RESUMO
Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.
Assuntos
Proteínas de Ligação a DNA , Chaperonas de Histonas , Animais , Humanos , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Cromatina/metabolismo , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
FAM111A is a replisome-associated protein and dominant mutations within its trypsin-like peptidase domain are linked to severe human developmental syndrome, the Kenny-Caffey syndrome. However, FAM111A functions remain unclear. Here, we show that FAM111A facilitates efficient activation of DNA replication origins. Upon hydroxyurea treatment, FAM111A-depleted cells exhibit reduced single-stranded DNA formation and a better survival rate. Unrestrained expression of FAM111A WT and patient mutants causes accumulation of DNA damage and cell death, only when the peptidase domain remains intact. Unrestrained expression of FAM111A WT also causes increased single-stranded DNA formation that relies on S phase entry, FAM111A peptidase activity but not its binding to proliferating cell nuclear antigen. Altogether, these data unveil how FAM111A promotes DNA replication under normal conditions and becomes harmful in a disease context.
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DNA de Cadeia Simples , Origem de Replicação , Humanos , Origem de Replicação/genética , Replicação do DNA/genética , Fase S , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Receptores Virais/metabolismoRESUMO
The initiation step of replication at replication origins determines when and where in the genome replication machines, replisomes, are generated. Tight control of replication initiation helps facilitate the two main tasks of genome replication, to duplicate the genome accurately and exactly once each cell division cycle. The regulation of replication initiation must ensure that initiation occurs during the S phase specifically, that no origin fires more than once per cell cycle, that enough origins fire to avoid non-replicated gaps, and that the right origins fire at the right time but only in favorable circumstances. Despite its importance for genetic homeostasis only the main molecular processes of eukaryotic replication initiation and its cellular regulation are understood. The MTBP protein (Mdm2-binding protein) is so far the last core replication initiation factor identified in metazoan cells. MTBP is the orthologue of yeast Sld7. It is essential for origin firing, the maturation of pre-replicative complexes (pre-RCs) into replisomes, and is emerging as a regulation focus targeted by kinases and by regulated degradation. We present recent insight into the structure and cellular function of the MTBP protein in light of recent structural and biochemical studies revealing critical molecular details of the eukaryotic origin firing reaction. How the roles of MTBP in replication and other cellular processes are mutually connected and are related to MTBP's contribution to tumorigenesis remains largely unclear.
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Faithful genome duplication requires appropriately controlled replication origin firing. The metazoan origin firing regulation hub Treslin/TICRR and its yeast orthologue Sld3 share the Sld3-Treslin domain and the adjacent TopBP1/Dpb11 interaction domain. We report a revised domain architecture model of Treslin/TICRR. Protein sequence analyses uncovered a conserved Ku70-homologous ß-barrel fold in the Treslin/TICRR middle domain (M domain) and in Sld3. Thus, the Sld3-homologous Treslin/TICRR core comprises its three central domains, M domain, Sld3-Treslin domain, and TopBP1/Dpb11 interaction domain, flanked by non-conserved terminal domains, the CIT (conserved in Treslins) and the C terminus. The CIT includes a von Willebrand factor type A domain. Unexpectedly, MTBP, Treslin/TICRR, and Ku70/80 share the same N-terminal domain architecture, von Willebrand factor type A and Ku70-like ß-barrels, suggesting a common ancestry. Binding experiments using mutants and the Sld3-Sld7 dimer structure suggest that the Treslin/Sld3 and MTBP/Sld7 ß-barrels engage in homotypic interactions, reminiscent of Ku70-Ku80 dimerization. Cells expressing Treslin/TICRR domain mutants indicate that all Sld3-core domains and the non-conserved terminal domains fulfil important functions during origin firing in human cells. Thus, metazoa-specific and widely conserved molecular processes cooperate during metazoan origin firing.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Origem de Replicação/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Conformação Proteica , Origem de Replicação/genética , Relação Estrutura-AtividadeRESUMO
Summary: The 10 known BRICHOS domain-containing proteins in humans have been linked to an unusually long list of pathologies, including cancer, obesity and two amyloid-like diseases. BRICHOS domains themselves have been described as intramolecular chaperones that act to prevent amyloid-like aggregation of their proteins' mature polypeptides. Using structural comparison of coevolution-based AlphaFold models and sequence conservation, we identified the Out at First (OAF) protein as a new member of the BRICHOS family in humans. OAF is an experimentally uncharacterized protein that has been proposed as a candidate biomarker for clinical management of coronavirus disease 2019 infections. Our analysis revealed how structural comparison of AlphaFold models can discover remote homology relationships and lead to a better understanding of BRICHOS domain molecular mechanism. Supplementary information: Supplementary data are available at Bioinformatics Advances online.
