is highly suppressed in G1 cells to ensure that mitotic recombination occurs solely between sister chromatids. Although many homologous recombination factors are cell-cycle regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 to constrain BRCA2 function to the S/G2 phases in human cells. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein, in complex with cullin-3 (CUL3)-RBX1 (ref. 6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA-end resection is sufficient to induce homologous recombination in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR-Cas9-based gene-targeting assay. We conclude that the mechanism prohibiting homologous recombination in G1 minimally consists of the suppression of DNA-end resection coupled with a multi-step block of the recruitment of BRCA2 to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce homologous recombination in G1 cells with defined factors could spur the development of gene-targeting applications in non-dividing cells.
Asunto(s)
Fase G1 , Recombinación Homóloga , Secuencia de Aminoácidos , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas Cullin/metabolismo , ADN/metabolismo , Daño del ADN , Reparación del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Fase G2 , Marcación de Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Recombinasa Rad51/metabolismo , Fase S , Tioléster Hidrolasas/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.
Asunto(s)
Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Secuencias de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase , Biocatálisis , Línea Celular , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestructura , Espectrometría de Masas , Microscopía Electrónica , Modelos Moleculares , Peso Molecular , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Dispersión de Radiación , Schizosaccharomyces/química , Relación Estructura-Actividad , Especificidad por Sustrato , Complejos de Ubiquitina-Proteína Ligasa/ultraestructura , UbiquitinaciónRESUMEN
Modification of proteins by ubiquitin (Ub) and Ub-like (Ubl) modifiers regulates a variety of cellular functions. The ability of Ub to form chains of eight structurally and functionally distinct types adds further complexity to the system. Ub-specific proteases (USPs) hydrolyse polyUb chains, and some have been suggested to be cross-reactive with Ubl modifiers, such as neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and interferon-stimulated gene 15 (ISG15). Here, we report that USP21 cleaves Ub polymers, and with reduced activity also targets ISG15, but is inactive against NEDD8. A crystal structure of USP21 in complex with linear diUb aldehyde shows how USP21 interacts with polyUb through a previously unidentified second Ub- and ISG15-binding surface on the USP domain core. We also rationalize the inability of USP21 to target NEDD8 and identify differences that allow USPs to distinguish between structurally related modifications.
Asunto(s)
Poliubiquitina/metabolismo , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Sitios de Unión/fisiología , Cristalografía por Rayos X , Citocinas/química , Citocinas/metabolismo , Humanos , Proteína NEDD8 , Unión Proteica , Ubiquitinas/química , Ubiquitinas/metabolismoRESUMEN
The structural determination of biological macromolecules has been transformative for understanding biochemical mechanisms and developing therapeutics. However, the ultimate goal of characterizing how structural dynamics underpin biochemical processes has been difficult. This is largely due to significant technical challenges that hinder data collection and analysis on the native timescales of macromolecular dynamics. Single-particle cryo-EM provides a powerful platform to approach this challenge, since samples can be frozen faster than the single-turnover timescales of most biochemical reactions. In order to enable time-resolved analysis, significant innovations in the handling and preparation of cryo-EM samples have been implemented, bringing us closer to the goal of the direct observation of protein dynamics in the milliseconds to seconds range. Here, the current state of time-resolved cryo-EM is reviewed and the most promising future research directions are discussed.
Asunto(s)
Microscopía por Crioelectrón , Sustancias Macromoleculares/químicaRESUMEN
Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. Here we report a time-resolved sample preparation method for cryo-electron microscopy (trEM) using a modular microfluidic device, featuring a 3D-mixing unit and variable delay lines that enables automated, fast, and blot-free sample vitrification. This approach not only preserves high-resolution structural detail but also substantially improves sample integrity and protein distribution across the vitreous ice. We validate the method by visualising reaction intermediates of early RecA filament growth across three orders of magnitude on sub-second timescales. The trEM method reported here is versatile, reproducible, and readily adaptable to a broad spectrum of fundamental questions in biology.
