RESUMO
Chalcone is a common simple fragment of natural products with anticancer activity. In a previous study, the research group discovered a series of chalcone derivatives with stronger anticancer activities. To find better anticancer drugs, novel chalcone derivatives A1-A14, B1-B14 have continuously been designed and synthesized. The antiproliferative activity of these compounds against breast cancer cells (MCF-7) was investigated by the Cell Counting Kit-8 (CCK-8) method with 5-fluorouracil (5-Fu) as the control drug. The results showed that compound A14 exhibited excellent antiproliferative ability compared to the control drug 5-Fu. Scratch experiments and cloning experiments further confirmed that compound A14 could inhibit the proliferation and colony formation activity of MCF-7 cells. In addition, molecular docking primarily explains the interaction between compound and protein. These results suggested that compound A14 could be a promising chalcone derivative for further anti-breast cancer research.
Assuntos
Antineoplásicos , Neoplasias da Mama , Chalcona , Chalconas , Humanos , Feminino , Chalcona/farmacologia , Chalconas/farmacologia , Chalconas/uso terapêutico , Relação Estrutura-Atividade , Simulação de Acoplamento Molecular , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias da Mama/tratamento farmacológico , Antineoplásicos/uso terapêutico , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Estrutura Molecular , Linhagem Celular TumoralRESUMO
The NAD+-dependent deacetylase Sirt1 has been implicated in the prevention of many age-related diseases, including cancer, type 2 diabetes, and cardiovascular disease. Resveratrol, a plant polyphenol, exhibits antiaging, antitumor, and vascular protection effects by activating Sirt1. However, the molecular mechanism of Sirt1 activation as induced by resveratrol remains unclear. By knockdown/rescue experiments, fluorometric Sirt1 activity assay, immunoprecipitation, and pull-down assays, we identify here that the tumor suppressor LKB1 (liver kinase B1) as a direct activator of Sirt1 elicited by resveratrol. Resveratrol promotes the binding between LKB1 and Sirt1, which we first reported, and this binding leads to LKB1-mediated phosphorylation of Sirt1 at three different serine residues in the C terminus of Sirt1. Mechanistically, LKB1-mediated phosphorylation increases intramolecular interactions in Sirt1, such as the binding of the C terminus to the deacetylase core domain, thereby eliminating DBC1 (Deleted in Breast Cancer 1, Sirt1 endogenous inhibitor) inhibition and promoting Sirt1-substrate interaction. Functionally, LKB1-dependent Sirt1 activation increases mitochondrial biogenesis and respiration through deacetylation and activation of the transcriptional coactivator PGC-1α. These results identify Sirt1 as a context-dependent target of LKB1 and suggest that a resveratrol-stimulated LKB1-Sirt1 pathway plays a vital role in mitochondrial metabolism, a key physiological process that contributes to numerous age-related diseases.
Assuntos
Resveratrol/farmacologia , Sirtuína 1 , Acetilação/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Biogênese de Organelas , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Protein lysine acetylation regulation is an important molecular mechanism for regulating cellular processes and plays critical physiological and pathological roles in cancers and diseases. Although massive acetylation sites have been identified through experimental identification and high-throughput proteomics techniques, their enzyme-specific regulation remains largely unknown. Here, we developed the deep learning-based protein lysine acetylation modification prediction (Deep-PLA) software for histone acetyltransferase (HAT)/histone deacetylase (HDAC)-specific acetylation prediction based on deep learning. Experimentally identified substrates and sites of several HATs and HDACs were curated from the literature to generate enzyme-specific data sets. We integrated various protein sequence features with deep neural network and optimized the hyperparameters with particle swarm optimization, which achieved satisfactory performance. Through comparisons based on cross-validations and testing data sets, the model outperformed previous studies. Meanwhile, we found that protein-protein interactions could enrich enzyme-specific acetylation regulatory relations and visualized this information in the Deep-PLA web server. Furthermore, a cross-cancer analysis of acetylation-associated mutations revealed that acetylation regulation was intensively disrupted by mutations in cancers and heavily implicated in the regulation of cancer signaling. These prediction and analysis results might provide helpful information to reveal the regulatory mechanism of protein acetylation in various biological processes to promote the research on prognosis and treatment of cancers. Therefore, the Deep-PLA predictor and protein acetylation interaction networks could provide helpful information for studying the regulation of protein acetylation. The web server of Deep-PLA could be accessed at http://deeppla.cancerbio.info.