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Motivation: Disrupted PERCC1 gene expression causes an intractable congenital diarrhoea in infants. However, this gene's molecular mechanism is unknown and no homologous proteins have been reported. Results: Our detailed evolutionary analysis of PERCC1 sequence reveals it to be a previously unappreciated member of the YAP/TAZ/FAM181 family of homologous transcriptional regulators. Like YAP and TAZ, PERCC1 likely interacts with DNA via binding to TEA/ATTS domain transcription factors (TEADs) using its conserved interface-2 and -3 sequences. We compare the expression patterns of PERCC1 with those of YAP, TAZ, TEADs. Our report provides the identification and first in-depth bioinformatic analysis of a YAP/TAZ homologue, and a likely new regulator of the YAP/TAZ-TEAD transcriptional complex. Availability and implementation: The data underlying this article are available in UniProt Database. Supplementary information: Supplementary data are available at Bioinformatics Advances online.
RESUMO
Protein posttranslational modifications add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms, and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologs. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than 1 billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared with products of likely endosymbiont gene transfer, mitochondrial proteins, and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification.
Assuntos
Cianobactérias , Transferência Genética Horizontal , Animais , Citrulinação , Sequência Conservada , Cianobactérias/genética , Evolução Molecular , FilogeniaRESUMO
Traditional sequence analysis algorithms fail to identify distant homologies when they lie beyond a detection horizon. In this review, we discuss how co-evolution-based contact and distance prediction methods are pushing back this homology detection horizon, thereby yielding new functional insights and experimentally testable hypotheses. Based on correlated substitutions, these methods divine three-dimensional constraints among amino acids in protein sequences that were previously devoid of all annotated domains and repeats. The new algorithms discern hidden structure in an otherwise featureless sequence landscape. Their revelatory impact promises to be as profound as the use, by archaeologists, of ground-penetrating radar to discern long-hidden, subterranean structures. As examples of this, we describe how triplicated structures reflecting longin domains in MON1A-like proteins, or UVR-like repeats in DISC1, emerge from their predicted contact and distance maps. These methods also help to resolve structures that do not conform to a "beads-on-a-string" model of protein domains. In one such example, we describe CFAP298 whose ubiquitin-like domain was previously challenging to perceive owing to a large sequence insertion within it. More generally, the new algorithms permit an easier appreciation of domain families and folds whose evolution involved structural insertion or rearrangement. As we exemplify with α1-antitrypsin, coevolution-based predicted contacts may also yield insights into protein dynamics and conformational change. This new combination of structure prediction (using innovative co-evolution based methods) and homology inference (using more traditional sequence analysis approaches) shows great promise for bringing into view a sea of evolutionary relationships that had hitherto lain far beyond the horizon of homology detection.
Assuntos
Proteínas/química , Algoritmos , Animais , Humanos , Modelos Moleculares , Conformação Proteica , Análise de Sequência de Proteína/métodos , Homologia de Sequência de AminoácidosRESUMO
Fam151b is a mammalian homologue of the C. elegans menorin gene, which is involved in neuronal branching. The International Mouse Phenotyping Consortium (IMPC) aims to knock out every gene in the mouse and comprehensively phenotype the mutant animals. This project identified Fam151b homozygous knock-out mice as having retinal degeneration. We show they have no photoreceptor function from eye opening, as demonstrated by a lack of electroretinograph (ERG) response. Histological analysis shows that during development of the eye the correct number of cells are produced and that the layers of the retina differentiate normally. However, after eye opening at P14, Fam151b mutant eyes exhibit signs of retinal stress and rapidly lose photoreceptor cells. We have mutated the second mammalian menorin homologue, Fam151a, and homozygous mutant mice have no discernible phenotype. Sequence analysis indicates that the FAM151 proteins are members of the PLC-like phosphodiesterase superfamily. However, the substrates and function of the proteins remains unknown.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Retina/fisiologia , Homologia de Sequência do Ácido Nucleico , Sequência de Aminoácidos , Animais , Contagem de Células , Técnicas de Inativação de Genes , Humanos , Camundongos , Modelos Moleculares , Mutação , Células Fotorreceptoras de Vertebrados/citologia , Conformação Proteica , Retina/citologiaRESUMO
