RESUMO
Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.
Assuntos
Chaperonina com TCP-1 , Ubiquitina-Proteína Ligases , Chaperonina com TCP-1/química , Ubiquitina-Proteína Ligases/genética , HumanosRESUMO
Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.
Assuntos
Antígenos de Histocompatibilidade Classe I , Neoplasias , Evasão Tumoral , Humanos , Apresentação de Antígeno , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos HLA , Neoplasias/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.
Assuntos
Síndromes de Imunodeficiência , Proteínas do Tecido Nervoso , Ubiquitinas , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Feminino , Masculino , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/genética , Inflamação/imunologia , Inflamação/genética , Linfócitos B/imunologia , Mutação com Perda de Função , Fibroblastos/metabolismo , Fibroblastos/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Camundongos , AlelosRESUMO
Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.
Assuntos
Dano ao DNA , Edição de Genes , Testes Genéticos , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas/genética , Camptotecina/farmacologia , Linhagem Celular , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Humanos , Mutação/genética , Fenótipo , Ligação Proteica , Domínios Proteicos , RNA Guia de Cinetoplastídeos/genética , Inibidores da Topoisomerase/farmacologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Stalled protein synthesis produces defective nascent chains that can harm cells. In response, cells degrade these nascent chains via a process called ribosome-associated quality control (RQC). Here, we review the irregularities in the translation process that cause ribosomes to stall as well as how cells use RQC to detect stalled ribosomes, ubiquitylate their tethered nascent chains, and deliver the ubiquitylated nascent chains to the proteasome. We additionally summarize how cells respond to RQC failure.
Assuntos
Escherichia coli/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Ribossomos/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Poli A/química , Poli A/genética , Poli A/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteólise , Splicing de RNA , Estabilidade de RNA , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Mutations in the BRCA1 and BRCA2 genes predispose afflicted individuals to breast, ovarian, and other cancers. The BRCA-encoded products form complexes with other tumor suppressor proteins and with the recombinase enzyme RAD51 to mediate chromosome damage repair by homologous recombination and also to protect stressed DNA replication forks against spurious nucleolytic attrition. Understanding how the BRCA tumor suppressor network executes its biological functions would provide the foundation for developing targeted cancer therapeutics, but progress in this area has been greatly hampered by the challenge of obtaining purified BRCA complexes for mechanistic studies. In this article, we review how recent effort begins to overcome this technical challenge, leading to functional and structural insights into the biochemical attributes of these complexes and the multifaceted roles that they fulfill in genome maintenance. We also highlight the major mechanistic questions that remain.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Redes Reguladoras de Genes , Rad51 Recombinase/genética , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , DNA/química , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Feminino , Genoma Humano , Instabilidade Genômica , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.
Assuntos
Autoimunidade , Citotoxicidade Imunológica , Imunoterapia Adotiva , Melanoma Experimental/terapia , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Neoplasias Cutâneas/terapia , Linfócitos T/transplante , Ubiquitina-Proteína Ligases/metabolismo , Animais , Feminino , Células HEK293 , Células HeLa , Humanos , Imunidade Humoral , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fenótipo , Ligação Proteica , Proteínas Repressoras/genética , Ribonucleases/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral , Ubiquitina-Proteína Ligases/genéticaRESUMO
The majority of newly diagnosed prostate cancers are slow growing, with a long natural life history. Yet a subset can metastasize with lethal consequences. We reconstructed the phylogenies of 293 localized prostate tumors linked to clinical outcome data. Multiple subclones were detected in 59% of patients, and specific subclonal architectures associate with adverse clinicopathological features. Early tumor development is characterized by point mutations and deletions followed by later subclonal amplifications and changes in trinucleotide mutational signatures. Specific genes are selectively mutated prior to or following subclonal diversification, including MTOR, NKX3-1, and RB1. Patients with low-risk monoclonal tumors rarely relapse after primary therapy (7%), while those with high-risk polyclonal tumors frequently do (61%). The presence of multiple subclones in an index biopsy may be necessary, but not sufficient, for relapse of localized prostate cancer, suggesting that evolution-aware biomarkers should be studied in prospective studies of low-risk tumors suitable for active surveillance.