Assuntos
Aprendizado Profundo , Histona Desacetilases/metabolismo , Lisina/metabolismo , Neoplasias/metabolismo , Acetilação , Conjuntos de Dados como Assunto , Humanos , Internet , Neoplasias/enzimologia , Neoplasias/patologiaRESUMO
The centromere is an evolutionarily conserved eukaryotic protein machinery essential for precision segregation of the parental genome into two daughter cells during mitosis. Centromere protein A (CENP-A) organizes the functional centromere via a constitutive centromere-associated network composing the CENP-T complex. However, how CENP-T assembles onto the centromere remains elusive. Here we show that CENP-T binds directly to Holliday junction recognition protein (HJURP), an evolutionarily conserved chaperone involved in loading CENP-A. The binding interface of HJURP was mapped to the C terminus of CENP-T. Depletion of HJURP by CRISPR-elicited knockout minimized recruitment of CENP-T to the centromere, indicating the importance of HJURP in CEPN-T loading. Our immunofluorescence analyses indicate that HJURP recruits CENP-T to the centromere in S/G2 phase during the cell division cycle. Significantly, the HJURP binding-deficient mutant CENP-T6L failed to locate to the centromere. Importantly, CENP-T insufficiency resulted in chromosome misalignment, in particular chromosomes 15 and 18. Taken together, these data define a novel molecular mechanism underlying the assembly of CENP-T onto the centromere by a temporally regulated HJURP-CENP-T interaction.
Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase G2/fisiologia , Fase S/fisiologia , Centrômero/genética , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , HumanosRESUMO
During cell division, accurate chromosome segregation is tightly regulated by Polo-like kinase 1 (PLK1) and opposing activities of Aurora B kinase and protein phosphatase 1 (PP1). However, the regulatory mechanisms underlying the aforementioned hierarchical signaling cascade during mitotic chromosome segregation have remained elusive. Sds22 is a conserved regulator of PP1 activity, but how it regulates PP1 activity in space and time during mitosis remains elusive. Here we show that Sds22 is a novel and cognate substrate of PLK1 in mitosis, and the phosphorylation of Sds22 by PLK1 elicited an inhibition of PP1-mediated dephosphorylation of Aurora B at threonine 232 (Thr232) in a dose-dependent manner. Overexpression of a phosphomimetic mutant of Sds22 causes a dramatic increase in mitotic delay, whereas overexpression of a non-phosphorylatable mutant of Sds22 results in mitotic arrest. Mechanistically, the phosphorylation of Sds22 by PLK1 strengthens the binding of Sds22 to PP1 and inhibits the dephosphorylation of Thr232 of Aurora B to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling hierarchy that orchestrates dynamic protein phosphorylation and dephosphorylation of critical mitotic regulators during chromosome segregation to guard chromosome stability.
Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos Humanos/metabolismo , Metáfase/fisiologia , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/genética , Instabilidade Cromossômica/fisiologia , Cromossomos Humanos/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Proteína Fosfatase 1/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Quinase 1 Polo-LikeRESUMO
ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4, named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34, which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration.
Assuntos
Membrana Celular/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/fisiologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Movimento Celular/genética , Fator de Crescimento Epidérmico/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fosforilação , Estrutura Terciária de Proteína , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.
