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1.
EMBO J ; 38(14): e101082, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31304626

RESUMO

Centrioles are core structural elements of both centrosomes and cilia. Although cytoplasmic granules called centriolar satellites have been observed around these structures, lack of a comprehensive inventory of satellite proteins impedes our understanding of their ancestry. To address this, we performed mass spectrometry (MS)-based proteome profiling of centriolar satellites obtained by affinity purification of their key constituent, PCM1, from sucrose gradient fractions. We defined an interactome consisting of 223 proteins, which showed striking enrichment in centrosome components. The proteome also contained new structural and regulatory factors with roles in ciliogenesis. Quantitative MS on whole-cell and centriolar satellite proteomes of acentriolar cells was performed to reveal dependencies of satellite composition on intact centrosomes. Although most components remained associated with PCM1 in acentriolar cells, reduced cytoplasmic and satellite levels were observed for a subset of centrosomal proteins. These results demonstrate that centriolar satellites and centrosomes form independently but share a substantial fraction of their proteomes. Dynamic exchange of proteins between these organelles could facilitate their adaptation to changing cellular environments during development, stress response and tissue homeostasis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Linfócitos/metabolismo , Animais , Autoantígenos/metabolismo , Galinhas , Células HEK293 , Homeostase , Humanos , Células Jurkat , Linfócitos/citologia , Proteômica
2.
Nat Commun ; 7: 11005, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26987684

RESUMO

Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215-HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome-spindle pole interface by the CEP215-HSET complex could promote survival of cancer cells containing supernumerary centrosomes.


Assuntos
Centrossomo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/metabolismo , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Polos do Fuso/metabolismo , Animais , Proteínas de Ciclo Celular , Linhagem Celular , Galinhas , Análise por Conglomerados , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Cinesinas/química , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/química , Ligação Proteica , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína
3.
Nat Protoc ; 11(2): 316-26, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26797456

RESUMO

Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2-3 d from the collection of material to results.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteínas/análise , Fatores de Tempo
4.
J Proteome Res ; 12(5): 2078-89, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23510160

RESUMO

The ThinPrep cervical smear is widely used in clinical practice for the cytological and molecular screening against abnormal cells and Human Papillomavirus (HPV) infection. Current advancements made to LC-MS proteomics include the use of stable isotope labeling for the in-depth analysis of proteins in complex clinical specimens. Such approaches have yet to be realized for ThinPrep clinical specimens. In this study, an LC-MS method based on isobaric (iTRAQ) labeling and high-resolution FT-Orbitrap mass spectrometry was used for the proteomic analysis of 23 human ThinPrep smear specimens. Tandem mass spectrometry analysis was performed with both nitrogen high collision dissociation (HCD MS/MS) and helium collision induced dissociation (CID MS/MS) peptide fragmentation modes. The analysis of three 8-plex sample sets yielded the identification of over 3200 unique proteins at FDR < 1%, of which over 2300 proteins were quantitatively profiled in at least one of the three experiments. The interindividual variability served to define the required sample size needed to identify significant protein expression differences. The degree of in-depth proteome coverage allowed the detection of 6 HPV-derived proteins including the high-risk HPV16 type in the specimens tested. The presence of the HPV strains of origin was also confirmed with PCR-hybridization molecular methods. This proof-of-principle study constitutes the first ever report on the nontargeted analysis of HPV proteins in human ThinPrep clinical specimens with high-resolution mass spectrometry. A further testament to the sensitivity and selectivity of the proposed study method was the confident detection of a significant number of phosphopeptides in these specimens.


Assuntos
Papillomavirus Humano 16/metabolismo , Infecções por Papillomavirus/metabolismo , Proteoma/metabolismo , Neoplasias do Colo do Útero/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Feminino , Humanos , Marcação por Isótopo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Infecções por Papillomavirus/diagnóstico , Proteoma/química , Proteômica , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal , Proteínas Virais/química
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