RESUMO
Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity.
Assuntos
Proteínas de Transporte/imunologia , Proteína DEAD-box 58/imunologia , Proteínas de Ligação a DNA/imunologia , Imunidade Inata , Mitocôndrias/imunologia , Proteínas Mitocondriais/imunologia , Complexos Multiproteicos/imunologia , Fosfoproteínas Fosfatases/imunologia , Transdução de Sinais/imunologia , ATPases Associadas a Diversas Atividades Celulares , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteína DEAD-box 58/genética , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Complexos Multiproteicos/genética , Fosfoproteínas Fosfatases/genética , Receptores Imunológicos , Transdução de Sinais/genética , Proteínas com Motivo Tripartido , Células Vero , Viroses/genética , Viroses/imunologia , Vírus/genética , Vírus/imunologiaRESUMO
PCV2 belongs to the genus Circovirus in the family Circoviridae, whose genome is replicated by rolling circle replication (RCR). PCV2 Rep is a multifunctional enzyme that performs essential functions at multiple stages of viral replication. Rep is responsible for nicking and ligating single-stranded DNA and unwinding double-stranded DNA (dsDNA). However, the structure and function of the Rep are still poorly understood, which significantly impedes viral replication research. This study successfully resolved the structure of the PCV2 Rep ATPase domain (PRAD) using X-ray crystallography. Homologous structure search revealed that Rep belonged to the superfamily 3 (SF3) helicase, and multiple conserved residues were identified during sequence alignment with SF3 family members. Simultaneously, a hexameric PRAD model was generated for analysing characteristic structures and sites. Mutation of the conserved site and measurement of its activity showed that the hallmark motifs of the SF3 family influenced helicase activity by affecting ATPase activity and ß-hairpin just caused the loss of helicase activity. The structural and functional analyses of the PRAD provide valuable insights for future research on PCV2 replication and antiviral strategies.
Assuntos
Circovirus , Suínos , Animais , Circovirus/genética , Adenosina Trifosfatases/genética , Cristalografia por Raios X , DNA Helicases/genética , Replicação do DNARESUMO
Organelle contact sites are multifunctional platforms for maintaining cellular homeostasis. Alternations of the mitochondria-associated membranes (MAM), one of the organelle contact sites where the endoplasmic reticulum (ER) is tethered to the mitochondria, have been involved in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the detailed mechanisms through which MAM integrity is disrupted in ALS have not been fully elucidated. Here, we examined whether AAA ATPase domain-containing protein 3A (ATAD3A), a mitochondrial membrane AAA ATPase accumulating at the MAM, is involved in ALS. We found that sigma-1 receptor (σ1R), an ER-resident MAM protein causative for inherited juvenile ALS, required ATAD3A to maintain the MAM. In addition, σ1R retained ATAD3A as a monomer, which is associated with an inhibition of mitochondrial fragmentation. ATAD3A dimerization and mitochondrial fragmentation were significantly induced in σ1R-deficient or SOD1-linked ALS mouse spinal cords. Overall, these observations indicate that MAM induction by σ1R depends on ATAD3A and that σ1R maintains ATAD3A as a monomer to inhibit mitochondrial fragmentation. Our findings suggest that targeting σ1R-ATAD3A axis would be promising for a novel therapeutic strategy to treat mitochondrial dysfunction in neurological disorders, including ALS.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Camundongos , Animais , Esclerose Lateral Amiotrófica/metabolismo , Mitocôndrias/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Doenças Neurodegenerativas/metabolismo , Proteínas Mitocondriais/metabolismo , Receptor Sigma-1RESUMO
The Snf2 proteins, comprising 53 different enzymes in humans, belong to the SF2 family. Many Snf2 enzymes possess chromatin-remodeling activity, requiring a functional ATPase domain consisting of conserved motifs named Q and I-VII. These motifs form two recA-like domains, creating an ATP-binding pocket. Little is known about the function of the conserved motifs in chromatin-remodeling enzymes. Here, we characterized the function of the Q and I (Walker I) motifs in hBRG1 (SMARCA4). The motifs are in close proximity to the bound ATP, suggesting a role in nucleotide binding and/or hydrolysis. Unexpectedly, when substituting the conserved residues Gln758 (Q motif) or Lys785 (I motif) of both motifs, all variants still bound ATP and exhibited basal ATPase activity similar to that of wildtype BRG1 (wtBRG1). However, all mutants