Assuntos
Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Pontos de Checagem do Ciclo Celular , Proteínas do Citoesqueleto , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas Nucleares/química , Fosforilação , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
BACKGROUND: In previous studies, authors have completed the total synthesis of several phloroglucinol natural products and synthesized a series of their derivatives, which were tested with good biological activities. OBJECTIVES: To discover anti-MRSA lead compound and study their mechanism of action. METHODS: Phloroglucinol derivatives were tested to investigate their activities against several gram-positive strains including Methicillin-resistant Staphylococcus aureus (MRSA). The mechanism study was conducted by determining extracellular potassium ion concentration, intracellular NADPH oxidase content, SOD activity, ROS amount in MRSA and MRSA survival rate under A5 treatment. The in vitro cytotoxicity test of A5 was conducted. RESULTS: The activity of monocyclic compounds was stronger than that of bicyclic compounds, and compound A5 showed the best MIC value of 0.98 µg/mL and MBC value of 1.95 µg/mL, which were 4-8 times lower than that of vancomycin. The mechanism study of A5 showed that it achieved anti-MRSA effect through membrane damage, which is proved by increased concentration of extracellular potassium ion after A5 treatment. Another possible mechanism is the over ROS production induced cell death, which is suggested by observed alternation of several reactive oxygen species (ROS) related indicators including NADPH concentration, superoxide dismutase (SOD) activity, ROS content and bacterial survival rate after A5 treatment. The cytotoxicity results in vitro showed that A5 was basically non-toxic to cells. CONCLUSION: Acylphloroglucinol derivative A5 showed good anti-MRSA activity, possibly via membrane damage and ROS-mediated oxidative stress mechanism. It deserves further exploration to be a potential lead for the development of new anti-MRSA agent.
Assuntos
Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Floroglucinol , Espécies Reativas de Oxigênio , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Floroglucinol/farmacologia , Floroglucinol/química , Floroglucinol/análogos & derivados , Antibacterianos/farmacologia , Antibacterianos/química , Espécies Reativas de Oxigênio/metabolismo , Humanos , Superóxido Dismutase/metabolismoRESUMO
Stable transmission of genetic information during cell division requires faithful chromosome segregation. Mounting evidence has demonstrated that polo-like kinase 1 (PLK1) dynamics at kinetochores control correct kinetochore-microtubule attachments and subsequent silencing of the spindle assembly checkpoint. However, the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive. Here, we identified a regulatory mechanism by which PLK1-elicited zeste white 10 (ZW10) phosphorylation regulates spindle checkpoint silencing in mitosis. ZW10 is a cognate substrate of PLK1, and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10-Zwint1 interactions. Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes, while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase, in which sister chromatids entangled as cells entered anaphase. These findings reveal the previously uncharacterized PLK1-ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis.
Assuntos
Proteínas de Ciclo Celular , Segregação de Cromossomos , Mitose , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fosforilação , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Células HeLa , Cinetocoros/metabolismo , Fuso Acromático/metabolismoRESUMO
Accurate chromosome segregation in mitosis depends on kinetochores that connect centromeric chromatin to spindle microtubules. Centromeres are captured by individual microtubules via a kinetochore constitutive centromere-associated network (CCAN) during chromosome segregation. CCAN contains 16 subunits, including CENP-W and CENP-T. However, the molecular recognition and mitotic regulation of the CCAN assembly remain elusive. Here, we revealed that CENP-W binds to the histone fold domain and an uncharacterized N-terminal region of CENP-T. Aurora B phosphorylates CENP-W at threonine 60, which enhances the interaction between CENP-W and CENP-T to ensure robust metaphase chromosome alignment and accurate chromosome segregation in mitosis. These findings delineate a conserved signaling cascade that integrates protein phosphorylation with CCAN integrity for the maintenance of genomic stability.
Assuntos
Aurora Quinase B , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Mitose , Aurora Quinase B/metabolismo , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Fosforilação , Células HeLa , Cinetocoros/metabolismo , Ligação Proteica , Centrômero/metabolismoRESUMO
As a reversible post-translational modification (PTM) discovered decades ago, protein lysine acetylation was known for its regulation of transcription through the modification of histones. Recent studies discovered that lysine acetylation targets broad substrates and especially plays an essential role in cellular metabolic regulation. Although acetylation is comparable with other major PTMs such as phosphorylation, an integrated resource still remains to be developed. In this work, we presented the compendium of protein lysine acetylation (CPLA) database for lysine acetylated substrates with their sites. From the scientific literature, we manually collected 7151 experimentally identified acetylation sites in 3311 targets. We statistically studied the regulatory roles of lysine acetylation by analyzing the Gene Ontology (GO) and InterPro annotations. Combined with protein-protein interaction information, we systematically discovered a potential human lysine acetylation network (HLAN) among histone acetyltransferases (HATs), substrates and histone deacetylases (HDACs). In particular, there are 1862 triplet relationships of HAT-substrate-HDAC retrieved from the HLAN, at least 13 of which were previously experimentally verified. The online services of CPLA database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0). The CPLA database is freely available for all users at: http://cpla.biocuckoo.org.
