RESUMO
Functional analyses of PDB (Paget's disease of bone)-associated mutants of the p62 [also known as SQSTM1 (sequestosome 1)] signalling adaptor protein represent an interesting paradigm for understanding not only the disease mechanism in this skeletal disorder, but also the critical determinants of ubiquitin recognition by an ubiquitin-binding protein. The 11 separate PDB mutations identified to date all affect the C-terminal region of p62 containing the UBA domain (ubiquitin-associated domain), a ubiquitin-binding element. All of these mutations have deleterious effects on ubiquitin binding by p62 in vitro, and there is evidence of an inverse relationship between ubiquitin-binding function and disease severity. The effects on ubiquitin-binding function of most of the mutations can be attributed to either reduced UBA domain stability, and/or the mutations affecting the presumed ubiquitin-binding interface of the UBA domain. However, a subset of the mutations are more difficult to rationalize; several of these affect sequences of p62 outside of the minimal ubiquitin-binding region, providing insights into non-UBA domain sequences within the host protein which mediate ubiquitin-binding affinity. The p62 mutations are presumed to result in activation of (osteoclast) NF-kappaB (nuclear factor kappaB) signalling. Understanding how loss of ubiquitin-binding function of p62 impacts on signal transduction events in osteoclasts will undoubtedly further our understanding of the disease mechanism in PDB at the molecular level.
Assuntos
Mutação , Osteíte Deformante/genética , Proteínas/genética , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Humanos , Fenótipo , Ligação Proteica , Proteínas/metabolismo , Proteína Sequestossoma-1RESUMO
Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget's disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.
Assuntos
Osso e Ossos/metabolismo , Mutação de Sentido Incorreto/genética , Osteíte Deformante/genética , Osteíte Deformante/metabolismo , Proteínas/genética , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação/genética , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Predisposição Genética para Doença/genética , Humanos , Osteíte Deformante/fisiopatologia , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas/química , Proteínas/metabolismo , Proteína Sequestossoma-1RESUMO
Mutations affecting the UBA (ubiquitin-associated) domain of SQSTM1 (Sequestosome 1) (p62) are a common cause of Paget's disease of bone. The missense mutations resolve into those which retain [P392L (Pro(392)-->Leu), G411S] or abolish (M404V, G425R) the ability of the isolated UBA domain to bind Lys-48-linked polyubiquitin. These effects can be rationalized with reference to the solution structure of the UBA domain, which we have determined by NMR spectroscopy. The UBA domain forms a characteristic compact three-helix bundle, with a hydrophobic patch equivalent to that previously implicated in ubiquitin binding by other UBA domains. None of the mutations affect overall folding of the UBA domain, but both M404V and G425R involve residues in the hydrophobic patch, whereas Pro-392 and Gly-411 are more remote. A simple model assuming the isolated UBA domain is functioning as a compact monomer can explain the effects of the mutations on polyubiquitin binding. The P392L and G411S mutations do however have subtle local effects on secondary structure, which may become more relevant in full-length SQSTM1. Identification of the in vivo ubiquitylated substrates of SQSTM1 will be most informative in determining the functional significance of the SQSTM1-ubiquitin interaction, and consequences of the disease-associated mutations.
Assuntos
Mutação , Osteíte Deformante/genética , Proteínas/química , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Glicina/química , Humanos , Espectroscopia de Ressonância Magnética , Mutação de Sentido Incorreto , Poliubiquitina/química , Prolina/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/fisiologia , Proteína Sequestossoma-1 , Relação Estrutura-Atividade , Ubiquitina/químicaRESUMO
Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.
Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/metabolismo , Transativadores , Transcrição Gênica , Proteínas de Peixe-Zebra , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lítio/farmacologia , Luciferases/genética , Microscopia de Fluorescência , Presenilina-1 , Transdução de Sinais , Frações Subcelulares/metabolismo , Proteínas Wnt , beta CateninaRESUMO
Monoclonal antibodies to myc, c-erbB-2 and epidermal growth factor-receptor (EGF-R) were raised using a synthetic peptide approach. The antibodies were characterised by ELISA, immunoblotting, immunoprecipitation and immunocytochemical procedures against cognate peptide and native proteins. All of the monoclonal antibodies detected peptide-blockable bands of appropriate molecular weight (myc-p62/66 kDa, c-erbB-2-185kDa; EGF-R-150/170 kDa) on immunoblots. The monoclonal antibodies to c-erbB-2 and EGF-R immunostained subpopulations of tumour cells on sections of formalin-fixed, paraffin wax embedded human infiltrating and invasive ductal carcinomas of breast. Intense blood cell staining was observed with the EGF-R antibody. This staining was shown to be peptide blockable and may reveal a true localisation for the EGF-receptor protein, a closely-related (erbB) protein or a degradation product. The monoclonal antibody to a common peptide from the myc protein family was epitope scanned using a modification of the Geysen pin technique. Hexapeptide sequence Ala-Pro-Ser-Glu-Asp-Ile was found to be bound most strongly by the myc monoclonal antibody, and amino acids Pro2 and Glu4 were found to be essential for antibody binding. The use of synthetic peptides for the production of monoclonal antibodies with predetermined specificity, which may be precisely identified using the epitope scanning technique, is discussed.
Assuntos
Anticorpos Monoclonais/biossíntese , Receptores ErbB/imunologia , Proteínas Oncogênicas Virais/imunologia , Proteínas Oncogênicas de Retroviridae/imunologia , Sequência de Aminoácidos , Epitopos/análise , Humanos , Hibridomas/imunologia , Dados de Sequência Molecular , Proteína Oncogênica p55(v-myc) , Proteínas Oncogênicas v-erbB , Peptídeos/imunologia , Células Tumorais Cultivadas , Vacinas SintéticasRESUMO
Although medulloblastoma and neuroblastoma share many common biological, histological and immunological features, the frequency of N-myc amplification differs markedly between the two tumours. In this study, Southern blot analysis revealed that the N-myc gene was not amplified in any of the nine medulloblastoma samples analysed. In contrast, over-expression of the gene was found in six of 11 samples as determined by immunocytochemistry and/or Western blot analysis, using an antiserum raised against a synthetic peptide representing a sequence unique to the N-myc gene product. The specificity of this reagent was demonstrated by studies on a variety of cell lines expressing N-myc and/or c-myc oncoproteins. Of the 12 medulloblastoma samples collected over a two-year period and analysed in the course of this project, a trend towards longer disease-free survival was noted in the patients having low levels of the N-myc protein in their tumour.