lost the nucleosome-dependent stimulation of the ATPase domain. Their chromatin-remodeling rates were impaired accordingly, but nucleosome binding was retained and still comparable with that of wtBRG1. Interestingly, a cancer-relevant substitution, L754F (Q motif), displayed defects similar to the Gln758 variant(s), arguing for a comparable loss of function. Because we excluded a mutual interference of ATP and nucleosome binding, we postulate that both motifs stimulate the ATPase and chromatin-remodeling activities upon binding of BRG1 to nucleosomes, probably via allosteric mechanisms. Furthermore, mutations of both motifs similarly affect the enzymatic functionality of BRG1 in vitro and in living cells. Of note, in BRG1-deficient H1299 cells, exogenously expressed wtBRG1, but not BRG1 Q758A and BRG1 K785R, exhibited a tumor suppressor-like function.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , DNA Helicases/genética , Humanos , Modelos Moleculares , Mutação , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Variants in the Structural Maintenance of Chromosomes flexible Hinge Domain-containing protein 1 (SMCHD1) can cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) and the unrelated Bosma arhinia microphthalmia syndrome (BAMS). In FSHD2, pathogenic variants are found anywhere in SMCHD1 while in BAMS, pathogenic variants are restricted to the extended ATPase domain. Irrespective of the phenotypic outcome, both FSHD2-associated and BAMS-associated SMCHD1 variants result in quantifiable local DNA hypomethylation. We compared FSHD2, BAMS and non-pathogenic SMCHD1 variants to derive genotype-phenotype relationships. METHODS: Examination of SMCHD1 variants and methylation of the SMCHD1-sensitive FSHD locus DUX4 in 187 FSHD2 families, 41 patients with BAMS and in control individuals. Analysis of variants in a three-dimensional model of the ATPase domain of SMCHD1. RESULTS: DUX4 methylation analysis is essential to establish pathogenicity of SMCHD1 variants. Although the FSHD2 mutation spectrum includes all types of variants covering the entire SMCHD1 locus, missense variants are significantly enriched in the extended ATPase domain. Identification of recurrent variants suggests disease-specific residues for FSHD2 and in BAMS, consistent with a largely disease-specific localisation of variants in SMCHD1. CONCLUSIONS: The localisation of missense variants within the ATPase domain of SMCHD1 may contribute to the differences in phenotypic outcome.
Assuntos
Atresia das Cóanas/genética , Proteínas Cromossômicas não Histona/genética , Microftalmia/genética , Distrofia Muscular Facioescapuloumeral/genética , Nariz/anormalidades , Adenosina Trifosfatases/genética , Metilação de DNA , Feminino , Variação Genética , Humanos , Masculino , Mutação , Mutação de Sentido Incorreto , Domínios ProteicosRESUMO
Δlon mutant of Escherichia coli becomes hypersensitive to DNA damaging agents and over-produce capsule due to stabilization of the Lon substrates, namely, SulA and RcsA, respectively. These phenotypes were earlier found to be suppressed in Δlon ssrA::cat/pUC4 K and Δlon faa (DnaJ, G232D) strains, called as "Alp" strains. We observed that a plasmid carrying an E. coli chromosomal fragment harboring few genes, a heat shock gene htpY and a portion of dnaK capable of encoding truncated N-terminal ATPase domain (244 aa) could suppress lon mutant phenotypes. Deletion of htpY did not affect the efficiency of suppression. Clones expressing DnaK' (244 aa) peptide alone could suppress both Δlon phenotypes in copy number dependent manner. Inactivation of clpQ did not affect the MMSR phenotype of Δlon strain carrying dnaK' clones indicating that ClpYQ protease does not degrade SulA. We hypothesize that the high levels of defective DnaK'-DnaJ chaperone complex formed in these strains might lead to aggregation of SulA and RcsA and, thereby the suppression of Δlon phenotypes. Systematic deletion analysis of dnaK' revealed that, â¼220 aa N-terminal DnaK peptide is required for suppression of cps-lac over-expression and â¼169 aa peptide is enough for the suppression of MMSS phenotype of Δlon mutant.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Protease La/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Expressão Gênica , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Metanossulfonato de Metila/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , Protease La/deficiência , Deleção de SequênciaRESUMO