SUMMARY: CPLANE is a protein complex required for assembly and maintenance of primary cilia. It contains several proteins, such as INTU, FUZ, WDPCP, JBTS17 and RSG1 (REM2- and RAB-like small GTPase 1), whose genes are mutated in ciliopathies. Using two contrasting evolutionary analyses, coevolution-based contact prediction and sequence conservation, we first identified the INTU/FUZ heterodimer as a novel member of homologous HerMon (Hermansky-Pudlak syndrome and MON1-CCZ1) complexes. Subsequently, we identified homologous Longin domains that are triplicated in each of these six proteins (MON1A, CCZ1, HPS1, HPS4, INTU and FUZ). HerMon complexes are known to be Rab effectors and Rab GEFs (Guanine nucleotide Exchange Factors) that regulate vesicular trafficking. Consequently, INTU/FUZ, their homologous complex, is likely to act as a GEF during activation of Rab GTPases involved in ciliogenesis. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Transdução de Sinais , Hexosaminidase A , Transporte Proteico , Proteínas rab de Ligação ao GTPRESUMO
Purpose: We previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modeled these mutations in mice and characterized the mutant eye phenotypes of these and Rwhs. Methods: The Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry, and electron microscopy techniques were used to study adult eyes. Results: An I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P, are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganized outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions. Conclusions: Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localized disruption of the laminar structure of the photoreceptors.
Assuntos
Proteínas de Membrana/genética , Microftalmia/genética , Mutação de Sentido Incorreto , Células Fotorreceptoras de Vertebrados/patologia , Doenças Retinianas/genética , Animais , Comprimento Axial do Olho/patologia , Sistemas CRISPR-Cas , Eletrorretinografia , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microftalmia/patologia , Microscopia Eletrônica de Transmissão , Oftalmoscopia , Reação em Cadeia da Polimerase , Doenças Retinianas/patologiaRESUMO
Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)-interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19-cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/metabolismo , Biologia Computacional/métodos , Ciclina C/genética , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/metabolismo , Quinase 8 Dependente de Ciclina/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Xia-Gibbs syndrome (XGS: OMIM # 615829) results from de novo truncating mutations within the AT-Hook DNA Binding Motif Containing 1 gene (AHDC1). To further define the phenotypic and molecular spectrum of this disorder, we established an XGS Registry and recruited patients from a worldwide pool of approximately 60 probands. Additional de novo truncating mutations were observed among 25 individuals, extending both the known number of mutation sites and the range of positions within the coding region that were sensitive to alteration. Detailed phenotypic examination of 20 of these patients via clinical records review and data collection from additional surveys showed a wider age range than previously described. Data from developmental milestones showed evidence for delayed speech and that males were more severely affected. Neuroimaging from six available patients showed an associated thinning of the corpus callosum and posterior fossa cysts. An increased risk of both scoliosis and seizures relative to the population burden was also observed. Data from a modified autism screening tool revealed that XGS shares significant overlap with autism spectrum disorders. These details of the phenotypic heterogeneity of XGS implicate specific genotype/phenotype correlations and suggest potential clinical management guidelines.
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Transtorno do Espectro Autista/etiologia , Proteínas de Ligação a DNA/genética , Deficiências do Desenvolvimento/etiologia , Mutação , Criança , Cognição/fisiologia , Corpo Caloso/diagnóstico por imagem , Corpo Caloso/patologia , Face/anormalidades , Feminino , Humanos , Masculino , Linhagem , Fenótipo , Sistema de Registros , Convulsões/etiologia , Síndrome , Adulto JovemRESUMO
Summary: The molecular functions of TMEM132 genes remain poorly understood and under-investigated despite their mutations associated with non-syndromic hearing loss, panic disorder and cancer. Here we show the full domain architecture of human TMEM132 family proteins solved using in-depth sequence and structural analysis. We reveal them to be five previously unappreciated cell adhesion molecules whose domain architecture has an early holozoan origin prior to the emergence of choanoflagellates and metazoa. The extra-cellular portions of TMEM132 proteins contain five conserved domains including three tandem immunoglobulin domains, and a cohesin domain homologue, the first such domain found in animals. These findings strongly predict a cellular adhesion function for TMEM132 family, connecting the extracellular medium with the intracellular actin cytoskeleton. Contact: luis.sanchez-pulido@igmm.ed.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online.
