Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
Cell ; 173(6): 1495-1507.e18, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29706546

RESUMO

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.


Assuntos
Ciclo Celular , DNA/análise , Proteoma/análise , Proteômica/métodos , Montagem e Desmontagem da Cromatina , Análise por Conglomerados , Células HeLa , Temperatura Alta , Humanos , Espectrometria de Massas , Mitose , Fosforilação , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , RNA Polimerase II/metabolismo , Solubilidade
2.
Cell ; 166(3): 664-678, 2016 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-27397507

RESUMO

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.


Assuntos
Embrião não Mamífero/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Oócitos/metabolismo , Animais , Blastoderma/metabolismo , Blastoderma/ultraestrutura , Drosophila , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Gastrulação , Oócitos/ultraestrutura
3.
Cell ; 159(5): 1056-1069, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25416945

RESUMO

Cdc42 is a highly conserved master regulator of cell polarity. Here, we investigated the mechanism by which yeast cells never re-establish polarity at cortical sites (cytokinesis remnants [CRMs]) that have previously supported Cdc42-mediated growth as a paradigm to mechanistically understand how Cdc42-inhibitory polarity cues are established. We revealed a two-step mechanism of loading the Cdc42 antagonist Nba1 into CRMs to mark these compartments as refractory for a second round of Cdc42 activation. Our data indicate that Nba1 together with a cortically tethered adaptor protein confers memory of previous polarization events to translate this spatial legacy into a biochemical signal that ensures the local singularity of Cdc42 activation. "Memory loss" mutants that repeatedly use the same polarity site over multiple generations display nuclear segregation defects and a shorter lifespan. Our work thus established CRMs as negative polarity cues that prevent Cdc42 reactivation to sustain the fitness of replicating cells.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Divisão Celular Assimétrica , Proteínas de Ciclo Celular/metabolismo , Polaridade Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo
4.
Cell ; 155(6): 1233-43, 2013 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-24315095

RESUMO

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.


Assuntos
Membrana Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Modelos Moleculares , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Polimerização
5.
Mol Cell ; 75(3): 483-497.e9, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31253574

RESUMO

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.


Assuntos
Proteína BRCA1/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Enzimas Desubiquitinantes/genética , Chaperonas de Histonas/genética , Neoplasias/genética , Sítios de Ligação/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Citoplasma/genética , Citoplasma/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/imunologia , Enzimas Desubiquitinantes/imunologia , Células HeLa , Humanos , Imunidade Celular/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Neoplasias/imunologia , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica/genética , Ubiquitina/genética , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética
6.
Nature ; 526(7571): 140-143, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26416747

RESUMO

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.


Assuntos
Microscopia Crioeletrônica , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Sítios de Ligação , Células HeLa , Humanos , Espectrometria de Massas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/ultraestrutura , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica
7.
Hepatology ; 60(3): 884-95, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24799195

RESUMO

UNLABELLED: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. CONCLUSION: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteína de Suscetibilidade a Apoptose Celular/antagonistas & inibidores , Proteína de Suscetibilidade a Apoptose Celular/fisiologia , Neoplasias Hepáticas/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologia , alfa Carioferinas/antagonistas & inibidores , Animais , Apoptose/genética , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Regulação para Baixo/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Fenótipo , Proteína Supressora de Tumor p53/toxicidade , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , alfa Carioferinas/metabolismo
8.
Mol Syst Biol ; 9: 648, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23511206

RESUMO

To understand the structure and function of large molecular machines, accurate knowledge of their stoichiometry is essential. In this study, we developed an integrated targeted proteomics and super-resolution microscopy approach to determine the absolute stoichiometry of the human nuclear pore complex (NPC), possibly the largest eukaryotic protein complex. We show that the human NPC has a previously unanticipated stoichiometry that varies across cancer cell types, tissues and in disease. Using large-scale proteomics, we provide evidence that more than one third of the known, well-defined nuclear protein complexes display a similar cell type-specific variation of their subunit stoichiometry. Our data point to compositional rearrangement as a widespread mechanism for adapting the functions of molecular machines toward cell type-specific constraints and context-dependent needs, and highlight the need of deeper investigation of such structural variants.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/análise , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Poro Nuclear/metabolismo , Calibragem , Linhagem Celular , Humanos , Espectrometria de Massas/métodos , Microscopia/métodos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteômica/métodos
9.
Hum Mol Genet ; 20(21): 4132-42, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21828076

RESUMO

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.