Therapeutic macromolecules are internalized into the cell by either clathrin-mediated endocytosis (CME) or clathrin-independent endocytosis (CIE). Although some chaperone proteins play an essential role in CME (e.g. Hsc70 in clathrin uncoating), relatively few of these proteins are functionally involved in CIE. We previously revealed a role for the mitochondrial chaperone protein GRP75 in heparan sulfate proteoglycan (HSPG)-mediated, membrane raft-associated macromolecule endocytosis. However, the mechanism underlying this process remains unclear. In this study, using a mitochondrial signal peptide-directed protein trafficking expression strategy, we demonstrate that wild-type GRP75 expression enhanced the uptakes of HSPG and CIE marker cholera toxin B subunit but impaired the uptake of CME marker transferrin. The endocytosis regulation function of GRP75 is largely mediated by its subcellular location in mitochondria and is essentially determined by its ATPase domain. Interestingly, the mitochondrial expression of GRP75 or its ATPase domain significantly stimulates increases in both RhoA and Cdc42 activation, remarkably induces stress fibers and enhances filopodia formation, which collectively results in the promotion of CIE, but the inhibition of CME. Furthermore, silencing of Cdc42 or RhoA impaired the ability of GRP75 overexpression to increase CIE. Therefore, these results suggest that endocytosis vesicle enrichment of GRP75 by mitochondria trafficking upregulates CIE through an actin cytoskeleton reorganization mechanism mediated by the concurrent activation of Cdc42 and RhoA. This finding provides novel insight into organelle-derived chaperone signaling and the regulation of different endocytosis pathways in cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Clatrina/metabolismo , Endocitose , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Biomarcadores/metabolismo , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Camundongos , Mitocôndrias/metabolismo , Células NIH 3T3 , Plasmídeos/metabolismo , Polimerização , Sinais Direcionadores de ProteínasRESUMO
Four new crystal structures of the ATPase domain of the GyrB subunit of Escherichia coli DNA gyrase have been determined. One of these, solved in the presence of K(+), is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K(+) ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na(+) ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.
Assuntos
Adenosina Trifosfatases/química , DNA Girase/química , Escherichia coli/enzimologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cátions Monovalentes/metabolismo , Cristalografia por Raios X , DNA Girase/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Modelos Moleculares , Potássio/metabolismo , Conformação Proteica , Sódio/metabolismoRESUMO
Gyrase ATPase domain, the pharmaceutical underexploited segment of DNA gyrase, the sole Type II topoisomerase present in Mycobacterium tuberculosis represents an attractive target for anti-tubercular drug discovery. Here we report, the development of a novel series of MTB DNA gyraseB inhibitor identified through a medium throughput screening (MTS) of BITS in-house chemical library (3000 compounds). The MTS hit was further remodeled by chemical synthesis to identify the most potent analogue 27 exhibiting an in vitro gyrB inhibitory IC50 of 0.15 µM. The series also demonstrated well correlating gyrase super coiling activity and in vitro anti-mycobacterial potency against MTB H37Rv strain. Furthermore the compounds displayed good safety profile in their subsequent cytotoxicity and hERG toxicity evaluations, to be worked out from a pharmaceutical point of view as potential anti-tubercular agents.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antituberculosos/farmacologia , DNA Girase/química , Tiazóis/farmacologia , Inibidores da Topoisomerase II/farmacologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Animais , Antituberculosos/química , DNA Girase/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Humanos , Modelos Moleculares , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tiazóis/química , Inibidores da Topoisomerase II/química , Peixe-ZebraRESUMO
Type II topoisomerase utilizes the energy from ATP hydrolysis to alter DNA topology during genome replication and transcription. The ATPase domain of this enzyme is required for ATP hydrolysis and plays a crucial role in coupling DNA binding and ATP turnover with the DNA strand passage reaction. The African swine fever virus (ASFV) specifically encodes a topoisomerase II (topo II), which is critical for viral replication and an attractive target for antiviral development. Here, we present a high-resolution crystal structure of the ASFV topo II ATPase domain complexed with the substrate analog AMPPNP. Structural comparison reveals that the ASFV topo II ATPase domain shares a conserved overall structure with its homologs from