Assuntos
Neoplasias da Próstata/patologia , Biomarcadores Tumorais/sangue , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Gradação de Tumores , Recidiva Local de Neoplasia , Polimorfismo de Nucleotídeo Único , Modelos de Riscos Proporcionais , Estudos Prospectivos , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.
Assuntos
Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Switching de Imunoglobulina/efeitos dos fármacos , Proteínas Mad2/antagonistas & inibidores , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a Telômeros/antagonistas & inibidores , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein ubiquitination is one of the most powerful posttranslational modifications of proteins, as it regulates a plethora of cellular processes in distinct manners. Simple monoubiquitination events coexist with more complex forms of polyubiquitination, the latter featuring many different chain architectures. Ubiquitin can be subjected to further posttranslational modifications (e.g., phosphorylation and acetylation) and can also be part of mixed polymers with ubiquitin-like modifiers such as SUMO (small ubiquitin-related modifier) or NEDD8 (neural precursor cell expressed, developmentally downregulated 8). Together, cellular ubiquitination events form a sophisticated and versatile ubiquitin code. Deubiquitinases (DUBs) reverse ubiquitin signals with equally high sophistication. In this review, we conceptualize the many layers of specificity that DUBs encompass to control the ubiquitin code and discuss examples in which DUB specificity has been understood at the molecular level. We further discuss the many mechanisms of DUB regulation with a focus on those that modulate catalytic activity. Our review provides a framework to tackle lingering questions in DUB biology.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Células Eucarióticas/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Acetilação , Regulação Alostérica , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Humanos , Modelos Moleculares , Proteína NEDD8 , Fosforilação , Ligação Proteica , Conformação Proteica , Proteólise , Especificidade por Substrato , Sumoilação , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ubiquitinas/genéticaRESUMO
Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.
Assuntos
Proteínas de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Drogas em Investigação/síntese química , Células Eucarióticas/microbiologia , Células Eucarióticas/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteólise , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.
Assuntos
Doença de Crohn , Humanos , Transcriptoma , Colo , Íleo , Inflamação/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The tumour suppressor breast cancer type 1 susceptibility protein (BRCA1) promotes DNA double-strand break (DSB) repair by homologous recombination and protects DNA replication forks from attrition. BRCA1 partners with BRCA1-associated RING domain protein 1 (BARD1) and other tumour suppressor proteins to mediate the initial nucleolytic resection of DNA lesions and the recruitment and regulation of the recombinase RAD51. The discovery of the opposing functions of BRCA1 and the p53-binding protein 1 (53BP1)-associated complex in DNA resection sheds light on how BRCA1 influences the choice of homologous recombination over non-homologous end joining and potentially other mutagenic pathways of DSB repair. Understanding the functional crosstalk between BRCA1-BARD1 and its cofactors and antagonists will illuminate the molecular basis of cancers that arise from a deficiency or misregulation of chromosome damage repair and replication fork maintenance. Such knowledge will also be valuable for understanding acquired tumour resistance to poly(ADP-ribose) polymerase (PARP) inhibitors and other therapeutics and for the development of new treatments. In this Review, we discuss recent advances in elucidating the mechanisms by which BRCA1-BARD1 functions in DNA repair, replication fork maintenance and tumour suppression, and its therapeutic relevance.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Proteínas Supressoras de Tumor/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Neoplasias da Mama/patologia , Reparo do DNA por Junção de Extremidades/genética , Replicação do DNA/genética , Feminino , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica/genética , Reparo de DNA por Recombinação/genéticaRESUMO
Ubiquitylation is an essential posttranslational modification that controls cell division, differentiation, and survival in all eukaryotes. By combining multiple E3 ligases (writers), ubiquitin-binding effectors (readers), and de-ubiquitylases (erasers) with functionally distinct ubiquitylation tags, the ubiquitin system constitutes a powerful signaling network that is employed in similar ways from yeast to humans. Here, we discuss conserved principles of ubiquitin-dependent signaling that illustrate how this posttranslational modification shapes intracellular signaling networks to establish robust development and homeostasis throughout the eukaryotic kingdom.
