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1.
Gene ; 784: 145584, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33753149

RESUMO

Sister chromatid cohesion (SCC) is essential for the maintenance of genome integrity. The establishment of SCC is coupled to DNA replication, and this is achieved in budding yeast Saccharomyces cerevisiae by a mechanism that is dependent on the interaction between Eco1 acetyltransferase and PCNA in the DNA replication complex. In vertebrates, the Eco1 homolog ESCO2 has been reported to interact with MCM complex in the DNA replication complex to establish DNA replication-dependent cohesion. Here we show that budding yeast Eco1 is also physically interacted with the MCM complex. We found that Eco1 was specifically bound to Mcm2 subunit in the MCM complex and they interacted via their N-terminal regions, using yeast two-hybrid system. The underlying mechanism of the interaction was different between yeast and vertebrates. Intensive molecular dissection of Eco1 identified residues important for interaction with Mcm2 and/or PCNA. Mutant forms of Eco1 (Eco1mWW and Eco1mGRK), where sets of the identified residues were substituted with alanine, resulted in impaired SCC, decreased level of acetylation of Smc3, and a reduction of Eco1 protein amount in yeast cells. We, hence, suggest that Eco1 is stabilized by its interactions with MCM complex and PCNA, which allows it to promote DNA replication-coupled SCC establishment.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilação , Acetiltransferases/química , Acetiltransferases/genética , Sítios de Ligação , Cromossomos Fúngicos/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estabilidade Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
3.
Nucleic Acids Res ; 49(5): 2959-2972, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619523

RESUMO

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.


Assuntos
DNA/química , Proteínas Nucleares/química , Fosfoproteínas/química , Cristalografia por Raios X , DNA/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica
4.
Nat Commun ; 12(1): 1028, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589610

RESUMO

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.


Assuntos
Proteínas de Transporte/química , DNA/química , Histonas/química , Proteínas Nucleares/química , Poli(ADP-Ribose) Polimerase-1/química , Serina/metabolismo , ADP-Ribosilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutamina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática
5.
Science ; 371(6531): 846-849, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33602856

RESUMO

Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/biossíntese , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Microscopia Crioeletrônica , Complexo IV da Cadeia de Transporte de Elétrons/química , Humanos , Proteínas de Membrana/química , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Ribossomos Mitocondriais/ultraestrutura , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Ribossomos/metabolismo
6.
Nucleic Acids Res ; 49(4): 2179-2191, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33533925

RESUMO

Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.


Assuntos
Arginina/química , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , DNA/biossíntese , Enzimas Multifuncionais/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Motivos de Aminoácidos , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Modelos Moleculares , Enzimas Multifuncionais/metabolismo , Ligação Proteica
7.
Science ; 371(6527)2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33479126

RESUMO

Dot1 (disruptor of telomeric silencing-1), the histone H3 lysine 79 (H3K79) methyltransferase, is conserved throughout evolution, and its deregulation is found in human leukemias. Here, we provide evidence that acetylation of histone H4 allosterically stimulates yeast Dot1 in a manner distinct from but coordinating with histone H2B ubiquitination (H2BUb). We further demonstrate that this stimulatory effect is specific to acetylation of lysine 16 (H4K16ac), a modification central to chromatin structure. We provide a mechanism of this histone cross-talk and show that H4K16ac and H2BUb play crucial roles in H3K79 di- and trimethylation in vitro and in vivo. These data reveal mechanisms that control H3K79 methylation and demonstrate how H4K16ac, H3K79me, and H2BUb function together to regulate gene transcription and gene silencing to ensure optimal maintenance and propagation of an epigenetic state.


Assuntos
Montagem e Desmontagem da Cromatina , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleossomos/enzimologia , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
8.
Nucleic Acids Res ; 49(3): 1411-1425, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33450002

RESUMO

DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA , Genes cdc , Mutação , Proteínas Nucleares/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Processamento de RNA , Estresse Fisiológico , Supressão Genética , Ubiquitinação
9.
Biochem J ; 478(1): 135-156, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33439989

RESUMO

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Transdução de Sinais/genética , Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/química , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
10.
Mol Cell ; 81(6): 1231-1245.e8, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33503405

RESUMO

ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte , Núcleo Celular , Proteínas de Ligação a DNA , Mutação de Sentido Incorreto , Proteínas Nucleares , Transdução de Sinais , Substituição de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinase 1 do Ponto de Checagem/química , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Sf9 , Spodoptera
11.
RNA ; 27(4): 496-512, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33483369

RESUMO

Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.


Assuntos
Proteínas Arqueais/química , Proteínas Cromossômicas não Histona/química , Proteínas Nucleares/química , Pyrococcus furiosus/genética , Ribonucleoproteínas Nucleolares Pequenas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Expressão Gênica , Metilação , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pyrococcus furiosus/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína
12.
Nat Commun ; 12(1): 121, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402676

RESUMO

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.