Asunto(s)
Microscopía por Crioelectrón/métodos , Microfluídica/métodos , Biofisica , Cinética , Microscopía FluorescenteRESUMEN
Parkinson's disease (PD) is a neurological disorder characterized by the progressive accumulation of neuronal α-synuclein (αSyn) inclusions called Lewy bodies. It is believed that Lewy bodies spread throughout the nervous system due to the cell-to-cell propagation of αSyn via cycles of secretion and uptake. Here, we investigated the internalization and intracellular accumulation of exogenous αSyn, two key steps of Lewy body pathogenesis, amplification and spreading. We found that stable αSyn fibrils substantially accumulate in different cell lines upon internalization, whereas αSyn monomers, oligomers, and dissociable fibrils do not. Our data indicate that the uptake-mediated accumulation of αSyn in a human-derived neuroblastoma cell line triggered an adaptive response that involved proteins linked to ubiquitin ligases of the S-phase kinase-associated protein 1 (SKP1), cullin-1 (Cul1), and F-box domain-containing protein (SCF) family. We found that SKP1, Cul1, and the F-box/LRR repeat protein 5 (FBXL5) colocalized and physically interacted with internalized αSyn in cultured cells. Moreover, the SCF containing the F-box protein FBXL5 (SCFFBXL5) catalyzed αSyn ubiquitination in reconstitution experiments in vitro using recombinant proteins and in cultured cells. In the human brain, SKP1 and Cul1 were recruited into Lewy bodies from brainstem and neocortex of patients with PD and related neurological disorders. In both transgenic and nontransgenic mice, intracerebral administration of exogenous αSyn fibrils triggered a Lewy body-like pathology, which was amplified by SKP1 or FBXL5 loss of function. Our data thus indicate that SCFFXBL5 regulates αSyn in vivo and that SCF ligases may constitute targets for the treatment of PD and other α-synucleinopathies.
Asunto(s)
Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Ubiquitina-Proteína Ligasas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Benzotiazoles/metabolismo , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Ratones , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Proteoma/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Ubiquitina/metabolismoRESUMEN
DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Reparación del ADN por Recombinación/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ciclo Celular , Reparación del ADN por Unión de Extremidades/fisiología , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/química , Endonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Meiosis/genética , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosforilación , Unión Proteica , Multimerización de Proteína , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Carcinoma Endometrioide/genética , Carcinosarcoma/genética , Neoplasias Endometriales/genética , Neoplasias Quísticas, Mucinosas y Serosas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Acetanilidas/farmacología , Adenocarcinoma de Células Claras/metabolismo , Animales , Apoptosis/efectos de los fármacos , Azepinas/farmacología , Carcinoma Endometrioide/metabolismo , Carcinosarcoma/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía Liquida , Proteínas Cullin/metabolismo , Resistencia a Antineoplásicos , Neoplasias Endometriales/metabolismo , Epigénesis Genética , Femenino , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Masculino , Espectrometría de Masas , Ratones Desnudos , Terapia Molecular Dirigida , Mutación , Trasplante de Neoplasias , Neoplasias Quísticas, Mucinosas y Serosas/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Neoplasias de la Próstata/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , UbiquitinaciónRESUMEN
Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3(KLHL21) E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3(KLHL21) activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células HeLa , Humanos , UbiquitinaciónRESUMEN
Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Cullin/metabolismo , Endocitosis , Endosomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transporte Biológico , Línea Celular , Humanos , Virus de la Influenza A/fisiología , Internalización del VirusRESUMEN
Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.
Asunto(s)
Proteínas Portadoras/metabolismo , Reparación del ADN por Unión de Extremidades , Recombinación Homóloga/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitinación/genética , Proteínas Portadoras/genética , Línea Celular , Proteínas Cullin/metabolismo , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Humanos , Mutación , Proteínas Nucleares/genética , Dominios y Motivos de Interacción de Proteínas/genética , ProteolisisRESUMEN
The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.
Asunto(s)
Proteínas Cullin/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multienzimáticos/metabolismo , Péptido Hidrolasas/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Microscopía por Crioelectrón , Humanos , Hidrólisis , Péptidos y Proteínas de Señalización Intracelular/química , Cinética , Espectrometría de Masas , Modelos Moleculares , Complejos Multienzimáticos/química , Proteína NEDD8 , Péptido Hidrolasas/química , Unión Proteica , Conformación ProteicaRESUMEN
Skp1-Cul1-Fbox (SCF) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCF(Skp2/Cks1) and SCF(Fbw7) and found that CSN occludes both SCF functional sites-the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Proteolisis , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Humanos , Complejos Multienzimáticos/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
The evolutionary conserved COP9 signalosome (CSN), a large multisubunit complex, plays a central role in regulating ubiquitination and cell signaling. Here we report recombinant insect cell expression and two-step purification of human CSN and demonstrate its functional assembly. We further obtain a three-dimensional structure of both native and recombinant CSN using electron microscopy and single particle analysis. Antibody labeling of CSN5 and segmentation of the structure suggest a likely subunit distribution and the architecture of its helical repeat subunits is revealed. We compare the structure of CSN with its homologous complexes, the 26S proteasome lid and eIF3, and propose a conserved architecture implying similar assembly pathways and/or conserved substrate interaction modes.