Assuntos
Processamento de Proteína Pós-Traducional/genética , Ubiquitina/genética , Ubiquitinação/genética , Células Eucarióticas/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
In a recent publication in Nature, Xu et al.1 discovered a role of CRL5-SPSB3 ubiquitin ligase in promoting ubiquitination and degradation of nuclear cGAS, which prevents aberrant cGAS activation by genomic DNA and contributes to the maintenance of immune homeostasis.
Assuntos
Homeostase , Nucleotidiltransferases , Ubiquitinação , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Núcleo Celular/metabolismo , Proteólise , AnimaisRESUMO
To maintain mitochondrial homeostasis, damaged or excessive mitochondria are culled in coordination with the physiological state of the cell. The integrated stress response (ISR) is a signaling network that recognizes diverse cellular stresses, including mitochondrial dysfunction. Because the four ISR branches converge to common outputs, it is unclear whether mitochondrial stress detected by this network can regulate mitophagy, the autophagic degradation of mitochondria. Using a whole-genome screen, we show that the heme-regulated inhibitor (HRI) branch of the ISR selectively induces mitophagy. Activation of the HRI branch results in mitochondrial localization of phosphorylated eukaryotic initiation factor 2, which we show is sufficient to induce mitophagy. The HRI mitophagy pathway operates in parallel with the mitophagy pathway controlled by the Parkinson's disease related genes PINK1 and PARKIN and is mechanistically distinct. Therefore, HRI repurposes machinery that is normally used for translational initiation to trigger mitophagy in response to mitochondrial damage.
Assuntos
Mitofagia , Proteínas Quinases , Mitofagia/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Autofagia/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de SinaisRESUMO
In a recent study in Nature, Haakonsen et al.1 identify the SIFI complex as a stress response silencer via its E3 ligase activity to target unimported mitochondrial proteins and stress response components for degradation via the proteasome.
Assuntos
Mitocôndrias , Complexo de Endopeptidases do Proteassoma , Sobrevivência Celular , Mitocôndrias/genética , Mitocôndrias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Phase separation is a vital mechanism that mediates the formation of biomolecular condensates and their functions. Necroptosis is a lytic form of programmed cell death mediated by RIPK1, RIPK3, and MLKL downstream of TNFR1 and has been implicated in mediating many human diseases. However, whether necroptosis is regulated by phase separation is not yet known. Here, we show that upon the induction of necroptosis and recruitment by the adaptor protein TAX1BP1, PARP5A and its binding partner RNF146 form liquid-like condensates by multivalent interactions to perform poly ADP-ribosylation (PARylation) and PARylation-dependent ubiquitination (PARdU) of activated RIPK1 in mouse embryonic fibroblasts. We show that PARdU predominantly occurs on the K376 residue of mouse RIPK1, which promotes proteasomal degradation of kinase-activated RIPK1 to restrain necroptosis. Our data demonstrate that PARdU on K376 of mouse RIPK1 provides an alternative cell death checkpoint mediated by phase separation-dependent control of necroptosis by PARP5A and RNF146.
Assuntos
Necroptose , Separação de Fases , Animais , Camundongos , Apoptose/fisiologia , Morte Celular , Fibroblastos/metabolismo , Necroptose/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.
Assuntos
Proteínas Culina , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Ubiquitinação , Ubiquitina/metabolismo , Poliubiquitina/metabolismo , Proteínas de Transporte/metabolismoRESUMO
The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.