Assuntos
Bases de Dados de Proteínas , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Humanos , Proteínas/química , Proteínas/metabolismo , Integração de SistemasRESUMO
In mitosis, accurate chromosome segregation depends on the kinetochore, a supermolecular machinery that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN) during mitosis remains uncharacterized. Building on our recent cryo-electron microscopic analyses of human CCAN structure, we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.
Assuntos
Proteína Quinase CDC2 , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Humanos , Proteína Quinase CDC2/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Mitose , FosforilaçãoRESUMO
We are entering the era of personalized genomics as breakthroughs in sequencing technology have made it possible to sequence or genotype an individual person in an efficient and accurate manner. Preliminary results from HapMap and other similar projects have revealed the existence of tremendous genetic variations among world populations and among individuals. It is important to delineate the functional implication of such variations, i.e. whether they affect the stability and biochemical properties of proteins. It is also generally believed that the genetic variation is the main cause for different susceptibility to certain diseases or different response to therapeutic treatments. Understanding genetic variation in the context of human diseases thus holds the promise for "personalized medicine." In this work, we carried out a genome-wide analysis of single nucleotide polymorphisms (SNPs) that could potentially influence protein phosphorylation characteristics in human. Here, we defined a phosphorylation-related SNP (phosSNP) as a non-synonymous SNP (nsSNP) that affects the protein phosphorylation status. Using an in-house developed kinase-specific phosphorylation site predictor (GPS 2.0), we computationally detected that approximately 70% of the reported nsSNPs are potential phosSNPs. More interestingly, approximately 74.6% of these potential phosSNPs might also induce changes in protein kinase types in adjacent phosphorylation sites rather than creating or removing phosphorylation sites directly. Taken together, we proposed that a large proportion of the nsSNPs might affect protein phosphorylation characteristics and play important roles in rewiring biological pathways. Finally, all phosSNPs were integrated into the PhosSNP 1.0 database, which was implemented in JAVA 1.5 (J2SE 5.0). The PhosSNP 1.0 database is freely available for academic researchers.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Fosforilação/genética , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional/genética , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Modelos Biológicos , Polimorfismo de Nucleotídeo Único/fisiologia , Proteínas Quinases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
During cell division/mitosis, a specific subset of proteins is spatially and temporally assembled into protein super complexes in three distinct regions, i.e. centrosome/spindle pole, kinetochore/centromere and midbody/cleavage furrow/phragmoplast/bud neck, and modulates cell division process faithfully. Although many experimental efforts have been carried out to investigate the characteristics of these proteins, no integrated database was available. Here, we present the MiCroKit database (http://microkit.biocuckoo.org) of proteins that localize in midbody, centrosome and/or kinetochore. We collected into the MiCroKit database experimentally verified microkit proteins from the scientific literature that have unambiguous supportive evidence for subcellular localization under fluorescent microscope. The current version of MiCroKit 3.0 provides detailed information for 1489 microkit proteins from seven model organisms, including Saccharomyces cerevisiae, Schizasaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Mus musculus and Homo sapiens. Moreover, the orthologous information was provided for these microkit proteins, and could be a useful resource for further experimental identification. The online service of MiCroKit database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0).
Assuntos
Centrossomo/química , Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Proteínas , Cinetocoros/química , Fuso Acromático , Algoritmos , Animais , Biologia Computacional/tendências , Genoma Fúngico , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Microscopia de Fluorescência/métodos , Software , Interface Usuário-ComputadorRESUMO
Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. In eukaryotic cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Although a list of mitotic kinases has been implicated in NEBD, how they coordinate their activity to dissolve the nuclear envelope and protein machinery such as nuclear pore complexes was unclear. Here, we identified a regulatory mechanism in which Nup62 is acetylated by TIP60 in human cell division. Nup62 is a novel substrate of TIP60, and the acetylation of Lys432 by TIP60 dissolves nucleoporin Nup62-Nup58-Nup54 complex during entry into mitosis. Importantly, this acetylation-elicited remodeling of nucleoporin complex promotes the distribution of Nup62 to the mitotic spindle, which is indispensable for orchestrating correct spindle orientation. Moreover, suppression of Nup62 perturbs accurate chromosome segregation during mitosis. These results establish a previously uncharacterized regulatory mechanism in which TIP60-elicited nucleoporin dynamics promotes chromosome segregation in mitosis.
