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1.
Mol Biol Cell ; 26(15): 2801-9, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26063733

RESUMO

Correct protein folding is fundamental for maintaining protein homeostasis and avoiding the formation of potentially cytotoxic protein aggregates. Although some proteins appear to fold unaided, actin requires assistance from the oligomeric molecular chaperone CCT. Here we report an additional connection between CCT and actin by identifying one of the CCT subunits, CCTε, as a component of the myocardin-related cotranscription factor-A (MRTF-A)/serum response factor (SRF) pathway. The SRF pathway registers changes in G-actin levels, leading to the transcriptional up-regulation of a large number of genes after actin polymerization. These genes encode numerous actin-binding proteins as well as actin. We show that depletion of the CCTε subunit by siRNA enhances SRF signaling in cultured mammalian cells by an actin assembly-independent mechanism. Overexpression of CCTε in its monomeric form revealed that CCTε binds via its substrate-binding domain to the C-terminal region of MRTF-A and that CCTε is able to alter the nuclear accumulation of MRTF-A after stimulation by serum addition. Given that the levels of monomeric CCTε conversely reflect the levels of CCT oligomer, our results suggest that CCTε provides a connection between the actin-folding capacity of the cell and actin expression.


Assuntos
Actinas/metabolismo , Chaperonina com TCP-1/metabolismo , Fator de Resposta Sérica/metabolismo , Animais , Células 3T3 BALB , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Chaperonina com TCP-1/genética , Humanos , Camundongos , Proteínas dos Microfilamentos/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transativadores/metabolismo
2.
BMC Res Notes ; 6: 429, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24156781

RESUMO

BACKGROUND: The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of ß-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. RESULTS: In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1-2843) and cancer-associated, truncated APC proteins, APC(1-1638) and APC(1-1311) were produced in Sf9 insect cells. CONCLUSIONS: Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.


Assuntos
Proteína da Polipose Adenomatosa do Colo/isolamento & purificação , Anticorpos Monoclonais/biossíntese , Cromatografia de Afinidade/métodos , Proteínas Recombinantes/isolamento & purificação , Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/administração & dosagem , Antígenos/química , Baculoviridae/genética , Cães , Expressão Gênica , Humanos , Células Madin Darby de Rim Canino , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera
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