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Moléculas de Adesão Celular/química , Proteínas de Membrana/química , Família Multigênica , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Humanos , Domínios de Imunoglobulina , Proteínas de Membrana/genética , Domínios Proteicos , Análise de Sequência de Proteína , CoesinasRESUMO
In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20) activates separase and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2, sister chromatids remain attached through centromeric/pericentromeric cohesin. We show here that, by promoting proteolysis of cyclins and Cdc25B at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1) delays the increase in Cdk1 activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase, an event crucial for the efficient resolution of chiasmata.
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Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos , Meiose , Animais , Subunidade Apc2 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Aurora Quinase B/metabolismo , Aurora Quinase C/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas Cdc20/fisiologia , Proteínas Cdh1/metabolismo , Centrômero , Proteínas Cromossômicas não Histona/metabolismo , Feminino , Centro Germinativo , Masculino , Camundongos , Camundongos Knockout , Modelos Teóricos , Separase/metabolismo , Fosfatases cdc25/fisiologia , CoesinasRESUMO
BACKGROUND: TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains. METHODS: We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements. RESULTS: Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila). CONCLUSIONS: Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Proteínas Associadas aos Microtúbulos/química , Proteínas Nucleares/química , Fosfoproteínas/química , Proteínas de Xenopus/química , Sequência de Aminoácidos , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dicroísmo Circular , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2- cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Reparo do DNA , DNA/metabolismo , Dictyostelium/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cisplatino/farmacologia , Dano ao DNA , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dictyostelium/efeitos dos fármacos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.
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Proteínas de Ciclo Celular/genética , Microtia Congênita/genética , Craniossinostoses/genética , Transtornos do Crescimento/genética , Micrognatismo/genética , Mutação , Patela/anormalidades , Adolescente , Adulto , Alelos , Processamento Alternativo/genética , Sequência de Aminoácidos , Âmnio/citologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Criança , Pré-Escolar , Análise Mutacional de DNA , Replicação do DNA , Exoma/genética , Éxons/genética , Feminino , Estudos de Associação Genética , Humanos , Masculino , Modelos Moleculares , Conformação Proteica , Síndrome , Adulto JovemRESUMO
UNLABELLED: Vasohibin-1 and Vasohibin-2 regulate angiogenesis, tumour growth and metastasis. Their molecular functions, however, were previously unknown, in large part owing to their perceived lack of homology to proteins of known structure and function. To identify their functional amino acids and domains, their molecular activity and their evolutionary history, we undertook an in-depth analysis of Vasohibin sequences. We find that Vasohibin proteins are previously undetected members of the transglutaminase-like cysteine protease superfamily, and all possess a non-canonical Cys-His-Ser catalytic triad. We further propose a calcium-dependent activation mechanism for Vasohibin proteins. These findings can now be used to design constructs for protein structure determination and to develop enzyme inhibitors as angiogenic regulators to treat metastasis and tumour growth. CONTACT: luis.sanchezpulido@dpag.ox.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Biocatálise , Sequência de Aminoácidos , Cisteína , Cisteína Proteases , TransglutaminasesRESUMO
Oxidative stress is a pathological feature of many neurological disorders; therefore, utilizing proteins that are protective against such cellular insults is a potentially valuable therapeutic approach. Oxidation resistance 1 (OXR1) has been shown previously to be critical for oxidative stress resistance in neuronal cells; deletion of this gene causes neurodegeneration in mice, yet conversely, overexpression of OXR1 is protective in cellular and mouse models of amyotrophic lateral sclerosis. However, the molecular mechanisms involved are unclear. OXR1 contains the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) domain, a motif present in a family of proteins including TBC1 domain family member 24 (TBC1D24), a protein mutated in a range of disorders characterized by seizures, hearing loss, and neurodegeneration. The TLDc domain is highly conserved across species, although the structure-function relationship is unknown. To understand the role of this domain in the stress response, we carried out systematic analysis of all mammalian TLDc domain-containing proteins, investigating their expression and neuroprotective properties in parallel. In addition, we performed a detailed structural and functional study of this domain in which we identified key residues required for its activity. Finally, we present a new mouse insertional mutant of Oxr1, confirming that specific disruption of the TLDc domain in vivo is sufficient to cause neurodegeneration. Our data demonstrate that the integrity of the TLDc domain is essential for conferring neuroprotection, an important step in understanding the functional significance of all TLDc domain-containing proteins in the cellular stress response and disease.