Assuntos
Segregação de Cromossomos , Lisina Acetiltransferase 5 , Complexo de Proteínas Formadoras de Poros Nucleares , Humanos , Acetilação , Mitose , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Lisina Acetiltransferase 5/metabolismoRESUMO
Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis. Although previously proposed to be an adaptor of retinoic acid receptor, here, we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and ensure accurate chromosome segregation in mitosis. We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore, suggesting that phosphorylation may regulate its localization. Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore-microtubule attachment at metaphase. Mechanistically, CENP-R phosphorylation disrupts its binding with CENP-U. Thus, we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore-microtubule attachment in mitosis. As CENP-R is absent from yeast, we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.
Assuntos
Segregação de Cromossomos , Cinetocoros , Animais , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose , FosforilaçãoRESUMO
In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.
RESUMO
Thiol groups on cysteines can undergo multiple post-translational modifications (PTMs), acting as a molecular switch to maintain redox homeostasis and regulating a series of cell signaling transductions. Identification of sophistical protein cysteine modifications is crucial for dissecting its underlying regulatory mechanism. Instead of a time-consuming and labor-intensive experimental method, various computational methods have attracted intense research interest due to their convenience and low cost. Here, we developed the first comprehensive deep learning based tool pCysMod for multiple protein cysteine modification prediction, including S-nitrosylation, S-palmitoylation, S-sulfenylation, S-sulfhydration, and S-sulfinylation. Experimentally verified cysteine sites curated from literature and sites collected by other databases and predicting tools were integrated as benchmark dataset. Several protein sequence features were extracted and united into a deep learning model, and the hyperparameters were optimized by particle swarm optimization algorithms. Cross-validations indicated our model showed excellent robustness and outperformed existing tools, which was able to achieve an average AUC of 0.793, 0.807, 0.796, 0.793, and 0.876 for S-nitrosylation, S-palmitoylation, S-sulfenylation, S-sulfhydration, and S-sulfinylation, demonstrating pCysMod was stable and suitable for protein cysteine modification prediction. Besides, we constructed a comprehensive protein cysteine modification prediction web server based on this model to benefit the researches finding the potential modification sites of their interested proteins, which could be accessed at http://pcysmod.omicsbio.info. This work will undoubtedly greatly promote the study of protein cysteine modification and contribute to clarifying the biological regulation mechanisms of cysteine modification within and among the cells.
RESUMO
Identification of protein phosphorylation sites with their cognate protein kinases (PKs) is a key step to delineate molecular dynamics and plasticity underlying a variety of cellular processes. Although nearly 10 kinase-specific prediction programs have been developed, numerous PKs have been casually classified into subgroups without a standard rule. For large scale predictions, the false positive rate has also never been addressed. In this work, we adopted a well established rule to classify PKs into a hierarchical structure with four levels, including group, family, subfamily, and single PK. In addition, we developed a simple approach to estimate the theoretically maximal false positive rates. The on-line service and local packages of the GPS (Group-based Prediction System) 2.0 were implemented in Java with the modified version of the Group-based Phosphorylation Scoring algorithm. As the first stand alone software for predicting phosphorylation, GPS 2.0 can predict kinase-specific phosphorylation sites for 408 human PKs in hierarchy. A large scale prediction of more than 13,000 mammalian phosphorylation sites by GPS 2.0 was exhibited with great performance and remarkable accuracy. Using Aurora-B as an example, we also conducted a proteome-wide search and provided systematic prediction of Aurora-B-specific substrates including protein-protein interaction information. Thus, the GPS 2.0 is a useful tool for predicting protein phosphorylation sites and their cognate kinases and is freely available on line.