RESUMO
The Ccr4-Not complex is evolutionarily conserved and important for regulation of mRNA synthesis and decay. The composition of the yeast complex has been well described. Orthologues of the yeast Ccr4-Not components have been identified in human cells including multiple subunits with mRNA deadenylase activity. In the present study, we examine the composition of the human Ccr4-Not complex in an in-depth proteomic approach using stable cell lines expressing tagged CNOT proteins. We find at least four different variants of the human complex, consisting of seven stable core proteins and mutually exclusive associated mRNA deadenylase subunits. Interestingly, human CNOT4 is in a separate approximately 200 kDa complex. Furthermore, analyses of associated proteins indicate involvement of Ccr4-Not complexes in splicing, transport and localization of RNA molecules. Taken together, human Ccr4-Not complexes are heterogeneous in composition owing to differences in their deadenylase subunits, which may reflect the multi-functionality of these complexes in cellular processes.
Assuntos
Fatores de Transcrição/metabolismo , Transporte Biológico , Linhagem Celular , Cromatografia em Gel , Biologia Computacional , Exorribonucleases/genética , Exorribonucleases/metabolismo , Células HeLa , Humanos , Ligação Proteica , Proteômica , Splicing de RNA/genética , Splicing de RNA/fisiologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Espectrometria de Massas em Tandem , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoAssuntos
Inibidores da Angiogênese/uso terapêutico , Ranibizumab/uso terapêutico , Degeneração Macular Exsudativa/diagnóstico , Degeneração Macular Exsudativa/tratamento farmacológico , Humanos , Injeções Intravítreas , Países Baixos , Estudos Retrospectivos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Acuidade Visual/fisiologia , Degeneração Macular Exsudativa/fisiopatologiaRESUMO
BACKGROUND: The evolutionarily conserved Ccr4-Not and Bur1/2 kinase complexes are functionally related in Saccharomyces cerevisiae. In this study, we further explore the relationship between the subunits Not4p and Bur2p. METHODOLOGY/PRINCIPAL FINDINGS: First, we investigated the presence of post-translational modifications on the Ccr4-Not complex. Using mass spectrometry analyses we identified several SP/TP phosphorylation sites on its Not4p, Not1p and Caf1p subunits. Secondly, the influence of Not4p phosphorylation on global H3K4 tri-methylation status was examined by immunoblotting. This histone mark is severely diminished in the absence of Not4p or of Bur2p, but did not require the five identified Not4p phosphorylation sites. Thirdly, we found that Not4p phosphorylation is not affected by the kinase-defective bur1-23 mutant. Finally, phenotypic analyses of the Not4p phosphomutant (not4S/T5A) and bur2Delta strains showed overlapping sensitivities to drugs that abolish cellular stress responses. The double-mutant not4S/T5A and bur2Delta strain even revealed enhanced phenotypes, indicating that phosphorylation of Not4p and BUR2 are active in parallel pathways for drug tolerance. CONCLUSIONS: Not4p is a phospho-protein with five identified phosphorylation sites that are likely targets of a cyclin-dependent kinase(s) other than the Bur1/2p complex. Not4p phosphorylation on the five Not4 S/T sites is not required for global H3K4 tri-methylation. In contrast, Not4p phosphorylation is involved in tolerance to cellular stresses and acts in pathways parallel to BUR2 to affect stress responses in Saccharomyces cerevisiae.
Assuntos
Ciclinas/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico , Ubiquitina-Proteína Ligases/fisiologia , Ciclinas/metabolismo , Histonas/metabolismo , Metilação , Complexos Multiproteicos , Mutação , Fosforilação , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
STUDY DESIGN: A prospective study of multifidus images. OBJECTIVE.: To compare the change of the cross-sectional area (CSA) of multifidus in subjects with and without chronic low back pain (LBP) at different postures. SUMMARY OF BACKGROUND DATA: Ultrasound imaging was shown to be useful in assessing acute LBP patients at rest only. METHODS AND MEASURES: Thirty-five male adults participated, including 19 normal and 16 chronic LBP patients. The CSAs of multifidus were assessed on ultrasound images acquired on both sides of the L4 and L5 levels with the subjects in prone lying, upright standing, and 25 degrees and 45 degrees forward stooping. RESULTS: In the control group, the CSA of multifidus increased from prone lying to upright standing and then gradually decreased from 25 degrees to 45 degrees forward stooping. However, a reverse pattern of the CSA changes was identified in patients with chronic LBP. CONCLUSION: Multifidus contracts maximally at upright standing in the normal group, while maximum contraction of the muscle occurs at 25 degrees forward stooping in the patient group. The role of multifidus may be altered in the stabilization of the lumbar spine of chronic LBP patients. Assessment of the change in muscle size reacting to different postures is found to be helpful.