RESUMO
The unfolded protein response (UPR) is an adaptive cellular program used by eukaryotic cells to cope with protein misfolding stress. During tumor development, cancer cells are facing intrinsic (oncogene activation) and extrinsic (limiting nutrient or oxygen supply) challenges, with which they must cope to survive. Moreover, chemotherapy represents an additional extrinsic challenge that cancer cells are facing and to which they adapt in the case of resistance. As of today, resistance to chemotherapy and targeted therapies is one of the important issues that oncologists have to deal with for treating cancer patients. In this review, we first describe the key molecular mechanisms controlling the UPR and their implication in solid cancers. Then, we review the literature that connects cancer chemotherapy resistance mechanisms and activation of the UPR. Finally, we discuss the possible applications of targeting the UPR to bypass drug resistance.
RESUMO
Quantitative proteomics represents a powerful approach for the comprehensive analysis of proteins expressed under defined conditions. These properties have been used to investigate the proteome of disease states, including cancer. It has become a major subject of studies to apply proteomics for biomarker and therapeutic target identification. In the last decades, technical advances in mass spectrometry have increased the capacity of protein identification and quantification. Moreover, the analysis of posttranslational modification (PTM), especially phosphorylation, has allowed large-scale identification of biological mechanisms. Even so, increasing evidence indicates that global protein quantification is often insufficient for the explanation of biology and has shown to pose challenges in identifying new and robust biomarkers. As a consequence, to improve the accuracy of the discoveries made using proteomics in human tumors, it is necessary to combine (i) robust and reproducible methods for sample preparation allowing statistical comparison, (ii) PTM analyses in addition to global proteomics for additional levels of knowledge, and (iii) use of bioinformatics for decrypting protein list. Herein, we present technical specificities for samples preparation involving isobaric tag labeling, TiO2-based phosphopeptides enrichment and hydrazyde-based glycopeptides purification as well as the key points for the quantitative analysis and interpretation of the protein lists. The method is based on our experience with tumors analysis derived from hepatocellular carcinoma, chondrosarcoma, human embryonic intervertebral disk, and chordoma experiments.
Assuntos
Proteínas de Neoplasias/química , Neoplasias/química , Proteoma/química , Animais , Cromatografia Líquida de Alta Pressão , Glicopeptídeos/química , Glicopeptídeos/isolamento & purificação , Humanos , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Proteoma/isolamento & purificação , Proteoma/metabolismo , Proteômica , Análise de Sequência de Proteína , Espectrometria de Massas em TandemRESUMO
In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.
Assuntos
Autofagia , Doenças do Sistema Nervoso , Deficiências na Proteostase/complicações , Resposta a Proteínas não Dobradas/fisiologia , Animais , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Doenças do Sistema Nervoso/complicações , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/terapia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
Inositol-requiring enzyme 1 (IRE1) is the most conserved transducer of the unfolded protein response (UPR), a homeostatic response that preserves proteostasis. Intriguingly, via its endoribonuclease activity, IRE1 produces either adaptive or death signals. This occurs through both unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay of mRNA (RIDD). Whereas XBP1 mRNA splicing is cytoprotective in response to endoplasmic reticulum (ER) stress, RIDD has revealed many unexpected features. For instance, RIDD cleaves RNA at an XBP1-like consensus site but with an activity divergent from XBP1 mRNA splicing and can either preserve ER homeostasis or induce cell death. Here we review recent findings on RIDD and propose a model of how IRE1 RNase activity might control cell fate decisions.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Endorribonucleases/metabolismo , Humanos , Ribonucleases/metabolismoRESUMO
Clinical studies have defined the core 'genetic blueprint' of a cancer cell, but this information does not necessarily predict the cancer phenotype. Signalling hubs that mediate such phenotype have been identified largely using OMICS platforms that measure dynamic molecular changes within the cancer cell landscape. The pro-oncogenic protein anterior gradient 2 (AGR2) is a case in point; AGR2 has been shown using a range of expression platforms to be involved in asthma, inflammatory bowel disease, cell transformation, cancer drug resistance and metastatic growth. AGR2 protein is also highly overexpressed in a diverse range of human cancers and can be secreted and detected in extracellular fluids, thus representing a compelling pro-oncogenic signalling intermediate in human cancer. AGR2 belongs to the protein disulphide isomerase family with all the key features of an endoplasmic reticulum-resident protein-this gives clues into how it might function as an oncoprotein through the regulation of protein folding, maturation and secretion that can drive metastatic cell growth. In this review, we will describe the known aspects of AGR2 molecular biology, including gene structure and regulation, emerging protein interaction networks and how its subcellular localization mediates its biological functions. We will finally review the cases of AGR2 expression in human cancers, the pathophysiological consequences of AGR2 overexpression, its potential role as a tumour biomarker that predicts the response to therapy and how the AGR2 pathway might form the basis for drug discovery programmes aimed at targeting protein folding/maturation pathways that mediate secretion and metastasis.