eukaryotes and prokaryotes but also has three characteristic regions, including the intra-molecular interface formed by the ATP-lid and QTK loop as well as helix α9, the K-loop in the transducer domain, and the antennae-like α-helix at the ATP binding domain. Mutating the key residues within these three regions impairs or abolishes the basal and DNA-stimulated ATPase activities and reduces or eliminates the relaxation activity of the holoenzyme. Our data indicate that all three regions are functionally important for the ATPase and relaxation activities and strongly suggest that ATP hydrolysis, DNA binding, and strand passage are highly coupled and managed by the allosteric coordination of multiple domains of the type II topoisomerase. Moreover, we find a promising druggable pocket in the dimeric interface of the ASFV topo II ATPase domain, which will benefit future anti-ASFV drug development. IMPORTANCE: The ATPase domain of type II topoisomerase provides energy by hydrolyzing ATP and coordinates with the DNA-binding/cleavage domain to drive and control DNA transport. The precise molecular mechanisms of how these domains respond to DNA binding and ATP hydrolysis signals and communicate with each other remain elusive. We determine the first high-resolution crystal structure of the ATPase domain of African swine fever virus (ASFV) topo II in complex with AMPPNP and biochemically investigate its function in ATPase and DNA relaxation activities. Importantly, we find that mutations at three characteristic regions of the ASFV ATPase domain produce parallel effects on the basal/DNA-stimulated ATPase and relaxation activities, implying the tight coupling of the ATP hydrolysis and strand passage process. Therefore, our data provide important implications for understanding the strand passage mechanism of the type II topoisomerase and the structural basis for developing ATPase domain-targeting antivirals against ASFV.
Assuntos
Vírus da Febre Suína Africana , Suínos , Animais , Vírus da Febre Suína Africana/genética , Adenilil Imidodifosfato/farmacologia , DNA Topoisomerases Tipo II/genética , DNA/metabolismo , Adenosina Trifosfatases/metabolismoRESUMO
Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47(C1) and Mtb-GyrB47(C2), have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P21 and diffracted to resolutions of 2.9 and 3.3 Å, respectively.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , DNA Girase/química , Mycobacterium tuberculosis/enzimologia , Adenosina Trifosfatases/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , DNA Girase/isolamento & purificaçãoRESUMO
BACKGROUND: The prospective uses of tryptanthrin and its analogues in cancer chemotherapy are well known, and they are also predicated on their capacity to reverse drug resistance in cancer therapy. OBJECTIVE: The current project entails developing a novel hybrid analogue that includes modifying the tryptanthrin molecule at the C-6 carbonyl position and is expected to exhibit substantial anticancer action. METHODS: In the ATPase domain of human topoisomerase II, a series of 162 substituted Schiff base analogues of tryptanthrin were developed, and molecular docking experiments were done using Gold 5.1 software interfaced with Hermes 1.6.2. (PDB ID: 1ZXM). RESULTS: Most of the compounds were found to have Goldscore above 100 and formed interactions with the residues like ASN91, ALA92, ASN95, ARG98, ASN120, ILE125, ILE141, PHE142, SER149, THR215, and ILE217. Compound RK-149 had highest Goldscore of 132.59, forming an interaction with ASN91 but had a lesser Goldscore as compared to the standard drug etoposide and had a better score than tryptanthrin. CONCLUSION: The nitrogen in the imine bond of the proposed compounds is responsible for significant interactions, demonstrating their anticancer potential.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Simulação de Acoplamento Molecular , Inibidores da Topoisomerase II/farmacologia , Inibidores da Topoisomerase II/química , Antineoplásicos/química , Quinazolinas/química , DNA Topoisomerases Tipo II/metabolismo , Neoplasias/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
Potato leafroll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the CP with ATPase domain exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. In addition, molecular docking study reveals that the PLRV CP protein has ATP-binding pocket at the interface of each monomer. This further confirms that knockdown of the CP harboring ATP-binding domain could hamper the process of viral genome packaging and assembly. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.
RESUMO
Human topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.