Assuntos
Coenzimas/química , Ditiocarb/análogos & derivados , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Nucleares/química , Compostos Organometálicos/química , Ubiquitina/química , Proteína com Valosina/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Clonagem Molecular , Coenzimas/genética , Coenzimas/metabolismo , Microscopia Crioeletrônica , Dissulfiram/química , Dissulfiram/metabolismo , Ditiocarb/química , Ditiocarb/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Compostos Organometálicos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina/metabolismo , Proteína com Valosina/antagonistas & inibidores , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Dedos de Zinco
13.
Methods Mol Biol ; 2231: 163-177, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33289893

RESUMO

The Database of Aligned Structural Homologs (DASH) is a tool for efficiently navigating the Protein Data Bank (PDB) by means of pre-computed pairwise structural alignments. We recently showed that, by integrating DASH structural alignments with the multiple sequence alignment (MSA) software MAFFT, we were able to significantly improve MSA accuracy without dramatically increasing manual or computational complexity. In the latest DASH update, such queries are not limited to PDB entries but can also be launched from user-provided protein coordinates. Here, we describe a further extension of DASH that retrieves intermolecular interactions of all structurally similar domains in the PDB to a query domain of interest. We illustrate these new features using a model of the NYN domain of the ribonuclease N4BP1 as an example. We show that the protein-nucleotide interactions returned are distributed on the surface of the NYN domain in an asymmetric manner, roughly centered on the known nuclease active site.


Assuntos
Proteínas de Ligação a RNA/química , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Software , Algoritmos , Sequência de Aminoácidos , Biologia Computacional , Bases de Dados de Proteínas , Proteínas Nucleares/química , Ligação Proteica , Domínios Proteicos , Ribonucleases/química
14.
Nat Commun ; 11(1): 5551, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144586

RESUMO

Genomic studies performed in cancer patients and tumor-derived cell lines have identified a high frequency of alterations in components of the mammalian switch/sucrose non-fermentable (mSWI/SNF or BAF) chromatin remodeling complex, including its core catalytic subunit, SMARCA4. Cells exhibiting loss of SMARCA4 rely on its paralog, SMARCA2, making SMARCA2 an attractive therapeutic target. Here we report the genomic profiling of solid tumors from 131,668 cancer patients, identifying 9434 patients with one or more SMARCA4 gene alterations. Homozygous SMARCA4 mutations were highly prevalent in certain tumor types, notably non-small cell lung cancer (NSCLC), and associated with reduced survival. The large sample size revealed previously uncharacterized hotspot missense mutations within the SMARCA4 helicase domain. Functional characterization of these mutations demonstrated markedly reduced remodeling activity. Surprisingly, a few SMARCA4 missense variants partially or fully rescued paralog dependency, underscoring that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations who may benefit from SMARCA2-targeted therapy.


Assuntos
DNA Helicases/genética , Mutação/genética , Neoplasias/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequenciamento Completo do Exoma , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Cromatina/metabolismo , Estudos de Coortes , DNA Helicases/química , Regulação Neoplásica da Expressão Gênica , Homozigoto , Humanos , Mutação de Sentido Incorreto/genética , Proteínas Nucleares/química , Nucleossomos/metabolismo , Domínios Proteicos , Fatores de Transcrição/química
15.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Genética
16.
Nat Commun ; 11(1): 5111, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037216

RESUMO

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , Estabilidade de RNA , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Eucariotos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
17.
Nat Commun ; 11(1): 5466, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122719

RESUMO

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares , Sítios de Ligação , Ciclo Celular , Cristalografia por Raios X , DNA/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular , Espectrometria de Fluorescência , Fatores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/metabolismo
18.
Nat Commun ; 11(1): 5425, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110077

RESUMO

Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein-protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.


Assuntos
Peptidomiméticos , Fatores de Transcrição/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Nature ; 585(7826): 609-613, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939087

RESUMO

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1-5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6-8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2-HPF1 bound to a nucleosome. This showed that PARP2-HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12-16.


Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Biocatálise , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica , DNA/metabolismo , Reparo do DNA , Ativação Enzimática , Humanos , Modelos Moleculares , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Nucleossomos/química , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/ultraestrutura , Domínios Proteicos
20.
Nucleic Acids Res ; 48(18): 10313-10328, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976585

RESUMO

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas Nucleares/ultraestrutura , Proteínas de Ligação a RNA/química , Transcrição Genética , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Epigênese Genética/genética , Inativação Gênica , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Agregados Proteicos/genética , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/ultraestrutura , Vírus/genética
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