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1.
Nature ; 596(7870): 138-142, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290405

RESUMO

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.


Assuntos
Proteína Quinase CDC2/química , Proteína Quinase CDC2/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Securina/química , Securina/metabolismo , Separase/química , Separase/metabolismo , Motivos de Aminoácidos , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/ultraestrutura , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/metabolismo , Quinases relacionadas a CDC2 e CDC28/ultraestrutura , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Microscopia Crioeletrônica , Ciclina B1/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Securina/ultraestrutura , Separase/antagonistas & inibidores , Separase/ultraestrutura , Especificidade por Substrato
2.
J Cell Biol ; 219(8)2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32614383

RESUMO

In the film Rashomon, four witnesses describe seemingly contradictory views of one event. In a recent analogy, an interaction between the master mitotic regulator cyclin B1 and the spindle checkpoint component Mad1 was independently described by three groups who propose strikingly different functions for this interaction. Here, we summarize their findings and present a perspective on reconciling the different views.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Mitose , Fuso Acromático/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Ciclina B1/química , Ciclina B1/genética , Humanos , Cinetocoros/metabolismo , Mutação , Poro Nuclear/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Fuso Acromático/genética
3.
Angew Chem Int Ed Engl ; 59(32): 13496-13501, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32346954

RESUMO

Triazole-based deubiquitylase (DUB)-resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain-specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB-resistant Ub probes is reported based on isopeptide-N-ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one-pot, ubiquitin-activating enzyme (E1)-catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi-milligram scale. Proteomic studies using label-free quantitative (LFQ) MS indicated that the isopeptide-N-ethylated Ub probes may complement the triazole-based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.


Assuntos
Sondas Moleculares/química , Poliubiquitina/química , Enzimas Ativadoras de Ubiquitina/química , Ciclina B1/química , Ciclina B1/genética , Células HEK293 , Células HeLa , Histonas/química , Histonas/genética , Humanos , Sondas Moleculares/síntese química , Mutação , Poliubiquitina/síntese química , Proteômica
4.
J Biol Chem ; 294(26): 10236-10252, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31101654

RESUMO

Proper cell division relies on the coordinated regulation between a structural component, the mitotic spindle, and a regulatory component, anaphase-promoting complex/cyclosome (APC/C). Hematopoietic PBX-interacting protein (HPIP) is a microtubule-associated protein that plays a pivotal role in cell proliferation, cell migration, and tumor metastasis. Here, using HEK293T and HeLa cells, along with immunoprecipitation and immunoblotting, live-cell imaging, and protein-stability assays, we report that HPIP expression oscillates throughout the cell cycle and that its depletion delays cell division. We noted that by utilizing its D box and IR domain, HPIP plays a dual role both as a substrate and inhibitor, respectively, of the APC/C complex. We observed that HPIP enhances the G2/M transition of the cell cycle by transiently stabilizing cyclin B1 by preventing APC/C-Cdc20-mediated degradation, thereby ensuring timely mitotic entry. We also uncovered that HPIP associates with the mitotic spindle and that its depletion leads to the formation of multiple mitotic spindles and chromosomal abnormalities, results in defects in cytokinesis, and delays mitotic exit. Our findings uncover HPIP as both a substrate and an inhibitor of APC/C-Cdc20 that maintains the temporal stability of cyclin B1 during the G2/M transition and thereby controls mitosis and cell division.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Ciclo Celular , Ciclina B1/química , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Mitose , Ciclossomo-Complexo Promotor de Anáfase/antagonistas & inibidores , Ciclossomo-Complexo Promotor de Anáfase/genética , Proteínas Cdc20/antagonistas & inibidores , Proteínas Cdc20/genética , Células HEK293 , Células HeLa , Humanos , Fuso Acromático , Especificidade por Substrato
5.
Oncotarget ; 7(14): 18076-84, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-26716515