Assuntos
Transtorno Autístico/enzimologia , Transtorno Autístico/genética , Síndrome do Hamartoma Múltiplo/enzimologia , Síndrome do Hamartoma Múltiplo/genética , Mutação/genética , PTEN Fosfo-Hidrolase/genética , Alanina/genética , Sequência de Aminoácidos , Ácido Aspártico/genética , Domínio Catalítico , Análise Mutacional de DNA , Mutação em Linhagem Germinativa/genética , Humanos , Dados de Sequência Molecular , Mutagênese/genética , PTEN Fosfo-Hidrolase/química , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
10.
Life Sci Alliance ; 5(6)2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35273078

RESUMO

Gene duplication enables the emergence of new functions by lowering the evolutionary pressure that is posed on the ancestral genes. Previous studies have highlighted the role of specific paralog genes during cell differentiation, for example, in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. Whereas ∼80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B members of the COPII complex. Altering the ratio between these two genes via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.


Assuntos
Evolução Biológica , Duplicação Gênica , Animais
11.
Sci Adv ; 8(35): eabq5206, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36044572

RESUMO

Nucleic acid and histone modifications critically depend on the tricarboxylic acid (TCA) cycle for substrates and cofactors. Although a few TCA cycle enzymes have been reported in the nucleus, the corresponding pathways are considered to operate in mitochondria. Here, we show that a part of the TCA cycle is operational also in the nucleus. Using 13C-tracer analysis, we identified activity of glutamine-to-fumarate, citrate-to-succinate, and glutamine-to-aspartate routes in the nuclei of HeLa cells. Proximity labeling mass spectrometry revealed a spatial vicinity of the involved enzymes with core nuclear proteins. We further show nuclear localization of aconitase 2 and 2-oxoglutarate dehydrogenase in mouse embryonic stem cells. Nuclear localization of the latter enzyme, which produces succinyl-CoA, changed from pluripotency to a differentiated state with accompanying changes in the nuclear protein succinylation. Together, our results demonstrate operation of an extended metabolic pathway in the nucleus, warranting a revision of the canonical view on metabolic compartmentalization.

12.
Mol Biol Cell ; 17(9): 4002-13, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16807353

RESUMO

The targeting of the tumor suppressor PTEN protein to distinct subcellular compartments is a major regulatory mechanism of PTEN function, by controlling its access to substrates and effector proteins. Here, we investigated the molecular basis and functional consequences of PTEN nuclear/cytoplasmic distribution. PTEN accumulated in the nucleus of cells treated with apoptotic stimuli. Nuclear accumulation of PTEN was enhanced by mutations targeting motifs in distinct PTEN domains, and it was dependent on an N-terminal nuclear localization domain. Coexpression of a dominant negative Ran GTPase protein blocked PTEN accumulation in the nucleus, which was also affected by coexpression of importin alpha proteins. The lipid- and protein-phosphatase activity of PTEN differentially modulated PTEN nuclear accumulation. Furthermore, catalytically active nuclear PTEN enhanced cell apoptotic responses. Our findings indicate that multiple nuclear exclusion motifs and a nuclear localization domain control PTEN nuclear localization by a Ran-dependent mechanism and suggest a proapoptotic role for PTEN in the cell nucleus.


Assuntos
Apoptose , Núcleo Celular/metabolismo , Sinais de Localização Nuclear/metabolismo , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Células 3T3 , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Catálise , Células Cultivadas , Chlorocebus aethiops , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Deleção de Sequência
13.
Cancer Res ; 67(20): 9731-9, 2007 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17942903

RESUMO

The signaling pathways involving class I phosphatidylinositol 3-kinases (PI3K) and the phosphatidylinositol-(3,4,5)-trisphosphate phosphatase PTEN regulate cell proliferation and survival. Thus, mutations in the corresponding genes are associated to a wide variety of human tumors. Heterologous expression of hyperactive forms of mammalian p110alpha and p110beta in Saccharomyces cerevisiae leads to growth arrest, which is counterbalanced by coexpression of mammalian PTEN. Using this in vivo yeast-based system, we have done an extensive functional analysis of germ-line and somatic human PTEN mutations, as well as a directed mutational analysis of discrete PTEN functional domains. A distinctive penetrance of the PTEN rescue phenotype was observed depending on the levels of PTEN expression in yeast and on the combinations of the inactivating PTEN mutations and the activating p110alpha or p110beta mutations analyzed, which may reflect pathologic differences found in tumors with distinct alterations at the p110 and PTEN genes or proteins. We also define the minimum length of the PTEN protein required for stability and function in vivo. In addition, a random mutagenesis screen on PTEN based on this system allowed both the reisolation of known clinically relevant PTEN mutants and the identification of novel PTEN loss-of-function mutations, which were validated in mammalian cells. Our results show that the PI3K/PTEN yeast-based system is a sensitive tool to test in vivo the pathologic properties and the functionality of mutations in the human p110 proto-oncogenes and the PTEN tumor suppressor and provide a framework for comprehensive functional studies of these tumor-related enzymes.


