RESUMO
Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a beta domain, which protrudes from the AAA domain, and a final C-terminal alpha-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the beta domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the beta domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Bases , Primers do DNA , Complexos Endossomais de Distribuição Requeridos para Transporte , Genótipo , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Plasmídeos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy.
Assuntos
Ciclo Celular , Proliferação de Células , Intestino Delgado/metabolismo , Mutação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fatores Etários , Animais , Diferenciação Celular , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Genótipo , Intestino Delgado/diagnóstico por imagem , Intestino Delgado/efeitos dos fármacos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Radiografia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
c-Met is a tyrosine receptor kinase which is activated by its ligand, the hepatocyte growth factor. Activation of c-Met leads to a wide spectrum of biological activities such as motility, angiogenesis, morphogenesis, cell survival and cell regeneration. c-Met is abnormally activated in many tumour types. Aberrant c-Met activation was found to induce tumour development, tumour cell migration and invasion, and the worst and final step in cancer progression, metastasis. In addition, c-Met activation in cells was also shown to confer resistance to apoptosis induced by UV damage or chemotherapeutic drugs. This study describes the development of monoclonal antibodies against c-Met as therapeutic molecules in cancer treatment/diagnostics. A panel of c-Met monoclonal antibodies was developed and characterised by epitope mapping, Western blotting, immunoprecipitation, agonist/antagonist effect in cell scatter assays and for their ability to recognise native c-Met by flow cytometry. We refer to these antibodies as Specifically Engaging Extracellular c-Met (seeMet). seeMet 2 and 13 bound strongly to native c-Met in flow cytometry and reduced SNU-5 cell growth. Interestingly, seeMet 2 binding was strongly reduced at 4oC when compared to 37oC. Detail mapping of the seeMet 2 epitope indicated a cryptic binding site hidden within the c-Met α-chain.