RESUMO

Follistatin-like 1 (FSTL1) was identified as a novel pro-inflammatory protein showing high-level expression in rheumatoid arthritis. The protective effect of FSTL1 via the inhibition of apoptosis was reported in myocardial injury. However, the functional mechanism of FSTL1 in cancer is poorly characterized, and its proliferative effects are ambiguous. Here, we examined the effects of FSTL1 on cellular proliferation and cell cycle checkpoints in lung cancer cells. FSTL1 inhibition induced the cellular portion of G2/M phase in human lung cancer cells via the accumulation of regulators of the transition through the G2/M phase, including the cyclin-dependent kinase 1 (Cdk1)-cyclin B1 complex. An increase in histone H3 phosphorylation (at Ser10), another hallmark of mitosis, indicated that the knockdown of FSTL1 in lung cancer cells stimulated a mitotic arrest. After that, apoptosis was promoted by the activation of caspase-3 and -9. Protein level of Bim, a BH3 domain-only, pro-apoptotic member and its isoforms, BimL, BimS, and BimEL were up-regulated by FSTL1 inhibition. Degradation of Bim was blocked in FSTL1-knockdown cells by decreased phosphorylation of Bim. Increased BimEL as well as decreased phosphorylated Erk1/2 is essential for cell death by FSTL1 inhibition in NCI-H460 cells. Taken together, our results suggest that the knockdown of FSTL1 induces apoptosis through a mitotic arrest and caspase-dependent cell death. FSTL1 plays the important roles in cellular proliferation and apoptosis in lung cancer cells, and thus can be a new target for lung cancer treatment.


Assuntos
Apoptose/genética , Proteína 11 Semelhante a Bcl-2/biossíntese , Carcinoma Pulmonar de Células não Pequenas/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Relacionadas à Folistatina/genética , Neoplasias Pulmonares/patologia , Células A549 , Proteína Quinase CDC2 , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina B1/química , Quinases Ciclina-Dependentes/química , Proteínas Relacionadas à Folistatina/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Histonas/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Mitose/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética
6.
Mol Cell ; 58(3): 495-506, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25921067

RESUMO

Ring-shaped cohesin keeps sister chromatids paired until cleavage of its Scc1/Rad21 subunit by separase triggers chromosome segregation in anaphase. Vertebrate separase is held inactive by mutually exclusive binding to securin or Cdk1-cyclin B1 and becomes unleashed only upon ubiquitin-dependent degradation of these regulators. Although most separase is usually found in association with securin, this anaphase inhibitor is dispensable for murine life while Cdk1-cyclin B1-dependent control of separase is essential. Here, we show that securin-independent inhibition of separase by Cdk1-cyclin B1 in early mitosis requires the phosphorylation-specific peptidyl-prolyl cis/trans isomerase Pin1. Furthermore, isomerization of previously securin-bound separase at the metaphase-to-anaphase transition renders it resistant to re-inhibition by residual securin. At the same time, isomerization also limits the half-life of separase's proteolytic activity, explaining how cohesin can be reloaded onto telophase chromatin in the absence of securin and cyclin B1 without being cleaved.


Assuntos
Segregação de Cromossomos/genética , Regulação Enzimológica da Expressão Gênica , Peptidilprolil Isomerase/genética , Separase/genética , Anáfase/genética , Proteína Quinase CDC2 , Cromátides/genética , Ciclina B1/química , Ciclina B1/genética , Ciclina B1/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Humanos , Immunoblotting , Metáfase/genética , Microscopia de Fluorescência , Mitose/genética , Modelos Genéticos , Modelos Moleculares , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Conformação Proteica , Interferência de RNA , Securina/genética , Securina/metabolismo , Separase/química , Separase/metabolismo
7.
ACS Chem Biol ; 10(4): 952-6, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25603287

RESUMO

The cis vs trans conformation, or shape, of phosphoserine-proline (pSer-Pro), a prevalent motif in cell cycle proteins, may play a significant role in regulating mitosis. We demonstrate that Cdk1-cyclin B, the central mitotic kinase, is specific for the trans conformation, not cis, of synthetic, locked Ser-Pro 11-residue peptide substrates, using LC-MSMS detection and sequencing of phosphorylated products. This substrate stereospecificity may contribute an additional level of mitotic regulation.


Assuntos
Ciclina B1/química , Ciclina B1/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Proteína Quinase CDC2 , Mitose , Peptidilprolil Isomerase de Interação com NIMA , Peptídeos/química , Peptídeos/metabolismo , Peptidilprolil Isomerase/metabolismo , Fosforilação , Conformação Proteica , Técnicas de Síntese em Fase Sólida , Estereoisomerismo , Espectrometria de Massas em Tandem
8.
Med Oncol ; 31(9): 107, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25106528

RESUMO

Pancreatic cancer (PC), a malignancy with very poor prognosis, presents many molecular alterations, including overexpression of Cyclin B1. However, the prognostic value of the protein in PC remains to be elucidated. In the present study, Cyclin B1 expression was detected immunohistochemically in specimens from 241 patients with PC and was correlated with clinicopathological features and patient survival. It was found that Cyclin B1 expression, located in nucleus and/or cytoplasm, was not statistically associated with clinicopathologic variables. However, overall survival of patients with high Cyclin B1 expression was significantly poorer than that of those with low Cyclin B1 expression (P = 0.010). Moreover, Cyclin B1 was identified as an independent prognostic factor by multivariate Cox regression test (P = 0.003). Finally, its independent implication for prognosis was proven in five subgroups of PC, i.e., males, patients aged ≤ 65 years, G1-2 and N0 tumors as well as those with perineural invasion (all P < 0.05). These data indicate that high expression of Cyclin B1 is a valuable molecular marker of unfavorable prognosis in PC.