Assuntos
Mutação em Linhagem Germinativa , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Células COS , Catálise , Linhagem Celular Tumoral , Chlorocebus aethiops , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Transfecção
14.
Annu Rev Biophys ; 48: 515-536, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30943044

RESUMO

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange. They are exceptionally large protein complexes that fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. About 30 different protein components, termed nucleoporins, assemble in multiple copies into an intricate cylindrical architecture. Here, we review our current knowledge of the structure of nucleoporins and how those come together in situ. We delineate architectural principles on several hierarchical organization levels, including isoforms, posttranslational modifications, nucleoporins, and higher-order oligomerization of nucleoporin subcomplexes. We discuss how cells exploit this modularity to faithfully assemble NPCs.


Assuntos
Poro Nuclear/química , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Processamento de Proteína Pós-Traducional
15.
Nat Commun ; 10(1): 2147, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089132

RESUMO

Cancer-relevant signalling pathways rely on bidirectional nucleocytoplasmic transport events through the nuclear pore complex (NPC). However, mechanisms by which individual NPC components (Nups) participate in the regulation of these pathways remain poorly understood. We discover by integrating large scale proteomics, polysome fractionation and a focused RNAi approach that Nup155 controls mRNA translation of p21 (CDKN1A), a key mediator of the p53 response. The underlying mechanism involves transcriptional regulation of the putative tRNA and rRNA methyltransferase FTSJ1 by Nup155. Furthermore, we observe that Nup155 and FTSJ1 are p53 repression targets and accordingly find a correlation between the p53 status, Nup155 and FTSJ1 expression in murine and human hepatocellular carcinoma. Our data suggest an unanticipated regulatory network linking translational control by and repression of a structural NPC component modulating the p53 pathway through its effectors.


Assuntos
Carcinoma Hepatocelular/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias Hepáticas/patologia , Metiltransferases/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Metiltransferases/metabolismo , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo
16.
Sci Rep ; 8(1): 11249, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30050042

RESUMO

The nuclear pore complex (NPC) is a large macromolecular assembly of around 30 different proteins, so-called nucleoporins (Nups). Embedded in the nuclear envelope the NPC mediates bi-directional exchange between the cytoplasm and the nucleus and plays a role in transcriptional regulation that is poorly understood. NPCs display modular arrangements with an overall structure that is generally conserved among many eukaryotic phyla. However, Nups of yeast or human origin show little primary sequence conservation with those from early-branching protozoans leaving those of the malaria parasite unrecognized. Here we have combined bioinformatic and genetic methods to identify and spatially characterize Nup components in the rodent infecting parasite Plasmodium berghei and identified orthologs from the human malaria parasite P. falciparum, as well as the related apicomplexan parasite Toxoplasma gondii. For the first time we show the localization of selected Nups throughout the P. berghei life cycle. Largely restricted to apicomplexans we identify an extended C-terminal poly-proline extension in SEC13 that is essential for parasite survival and provide high-resolution images of Plasmodium NPCs obtained by cryo electron tomography. Our data provide the basis for full characterization of NPCs in malaria parasites, early branching unicellular eukaryotes with significant impact on human health.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/análise , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Plasmodium berghei/enzimologia , Biologia Computacional , Genes Essenciais , Biologia Molecular , Plasmodium berghei/genética , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Toxoplasma/enzimologia , Toxoplasma/genética
17.
Genome Biol ; 17: 47, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26975353

RESUMO

BACKGROUND: Recent large-scale studies revealed cell-type specific proteomes. However, protein complexes, the basic functional modules of a cell, have been so far mostly considered as static entities with well-defined structures. The co-expression of their members has not been systematically charted at the protein level. RESULTS: We used measurements of protein abundance across 11 cell types and five temporal states to analyze the co-expression and the compositional variations of 182 well-characterized protein complexes. We show that although the abundance of protein complex members is generally co-regulated, a considerable fraction of all investigated protein complexes is subject to stoichiometric changes. Compositional variation is most frequently seen in complexes involved in chromatin regulation and cellular transport, and often involves paralog switching as a mechanism for the regulation of complex stoichiometry. We demonstrate that compositional signatures of variable protein complexes have discriminative power beyond individual cell states and can distinguish cancer cells from healthy ones. CONCLUSIONS: Our work demonstrates that many protein complexes contain variable members that cause distinct stoichometries and functionally fine-tune complexes spatiotemporally. Only a fraction of these compositional variations is mediated by changes in transcription and other mechanisms regulating protein abundance contribute to determine protein complex stoichiometries. Our work highlights the superior power of proteome profiles to study protein complexes and their variants across cell states.


