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1.
Curr Opin Genet Dev ; 71: 106-113, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34340015

RESUMO

PARP1 and PARP2 govern the DNA-damage response by catalysing the reversible post-translational modification ADP-ribosylation. During the repair of DNA lesions, PARP1 and PARP2 combine with an accessory factor HPF1, which is required for the modification of target proteins on serine residues. Although the physiological role of individual ADP-ribosylation sites is still unclear, serine ADP-ribosylation at damage sites leads to the recruitment of chromatin remodellers and repair factors to ensure efficient DNA repair. ADP-ribosylation signalling is tightly controlled by the coordinated activities of (ADP-ribosyl)glycohydrolases PARG and ARH3 that, by reversing the modification, guarantee proper kinetics of DNA repair and cell cycle re-entry. The recent advances in the structural and mechanistic understanding of ADP-ribosylation provide new insights into human physiopathology and cancer therapy.

2.
Nat Commun ; 12(1): 4055, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210965

RESUMO

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.


Assuntos
Proteínas de Transporte/metabolismo , Dano ao DNA , Reparo do DNA , Neoplasias/tratamento farmacológico , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Serina/metabolismo , ADP-Ribosilação , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional
3.
Elife ; 102021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34328418

RESUMO

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labeling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labeling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The 'closed' octamer interconverts with other oligomers in a phosphorylation-dependent manner and, unlike these 'open' forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fosforilação , Proteínas Quinases/química
4.
Mol Cell ; 81(12): 2640-2655.e8, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34019811

RESUMO

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.


Assuntos
Glicosídeo Hidrolases/metabolismo , Poli ADP Ribosilação/fisiologia , ADP-Ribosilação , Adenosina Difosfato Ribose/metabolismo , Linhagem Celular Tumoral , Cromatina , DNA , Dano ao DNA , Fibroblastos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/fisiologia , Células HEK293 , Células HeLa , Humanos , Poli Adenosina Difosfato Ribose/metabolismo , Cultura Primária de Células
5.
FEBS J ; 288(7): 2131-2142, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32785980

RESUMO

Despite decades of research on ADP-ribosyltransferases (ARTs) from the poly(ADP-ribose) polymerase (PARP) family, one key aspect of these enzymes - their substrate specificity - has remained unclear. Here, we briefly discuss the history of this area and, more extensively, the recent breakthroughs, including the identification of protein serine residues as a major substrate of PARP1 and PARP2 in human cells and of cysteine and tyrosine as potential targets of specific PARPs. On the molecular level, the modification of serine residues requires a composite active site formed by PARP1 or PARP2 together with a specificity-determining factor, HPF1; this represents a new paradigm not only for PARPs but generally for post-translational modification (PTM) catalysis. Additionally, we discuss the identification of DNA as a substrate of PARP1, PARP2 and PARP3, and some bacterial ARTs and the discovery of noncanonical RNA capping by several PARP family members. Together, these recent findings shed new light on PARP-mediated catalysis and caution to 'expect the unexpected' when it comes to further potential substrates.


Assuntos
Proteínas de Ciclo Celular/genética , DNA/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , ADP Ribose Transferases/genética , Proteínas de Transporte/genética , Catálise , Proteínas de Ciclo Celular/química , Humanos , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerases/química , Capuzes de RNA/genética , Especificidade por Substrato/genética
6.
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
7.
Nature ; 579(7800): 598-602, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32028527

RESUMO

The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. After binding to genomic lesions, these enzymes use NAD+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues5-10. Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.


Assuntos
ADP-Ribosilação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biocatálise , Proteínas de Transporte/genética , Domínio Catalítico , Células HEK293 , Humanos , Modelos Moleculares , Mutação , NAD/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Anêmonas-do-Mar
8.
Nat Chem Biol ; 15(5): 510-518, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962626

RESUMO

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.


Assuntos
Arginina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Arginina/química , Modelos Moleculares , Fosforilação
9.
Protein Sci ; 20(8): 1285-97, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21574196

RESUMO

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.


Assuntos
Estabilidade Proteica , Proteínas/química , Proteínas/metabolismo , Proteólise , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dobramento de Proteína
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