Assuntos
Biomarcadores Tumorais/análise , Ciclina B1/análise , Neoplasias Pancreáticas/epidemiologia , Neoplasias Pancreáticas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/mortalidade , Prognóstico
9.
Mol Med Rep ; 10(4): 2160-4, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25070000

RESUMO

Small non­coding RNAs from the microRNA family (miRs) are important elements in the posttranscriptional control of gene expression. miRs are known to regulate numerous cellular processes and are of crucial importance during development and in pathological conditions, including tumor initiation and progression. In the present study, the expression level of miR­181 was reduced in glioma tissues compared with the adjacent normal tissues. The enforced expression of miR­181 was able to inhibit cell proliferation in U251 and SHG­44 cells, while antisense miR­181 oligonucleotides (antisense miR­181) enhanced cell proliferation. At the molecular level, these results further revealed that the expression of cyclin B1, a positive cell­cycle regulator, was negatively regulated by miR­181. Therefore, the data reported in the present study demonstrates that miR­181 is an important regulator in glioma. These results may contribute to improving the understanding of the key misregulated miRNAs in glioma.


Assuntos
Neoplasias Encefálicas/patologia , Ciclina B1/metabolismo , Glioma/patologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Sequência de Bases , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina B1/química , Ciclina B1/genética , Glioma/metabolismo , Humanos , MicroRNAs/antagonistas & inibidores , Oligonucleotídeos Antissenso/metabolismo , Alinhamento de Sequência
10.
PLoS One ; 8(3): e59169, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23505570

RESUMO

Cyclin B1-CDK1 activity is essential for mitotic entry, but questions remain regarding how the activity of this kinase is spatially regulated. Previous studies showed that the cyclin B1 subunit localizes to several compartments of a mitotic cell, including the centrosomes, mitotic spindle, kinetochores and chromosomes via distinct sequence elements. Mitotic chromosome association occurs through the unstructured N-terminal domain of cyclin B1 and is independent of CDK1 binding. Here, we use live cell imaging of human cyclin B1 fused to GFP to precisely define the sequence elements within cyclin B1 that mediate its association with condensed mitotic chromosomes. We find that a short, evolutionarily conserved N-terminal motif is required for cyclin B1 to localize to mitotic chromosomes. We further reveal a role for arginine residues within and near the destruction box sequence in the chromosome association of cyclin B1. Additionally, our data suggest that sequences further downstream in cyclin B1, such as the cytoplasmic retention sequence and the cyclin box, may negatively modulate chromosome association. Because multiple basic residues are required for cyclin B1 association with mitotic chromosomes, electrostatic interactions with DNA may facilitate cyclin B1 localization to chromosomes.


Assuntos
Cromossomos Humanos/metabolismo , Ciclina B1/metabolismo , Mitose/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Ciclina B1/química , Ciclina B1/genética , Células HeLa , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Alinhamento de Sequência
11.
Biomed Res Int ; 2013: 732307, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24416725

RESUMO

The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB) is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP) at C-terminus and red fluorescent protein (RFP, DsRed) at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future.


Assuntos
Sequência de Aminoácidos/genética , Ciclo Celular/genética , Ciclina B1/química , Genes Reporter/genética , Linhagem Celular Tumoral , Ciclina B1/genética , Ciclina B1/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Deleção de Sequência , Proteína Vermelha Fluorescente
13.
Theriogenology ; 78(6): 1171-81.e1, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22901768