Assuntos
Linhagem da Célula/genética , Cromatina/genética , Complexos Multiproteicos/química , Proteoma/genética , Animais , Cromatina/química , Humanos , Mamíferos , Complexos Multiproteicos/genética , Estrutura Terciária de Proteína , Transcrição Gênica
18.
Cancer Lett ; 223(2): 303-12, 2005 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-15896465

RESUMO

The binding of PTEN to PDZ-domain-containing proteins appears to play an important role in the control of cell growth, motility and apoptosis. In turn, this binding can be abrogated by cleavage of the PTEN C-terminal region by caspase-3. We have generated and characterized monoclonal antibodies (mAb) directed against distinct epitopes at the C-terminal region of PTEN, and used them to define protein-binding epitopes on PTEN and to study its cleavage by caspase-3. mAb directed against epitopes at the far C-terminus of PTEN blocked binding to PTEN cognate PDZ domains and did not recognize the caspase-3 cleaved PTEN fragments. On the other hand, mAb that recognized an epitope within the C2 domain of PTEN did not prevent binding to PDZ domains, but could detect the caspase-3 cleaved PTEN fragments. The analysis of PTEN cleavage by caspase-3 revealed that the lipid phosphatase activity of PTEN controls its own degradation by interfering with the PI3-K anti-apoptotic activity. Our results define protein-binding sites on the PTEN tumor suppressor at the immunochemical level, and suggest a regulatory link between PTEN phosphatase activity, caspase-3 sensitivity and PTEN-protein interactions.


Assuntos
Sítios de Ligação , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Caspase 3 , Caspases/metabolismo , Epitopos , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Proteínas de Neoplasias , Neoplasias/fisiopatologia , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Supressoras de Tumor/metabolismo
19.
Methods Cell Biol ; 122: 117-46, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24857728

RESUMO

Accurate knowledge of the stoichiometry of protein complexes is a crucial prerequisite for understanding their structure and function. To purify or enrich large and intricate protein complexes such that their structure is preserved and to absolutely quantify all of their protein components is an enormous technical challenge. In this chapter, we describe how to purify nuclear envelopes from human tissue culture cells that are highly enriched for nuclear pore complexes. We use the nuclear pore as an example to discuss how the structural preservation of such preparations can be controlled. Furthermore, we give a practical guide how to develop and employ targeted proteomic assays for both, the absolute quantification of stoichiometries and the relative quantification of protein complex composition across multiple biological conditions. The concept discussed here is universally applicable to any protein complex.


Assuntos
Complexos Multiproteicos/isolamento & purificação , Complexo de Proteínas Formadoras de Poros Nucleares/isolamento & purificação , Poro Nuclear/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Complexos Multiproteicos/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
20.
Cell Signal ; 24(2): 577-587, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22036806

RESUMO

The tumor suppressor activity of p27Kip1 takes place in the cell nucleus by inhibitory binding to cyclin/CDK complexes. p27Kip1 can also be localized in the cytoplasm, where it has been proposed to have oncogenic properties. Here, we describe a novel role for cytoplasmic p27Kip1 which could account for its activity as an oncoprotein by negative regulation of the PTEN tumor suppressor. p27Kip1 physically interacted with the open conformation of PTEN, which is competent to enter the nucleus. In mammalian cells, cytoplasmic p27Kip1 retained to nuclear-targeted PTEN in the cytoplasm. This retention was exerted by the C-terminal p27Kip1 region, and was independent of cyclin/CDK-binding. The nuclear accumulation of PTEN triggered by pro-apoptotic TNFα treatment was abolished by cytoplasmic p27Kip1. Furthermore, conformationally-open PTEN displayed diminished protein stability and pro-apoptotic activity in the presence of cytoplasmic p27Kip1. Our results support a conformationally-dependent model of cytoplasmic retention and negative regulation of the activity of nuclear PTEN by oncogenic cytoplasmic p27Kip1, and suggest the existence of reciprocal mechanisms to regulate the levels of both p27Kip1 and PTEN.


Assuntos
Apoptose/genética , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Citosol/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Animais , Apoptose/efeitos dos fármacos , Células COS , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p27/genética , Células HEK293 , Humanos , Células K562 , PTEN Fosfo-Hidrolase/genética , Fosforilação/efeitos dos fármacos , Plasmídeos , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA