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1.
J Biol Chem ; 292(10): 4336-4349, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28126905

RESUMO

Microtubule dynamics involves the polymerization and depolymerization of tubulin dimers and is an essential and highly regulated process required for cell viability, architecture, and division. The regulation of the microtubule network also depends on the maintenance of a pool of αß-tubulin heterodimers. These dimers are the end result of complex folding and assembly events, requiring the TCP1 Ring Complex (TriC or CCT) chaperonin and five tubulin-specific chaperones, tubulin binding cofactors A-E (TBCA-TBCE). However, models of the actions of these chaperones are incomplete or inconsistent. We previously purified TBCD from bovine tissues and showed that it tightly binds the small GTPase ARL2 but appears to be inactive. Here, in an effort to identify the functional form of TBCD and using non-denaturing gels and immunoblotting, we analyzed lysates from a number of mouse tissues and cell lines to identify the quaternary state(s) of TBCD and ARL2. We found that both proteins co-migrated in native gels in a complex of ∼200 kDa that also contained ß-tubulin. Using human embryonic kidney cells enabled the purification of the TBCD·ARL2·ß-tubulin trimer found in cell and tissue lysates as well as two other novel TBCD complexes. Characterization of ARL2 point mutants that disrupt binding to TBCD suggested that the ARL2-TBCD interaction is critical for proper maintenance of microtubule densities in cells. We conclude that the TBCD·ARL2·ß-tubulin trimer represents a functional complex whose activity is fundamental to microtubule dynamics.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Bovinos , Proteínas de Ligação ao GTP/química , Células HeLa , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/química , Células NIH 3T3 , Ligação Proteica , Dobramento de Proteína , Tubulina (Proteína)/química
2.
Mol Cell Biol ; 26(21): 8022-31, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16954387

RESUMO

Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a gamma-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with gamma-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.


Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia de Células T/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Notch1/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Receptor Notch1/genética , Retroviridae/genética , Retroviridae/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T
3.
Mol Biol Cell ; 20(10): 2593-604, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19321666

RESUMO

Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus.


Assuntos
Conexina 43/química , Conexina 43/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Hexaclorocicloexano/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos
4.
J Biol Chem ; 283(11): 7155-65, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18171676

RESUMO

Microtubules are highly dynamic structures, composed of alpha/beta-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the full-length protein or either of two centrosome localization domains leads to the loss of anchoring of the gamma-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of alpha-or beta-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.


Assuntos
Centrossomo/metabolismo , Centrossomo/ultraestrutura , Fuso Acromático , Tubulina (Proteína)/metabolismo , Animais , Bovinos , Células HeLa , Humanos , Microscopia de Fluorescência , Microssomos/metabolismo , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo
5.
Hum Mol Genet ; 12(9): 1029-37, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12700171

RESUMO

Corneal disease is the most common cause of bilateral blindness in the world. Visual loss in this condition is often due to changes in morphology and function of the corneal epithelial surface. Corneal disease-1 (corn1) and corn1(2J) are spontaneous mouse mutants that develop irregular thickening of the corneal epithelium, similar to that observed in human corneal surface disease. These autosomal-recessive mutations cause an increase in the rate of proliferation of the corneal epithelial cells. Here, we report that the phenotypes in both mutants are caused by mutations within the destrin gene (also known as actin-depolymerizing factor). By positional cloning, we identified a deletion encompassing the entire coding sequence of the destrin gene in corn1 mice, and a point mutation (Pro106Ser) in the coding sequence of destrin in corn1(2J) mice. In situ analysis showed that destrin is highly expressed in the corneal epithelium. Consistent with the cellular roles for destrin, an essential regulator of actin filament turnover that acts by severing and enhancing depolymerization of actin filament, we observed that the corn1 mutations increased the content of filamentous actin in corneal epithelial cells. Our results suggest an in vivo connection between remodeling of the actin cytoskeleton and the control of cell proliferation, and a new pathway through which an aberrant actin cytoskeleton can cause epithelial hyperproliferation.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Divisão Celular/fisiologia , Córnea/metabolismo , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Fatores de Despolimerização de Actina , Animais , Destrina , Epitélio/metabolismo , Camundongos , Deleção de Sequência
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