RESUMO

During mammalian oocyte maturation, two consecutive meiotic divisions are required to form a haploid gamete. For each meiotic division, oocytes must transfer from metaphase to anaphase, but maturation promoting factor (cyclin-dependent kinase 1/cyclin B1) activity would keep the oocytes at metaphase. Therefore, inactivation of maturation promoting factor is needed to finish the transition and complete both these divisions; this is provided through anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. The objective of this study was to examine meiotic divisions in bovine oocytes after expression of a full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion, coupled with the fluorochrome Venus, by microinjecting their complementary RNA (cRNA). Overexpression of full-length cyclin B1-Venus inhibited homologue disjunction and first polar body formation in maturing oocytes (control 70% vs. overexpression 16%; P < 0.05). However at the same levels of expression, it did not block second meiotic metaphase and cleavage of eggs after parthenogenetic activation (control: 82% pronuclei and 79% cleaved; overexpression: 91% pronuclei and 89% cleaved). The full length cyclin B1 and a nondegradable N-terminal 87 amino acid deletion caused metaphase arrest in both meiotic divisions, whereas degradation of securin was unaffected. Roscovitine, a potent cyclin-dependent kinase 1 (CDK1) inhibitor, overcame this metaphase arrest in maturing oocytes at 140 µM, but higher doses (200 µM) were needed to overcome arrest in eggs. In conclusion, because metaphase I (MI) blocked by nondegradable cyclin B1 was distinct from metaphase II (MII) in their different sensitivities to trigger CDK1 inactivation, we concluded that mechanisms of MI arrest differed from MII arrest.


Assuntos
Bovinos , Ciclina B1/genética , Ciclina B1/metabolismo , Meiose/genética , Meiose/fisiologia , Oócitos/fisiologia , Anáfase , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Ciclina B1/química , Ativação Enzimática , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Transferência de Genes/veterinária , Metáfase/efeitos dos fármacos , Metáfase/fisiologia , Mutação/genética , Oócitos/ultraestrutura , Partenogênese , Purinas/farmacologia , RNA Complementar/genética , Roscovitina , Complexos Ubiquitina-Proteína Ligase/metabolismo , Complexos Ubiquitina-Proteína Ligase/fisiologia
14.
Mol Cell ; 43(3): 406-17, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21816347

RESUMO

Cyclin-dependent kinases comprise the conserved machinery that drives progress through the cell cycle, but how they do this in mammalian cells is still unclear. To identify the mechanisms by which cyclin-cdks control the cell cycle, we performed a time-resolved analysis of the in vivo interactors of cyclins E1, A2, and B1 by quantitative mass spectrometry. This global analysis of context-dependent protein interactions reveals the temporal dynamics of cyclin function in which networks of cyclin-cdk interactions vary according to the type of cyclin and cell-cycle stage. Our results explain the temporal specificity of the cell-cycle machinery, thereby providing a biochemical mechanism for the genetic requirement for multiple cyclins in vivo and reveal how the actions of specific cyclins are coordinated to control the cell cycle. Furthermore, we identify key substrates (Wee1 and c15orf42/Sld3) that reveal how cyclin A is able to promote both DNA replication and mitosis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Ciclina A2/metabolismo , Ciclina B1/metabolismo , Quinases Ciclina-Dependentes/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Ciclina A2/química , Ciclina A2/fisiologia , Ciclina B1/química , Ciclina B1/fisiologia , Ciclina E/química , Ciclina E/metabolismo , Ciclina E/fisiologia , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/fisiologia , Fosforilação , Proteômica/métodos , Alinhamento de Sequência , Especificidade por Substrato
15.
Cancer Immunol Immunother ; 60(2): 227-34, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20981424

RESUMO

With the aim to identify cyclin B1-derived peptides with high affinity for HLA-A2, we used three in silico prediction algorithms to screen the protein sequence for possible HLA-A2 binders. One peptide scored highest in all three algorithms, and the high HLA-A2-binding affinity of this peptide was verified in an HLA stabilization assay. By stimulation with peptide-loaded dendritic cells a CTL clone was established, which was able to kill two breast cancer cell lines in an HLA-A2-dependent and peptide-specific manner, demonstrating presentation of the peptide on the surface of cancer cells. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors.


Assuntos
Anticorpos/sangue , Anticorpos/imunologia , Ciclina B1/imunologia , Epitopos de Linfócito T/imunologia , Saúde , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Células Cultivadas , Ciclina B1/química , Ensaio de Imunoadsorção Enzimática , Antígeno HLA-A2/imunologia , Humanos , Pessoa de Meia-Idade , Neoplasias/sangue
16.
FEBS Lett ; 584(22): 4505-10, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20965177

RESUMO

Hip2, a ubiquitin conjugating enzyme, is involved in the suppression of cell death. The present study revealed that Hip2 regulates the stability of the apoptotic and cell cycle regulator cyclin B1. Hip2 was found to interact with cyclin B1 to promote its degradation through the ubiquitin proteasome pathway. As a result, Hip2 significantly blocked cell death induced by the cyclin B1 protein, suggesting that Hip2 is involved in the regulation of cyclin B1-mediated cell death.


