RESUMO
In mammals, â¼100 deubiquitinases act on â¼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.
Assuntos
Proteína BRCA1/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Enzimas Desubiquitinantes/genética , Chaperonas de Histonas/genética , Neoplasias/genética , Sítios de Ligação/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Citoplasma/genética , Citoplasma/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/imunologia , Enzimas Desubiquitinantes/imunologia , Células HeLa , Humanos , Imunidade Celular/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Neoplasias/imunologia , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica/genética , Ubiquitina/genética , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genéticaRESUMO
Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we revealed the expression of RPL39L protein beyond mouse germ cells, in human pluripotent cells, cancer cell lines and tissue samples. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread autophagy. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.
RESUMO
The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the Ë29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.
Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Proteínas Culina/metabolismo , Humanos , Modelos Moleculares , Mutação , Domínios Proteicos , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoRESUMO
In this Article, in Fig. 1a, the 5' and 3' labels were reversed in the DNA sequence, and Fig. 4 was missing panel labels a-e. These errors have been corrected online.
RESUMO
Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , DNA/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Dímeros de Pirimidina/análise , Microscopia Crioeletrônica , DNA/química , DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Histonas/química , Histonas/metabolismo , Histonas/ultraestrutura , Humanos , Modelos Moleculares , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Dímeros de Pirimidina/química , Dímeros de Pirimidina/genética , Termodinâmica , Raios Ultravioleta/efeitos adversosRESUMO
Heterogenous nuclear ribonucleoproteins (hnRNPs) are abundant proteins implicated in various steps of RNA processing that assemble on nuclear RNA into larger complexes termed 40S hnRNP particles. Despite their initial discovery 55 years ago, our understanding of these intriguing macromolecular assemblies remains limited. Here, we report the biochemical purification of native 40S hnRNP particles and the determination of their complete protein composition by label-free quantitative mass spectrometry, identifying A-group and C-group hnRNPs as the major protein constituents. Isolated 40S hnRNP particles dissociate upon RNA digestion and can be reconstituted in vitro on defined RNAs in the presence of the individual protein components, demonstrating a scaffolding role for RNA in nucleating particle formation. Finally, we revealed their nanometer scale, condensate-like nature, promoted by intrinsically disordered regions of A-group hnRNPs. Collectively, we identify nuclear 40S hnRNP particles as novel dynamic biomolecular condensates.
Assuntos
Condensados Biomoleculares , Ribonucleoproteínas Nucleares Heterogêneas , Núcleo Celular/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , RNA/metabolismoRESUMO
The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remain poorly understood. Using biochemical assays, cross-linking mass spectrometry, and cryo-electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end degron. GID4 substrate binding and ligase activity is regulated by ARMC8α, while the shorter ARMC8ß isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring-like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity.
Assuntos
Proteínas de Grupo de Alta Mobilidade , Ubiquitina-Proteína Ligases , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Substrates enter the cylindrical 20S proteasome through a gated channel that is regulated by the ATPases in the 19S regulatory particle in eukaryotes or the homologous PAN ATPase complex in archaea. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X (HbYX) motif that triggers gate opening upon ATP binding. Using cryo-electron microscopy, we identified the sites in the archaeal 20S where PAN's C-terminal residues bind and determined the structures of the gate in its closed and open forms. Peptides containing the HbYX motif bind to 20S in the pockets between neighboring alpha subunits where they interact with conserved residues required for gate opening. This interaction induces a rotation in the alpha subunits and displacement of a reverse-turn loop that stabilizes the open-gate conformation. This mechanism differs from that of PA26/28, which lacks the HbYX motif and does not cause alpha subunit rotation. These findings demonstrated how the ATPases' C termini function to facilitate substrate entry.
Assuntos
Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Conformação Proteica , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Animais , Proteínas Arqueais/genética , Sítios de Ligação , Microscopia Crioeletrônica , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Thermoplasma/química , Thermoplasma/metabolismoRESUMO
The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to dynamics. Solid-state NMR characterization of the capsid structure reveals few conformational differences between the quasi-equivalent subunits of the ACNDV capsid and an overall higher capsid structural disorder compared to HBV. Despite these differences, the capsids of ACNDV and HBV are structurally highly similar despite the 400 million years since their separation.
Assuntos
Proteínas do Capsídeo , Hepadnaviridae , Animais , Proteínas do Capsídeo/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Capsídeo/metabolismo , Hepadnaviridae/metabolismo , Mamíferos/metabolismoRESUMO
The K63-linkage specific deubiquitinase BRCC36 forms the core of two multi-subunit deubiquitination complexes: BRCA1-A and BRISC. BRCA1-A is recruited to DNA repair foci, edits ubiquitin signals on chromatin, and sequesters BRCA1 away from the site of damage, suppressing homologous recombination by limiting resection. BRISC forms a complex with metabolic enzyme SHMT2 and regulates the immune response, mitosis, and hematopoiesis. Almost two decades of research have revealed how BRCA1-A and BRISC use the same core of subunits to perform very distinct biological tasks.
Assuntos
Proteína BRCA1/genética , Reparo do DNA/genética , Enzimas Desubiquitinantes/genética , Glicina Hidroximetiltransferase/genética , Cromatina/genética , Proteínas de Ligação a DNA/genética , Hematopoese/genética , Chaperonas de Histonas/genética , Humanos , Imunidade/genética , Mitose/genética , Proteína SUMO-1/genética , Ubiquitina/genéticaRESUMO
Eukaryotic translation initiation factors (eIFs) 1A and 1 are central players in the complex process of start-codon recognition. To improve mechanistic understanding of this process, we determined the crystal structure of the 40S ribosomal subunit in complex with eIF1A and eIF1 from Tetrahymena thermophila at a resolution of 3.7 Å. It reveals the positions of the two factors on the 40S and the conformational changes that accompany their binding.
Assuntos
Fatores de Iniciação em Eucariotos/química , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Subunidades Ribossômicas Menores de Eucariotos/química , Tetrahymena thermophila/química , Clonagem Molecular , Cristalização , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Eukaryotic ribosomes are substantially larger and more complex than their bacterial counterparts. Although their core function is conserved, bacterial and eukaryotic protein synthesis differ considerably at the level of initiation. The eukaryotic small ribosomal subunit (40S) plays a central role in this process; it binds initiation factors that facilitate scanning of messenger RNAs and initiation of protein synthesis. We have determined the crystal structure of the Tetrahymena thermophila 40S ribosomal subunit in complex with eukaryotic initiation factor 1 (eIF1) at a resolution of 3.9 angstroms. The structure reveals the fold of the entire 18S ribosomal RNA and of all ribosomal proteins of the 40S subunit, and defines the interactions with eIF1. It provides insights into the eukaryotic-specific aspects of protein synthesis, including the function of eIF1 as well as signaling and regulation mediated by the ribosomal proteins RACK1 and rpS6e.