Assuntos
Adenosina Trifosfatases , Trifosfato de Adenosina , DNA Topoisomerases Tipo II , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , Humanos , Hidrólise , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
ATAD2 is a promising oncoprotein with tumor-promoting functions in many cancers. It is a valid cancer drug-target and a potential cancer-biomarker for multiple malignancies. As a cancer/testis antigen (CTA), ATAD2 could also be a probable candidate for immunotherapy. It is a unique CTA that belongs to both AAA+ ATPase and bromodomain family proteins. Since 2007, several research groups have been reported on the pleiotropic oncogenic functions of ATAD2 in diverse signaling pathways, including Rb/E2F-cMyc pathway, steroid hormone signaling pathway, p53 and p38-MAPK-mediated apoptotic pathway, AKT pathway, hedgehog signaling pathway, HIF1α signaling pathway, and Epithelial to Mesenchymal Transition (EMT) pathway in various cancers. In all these pathways, ATAD2 participates in chromatin dynamics, DNA replication, and gene transcription, demonstrating its role as an epigenetic reader and transcription factor or coactivator to promote tumorigenesis. However, despite the progress, an overall mechanism of ATAD2-mediated oncogenesis in diverse origin is elusive. In this review, we summarize the accumulated evidence to envision the overall ATAD2 signaling networks during carcinogenesis and highlight the area where missing links await further research. Besides, the structure-function aspect of ATAD2 is also discussed. Since the efforts have already been initiated to explore targeted drug molecules and RNA-based therapeutic alternatives against ATAD2, their potency and prospects have been elucidated. Together, we believe this is a well-rounded review on ATAD2, facilitating a new drift in ATAD2 research, essential for its clinical implication as a biomarker and/or cancer drug-target.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Terapia de Alvo Molecular , Neoplasias/patologia , ATPases Associadas a Diversas Atividades Celulares/genética , Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismoRESUMO
In yeast Saccharomyces cerevisiae, the Dhh1 protein, a member of the DEAD-box RNA helicase, stimulates Dcp2/Dcp1-mediated mRNA decapping and functions as a general translation repressor. Dhh1 also positively regulates translation of a selected set of mRNAs, including Ste12, a transcription factor for yeast mating and pseudohyphal growth. Given the diverse functions of Dhh1, we investigated whether the putative phosphorylation sites or the conserved motifs for the DEAD-box RNA helicases were crucial in the regulatory roles of Dhh1 during pseudohyphal growth. Mutations in the ATPase A or B motif (DHH1-K96R or DHH1-D195A) showed significant defects in pseudohyphal colony morphology and agar invasive phenotypes. The N-terminal phospho-mimetic mutation, DHH1-T16E, showed defects in pseudohyphal phenotypes. Decreased levels of Ste12 protein were also observed in these pseudohyphal-defective mutant cells under filamentous-inducing low nitrogen conditions. We suggest that the ATPase motifs and the Thr16 phosphorylation site of Dhh1 are crucial to its regulatory roles in pseudohyphal growth under low nitrogen conditions.
Assuntos
Adenosina Trifosfatases/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Hifas/genética , Fosforilação , Biossíntese de Proteínas/genética , Domínios Proteicos/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
The ATP-dependent chromatin remodeler CSB is implicated in a variety of different DNA repair mechanisms, including transcription-coupled nucleotide excision repair (TC-NER), base excision repair and DNA double strand break (DSB) repair. However, how CSB is regulated in these various repair processes is not well understood. Here we report that the first 30 amino acids of CSB along with two phosphorylation events on S10 and S158, previously reported to be required for CSB function in homologous recombination (HR)-mediated repair, are dispensable for repairing UV-induced DNA damage, suggesting that the regulation of CSB in these two types of repair are carried out by distinct mechanisms. In addition, we show that although the central ATPase domain of CSB is engaged in interactions with both the N- and C-terminal regions, these interactions are disrupted following UV-induced DNA damage. The UV-induced disengagement of the C-terminal region of CSB from the ATPase domain requires two conserved amino acids W1486 and L1488, which are thought to contribute to the hydrophobic core formation of the winged helix domain (WHD) at its C-terminus. Failure to undergo UV-induced dissociation of the C-terminal region of CSB from the ATPase domain is associated with impairment in its UV-induced chromatin association, its UV-induced post-translational modification as well as cell survival. Collectively, these findings suggest that UV-induced dissociation of CSB domain interactions is a necessary step in repairing UV-induced DNA damage and that the WHD of CSB plays a key role in this dissociation.
Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Domínios Proteicos , Raios Ultravioleta , Adenosina Trifosfatases , Linhagem Celular , Síndrome de Cockayne , DNA/metabolismo , DNA/efeitos da radiação , DNA Helicases/efeitos da radiação , Enzimas Reparadoras do DNA/efeitos da radiação , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/efeitos da radiação , Processamento de Proteína Pós-TraducionalRESUMO
The innate immune sensor retinoic acid-inducible gene I (RIG-I) detects cytosolic viral RNA and requires a conformational change caused by both ATP and RNA binding to induce an active signaling state and to trigger an immune response. Previously, we showed that ATP hydrolysis removes RIG-I from lower-affinity self-RNAs (
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Doenças da Aorta/genética , Proteína DEAD-box 58/química , Hipoplasia do Esmalte Dentário/genética , Metacarpo/anormalidades , Doenças Musculares/genética , Odontodisplasia/genética , Osteoporose/genética , Calcificação Vascular/genética , Proteínas AAA/química , Proteínas AAA/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Doenças da Aorta/enzimologia , Doenças da Aorta/patologia , Citosol/virologia , Proteína DEAD-box 58/genética , Hipoplasia do Esmalte Dentário/enzimologia , Hipoplasia do Esmalte Dentário/patologia , Humanos , Hidrólise , Imunidade Inata/genética , Metacarpo/enzimologia , Metacarpo/patologia , Doenças Musculares/enzimologia , Doenças Musculares/patologia , Mutação , Odontodisplasia/enzimologia , Odontodisplasia/patologia , Osteoporose/enzimologia , Osteoporose/patologia , Ligação Proteica , Conformação Proteica , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética , Receptores Imunológicos , Calcificação Vascular/enzimologia , Calcificação Vascular/patologiaRESUMO
We developed an insertion sequence transposition detection system called the "jumping cat assay" and applied it to the Bacillus subtilis chromosome using IS256Bsu1 derived from B. subtilis natto. The high frequency of transposition enabled us to explore host factors; combining the assay and genetic analyses revealed that recA is essential for the transposition of IS256Bsu1. Detailed analyses using various domain mutants of recA demonstrated that this essentiality is not related to the function of recA in homologous recombination. Instead, the ATP binding and hydrolysis function seemed to be crucial for IS transposition. To elucidate the role of recA, we focused on the muB gene of the enterobacteriophage Mu. Based on information from the NCBI Conserved Domain Database, both MuB and RecA belong to the P-loop dNTPase superfamily. Further experiments revealed that muB complements the transposition-defective phenotype of a recA deletant, although it could not rescue UV sensitivity. These results suggest that recA shares a common function with muB that helps the transposition of IS256Bsu1 in B. subtilis.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Recombinases Rec A/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga , Mutação , Ligação Proteica , Recombinases Rec A/genética , Proteínas Virais/genéticaRESUMO
Dhh1 and Dhh1 homologues (RCK/p54/DDX6) are members of the DEAD-box protein family of RNA helicases. These proteins display conserved sequence motifs for ATPase and RNA binding activities. Dhh1 is a component of the P-bodies (processing bodies) of mRNA granules and functions as an mRNA decapping activator in Saccharomyces cerevisiae. Dhh1 also contributes to gene-specific regulation during yeast mating. The dhh1 deletion mutation results in a significant decrease in the expression of Ste12, a mating-specific transcription factor, showing severe mating defects. Here, we introduced amino-acid substitution mutations in the ATPase and RNA binding domains of Dhh1 and also constructed a deletion of 79 amino acids at the Q/P-rich C-terminal region. The mutations in ATPase A and B motif (K96R, D195A) and C-terminus deletion showed reduced levels of mating efficiency as well as Ste12 protein expression. The Q/P-rich C-terminal region of Dhh1 was dispensable for growth at nonpermissive temperature 37°C but appeared to play an important role in regulating the Ste12 protein expression and mating processes. The P-body accumulation induced by treatment with α-mating factor required ATPase, RNA-binding and the Q/P-rich C-terminal domains of Dhh1.