Assuntos
Ciclina B1/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Apoptose , Ciclina B1/química , Células HCT116 , Células HEK293 , Humanos , Ligação Proteica , Estabilidade Proteica , Ubiquitinação
17.
J Cell Biol ; 191(2): 313-29, 2010 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-20956380

RESUMO

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclina B1/biossíntese , Mitose , Regiões 3' não Traduzidas , Aneuploidia , Animais , Proteínas Cdc20 , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/genética , Células Cultivadas , Segregação de Cromossomos , Cromossomos de Mamíferos/metabolismo , Ciclina B1/química , Ciclina B1/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Cinetocoros/metabolismo , Camundongos , Neoplasias/genética , Neurogênese/genética , Biossíntese de Proteínas
18.
Oncogene ; 29(24): 3501-8, 2010 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-20418911

RESUMO

The migration and invasion inhibitor protein (MIIP, also known as IIp45) was discovered as a negative regulator of cell migration and invasion in glioma. Our previous studies have shown that the MIIP protein was reduced or undetectable in some tissue samples obtained from patients with glioblastoma. The significance of MIIP in gliomagenesis is unknown. In this study, we report that MIIP has an important role in the inhibition of gliomagenesis and attenuation of mitotic transition. Increased MIIP expression levels inhibited colony formation and cell growth of glioma cell lines in vitro, whereas decreased expression by specific small interfering RNA for MIIP resulted in increased cell growth. Expression of MIIP in a glial-specific mouse model blocked glioma development and progression, thus showing that MIIP is an inhibitor of gliomagenesis. Furthermore, we show that MIIP attenuates mitotic transition and results in increased mitotic catastrophe. The biochemical mechanism of MIIP in this process is associated with its regulation of anaphase-promoting complex (APC/C) activity. MIIP interacts directly with Cdc20, and the interaction of MIIP with Cdc20 inhibits APC/C-mediated degradation of cyclin B1. Thus, MIIP attenuates mitotic transition and increases mitotic catastrophe, thereby inhibiting glioma development and progression.


Assuntos
Proteínas de Transporte/metabolismo , Glioma/metabolismo , Glioma/patologia , Mitose , Animais , Proteínas de Transporte/genética , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ciclina B1/química , Ciclina B1/metabolismo , Progressão da Doença , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Neuroglia/patologia , Especificidade de Órgãos , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo
19.
PLoS One ; 2(2): e247, 2007 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-17327911

RESUMO

M-phase Promoting Factor (MPF; the cyclin B-cdk 1 complex) is activated at M-phase onset by removal of inhibitory phosphorylation of cdk1 at thr-14 and tyr-15. At M-phase exit, MPF is destroyed by ubiquitin-dependent cyclin proteolysis. Thus, control of MPF activity via inhibitory phosphorylation is believed to be particularly crucial in regulating transition into, rather than out of, M-phase. Using the in vitro cell cycle system derived form Xenopus eggs, here we show, however, that inhibitory phosphorylation of cdk1 contributes to control MPF activity during M-phase exit. By sampling extracts at very short intervals during both meiotic and mitotic exit, we found that cyclin B1-associated cdk1 underwent transient inhibitory phosphorylation at tyr-15 and that cyclin B1-cdk1 activity fell more rapidly than the cyclin B1 content. Inhibitory phosphorylation of MPF correlated with phosphorylation changes of cdc25C, the MPF phosphatase, and physical interaction of cdk1 with wee1, the MPF kinase, during M-phase exit. MPF down-regulation required Ca(++)/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase (PKA) activities at meiosis and mitosis exit, respectively. Treatment of M-phase extracts with a mutant cyclin B1-cdk1AF complex, refractory to inhibition by phosphorylation, impaired binding of the Anaphase Promoting Complex/Cyclosome (APC/C) to its co-activator Cdc20 and altered M-phase exit. Thus, timely M-phase exit requires a tight coupling of proteolysis-dependent and proteolysis-independent mechanisms of MPF inactivation.


Assuntos
Proteína Quinase CDC2/fisiologia , Ciclina B1/fisiologia , Metáfase/fisiologia , Oócitos/citologia , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/química , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/fisiologia , Ciclina B1/química , Ciclina B1/genética , Ativação Enzimática , Feminino , Humanos , Meiose , Mitose , Fosforilação , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis , Fosfatases cdc25/fisiologia
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