RESUMO
Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification1-3. The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome4, counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1)5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6-9). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3-H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A-H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1.
Assuntos
Enzimas Desubiquitinantes , Histonas , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Humanos , Microscopia Crioeletrônica , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Complexo Repressor Polycomb 1/química , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/ultraestrutura , Proteínas do Grupo Polycomb/química , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/ultraestrutura , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/ultraestrutura , Ubiquitinação , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Domínio Catalítico , Enzimas Desubiquitinantes/classificação , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/ultraestrutura , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestruturaRESUMO
Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.
Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/ultraestrutura , Proteínas Nucleares/ultraestrutura , Proteases Específicas de Ubiquitina/ultraestrutura , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/ultraestrutura , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Ligação Proteica/genética , Conformação Proteica , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genéticaRESUMO
Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Interferon Tipo I/imunologia , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/metabolismo , Transdução de Sinais/imunologia , Microscopia Crioeletrônica , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/ultraestrutura , Glicina Hidroximetiltransferase/ultraestrutura , Células HEK293 , Humanos , Inflamação/imunologia , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Fosfato de Piridoxal/metabolismoRESUMO
BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.