Assuntos
Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Proteínas/genética , Proteínas/metabolismo , Motivos de Aminoácidos , Androgênios/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Resistencia a Medicamentos Antineoplásicos , Estrogênios/metabolismo , Líquido Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mucoproteínas , Família Multigênica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Proteínas Oncogênicas , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas , Tamoxifeno/farmacologiaRESUMO
Orthotopic liver transplantation (OLT) continues to be the only remedy for end-stage liver disease. In an attempt to decrease the ever-widening gap between organ donor and recipient numbers, and ultimately make more livers amenable to transplantation, we characterized the healthy human liver's response to ischemia and reperfusion-induced injury during transplantation. This was carried out by transcriptional profiling using cDNA microarray to identify genes whose expression was modulated at the 1-h postreperfusion time point. We observed that the map kinase phosphatase-1/dual-specificity phosphatase-1 (MKP-1/DUSP1) mRNA was strongly and significantly upregulated. Validation of this observation was carried out using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunohistochemistry. In addition, we characterized the signaling pathways regulating MKP-1 expression using the human hepatoma cell line HepG2. Finally, by combining MKP-1 silencing with reperfusion-associated stresses, we reveal the preferential role of this protein in attenuating the activity of the JNK and p38(MAPK) pathways, and the resulting apoptosis, making MKP-1 a potential target for therapeutic intervention.
Assuntos
Fosfatase 1 de Especificidade Dupla/metabolismo , Transplante de Fígado/fisiologia , Fígado/metabolismo , Traumatismo por Reperfusão/metabolismo , Apoptose/fisiologia , Biópsia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Fosfatase 1 de Especificidade Dupla/genética , Humanos , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , MAP Quinase Quinase 4/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The lysosomal cysteine proteinase cathepsin L is involved in proteolytic processing of internalized proteins. In transformed cells, where it is frequently overexpressed, its intracellular localization and functions can be altered. Previously, we reported that treatment of highly metastatic, murine carcinoma H-59 cells with small molecule cysteine proteinase inhibitors altered the responsiveness of the type I insulin-like growth factor (IGF-I) receptor and consequently reduced cell invasion and metastasis. To assess more specifically the role of cathepsin L in IGF-I-induced signaling and tumorigenicity, we generated H-59 subclones with reduced cathepsin L expression levels. These clonal lines showed an altered responsiveness to IGF-I in vitro, as evidenced by (i) loss of IGF-I-induced receptor phosphorylation and Shc recruitment, (ii) reduced IGF-I (but not IGF-II)-induced cellular proliferation and migration, (iii) decreased anchorage-independent growth and (iv) reduced plasma membrane levels of IGF-IR. These changes resulted in increased apoptosis in vivo and an impaired ability of the cells to form liver metastases. The results demonstrate that cathepsin L expression levels regulate cell responsiveness to IGF-I and thereby identify a novel function for cathepsin L in the control of the tumorigenic/metastatic phenotype.
Assuntos
Carcinoma/patologia , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Fator de Crescimento Insulin-Like I/uso terapêutico , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/patologia , Animais , Carcinoma/metabolismo , Catepsina L , Catepsinas/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Regulação para Baixo , Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Transplante de Neoplasias , RNA Interferente Pequeno/farmacologia , Células Tumorais CultivadasRESUMO
The endoplasmic reticulum (ER) has evolved specific mechanisms to ensure protein folding as well as the maintenance of its own homeostasis. When these functions are not achieved, specific ER stress signals are triggered to activate either adaptive or apoptotic responses. Here, we demonstrate that MCF-7 cells are resistant to tunicamycin-induced apoptosis. We show that the expression level of the ER chaperone calnexin can directly influence tunicamycin sensitivity in this cell line. Interestingly, the expression of a calnexin lacking the chaperone domain (DeltaE) partially restores their sensitivity to tunicamycin-induced apoptosis. Indeed, we show that DeltaE acts as a scaffold molecule to allow the cleavage of Bap31 and thus generate the proapoptotic p20 fragment. Utilizing the ability of MCF-7 cells to resist tunicamycin-induced apoptosis, we have characterized a molecular mechanism by which calnexin regulates ER-stress-mediated apoptosis in a manner independent of its chaperone functions but dependent of its binding to Bap31.
Assuntos
Neoplasias da Mama/metabolismo , Calnexina/metabolismo , Carcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Tunicamicina/farmacologia , Sequência de Aminoácidos , Apoptose , Calnexina/genética , Calnexina/fisiologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Células Clonais , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Humanos , Dados de Sequência MolecularRESUMO
Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C(6)-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Caenorhabditis elegans/química , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/química , Citoesqueleto/metabolismo , Desenvolvimento Embrionário , Endossomos/química , Endossomos/genética , Endossomos/metabolismo , Células Epiteliais/química , Células Epiteliais/citologia , Camundongos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação/genética , Células NIH 3T3 , Filogenia , Alinhamento de Sequência , Proteínas rho de Ligação ao GTP/genética , Rede trans-Golgi/química , Rede trans-Golgi/metabolismoRESUMO
BACKGROUND: The cellular events following liver ischemia/reperfusion (I/R) during transplantation are largely unknown. The spectrum of I/R damage to the liver can be clinically revealed by the development of primary graft dysfunction or nonfunction. Because viral-induced liver necrosis has been associated with the development of calcifications in an animal model, we investigated the spectrum of I/R changes identified at an ultrastructural level among livers after liver transplant (LT). MATERIALS AND METHODS: Random liver biopsies from five recipients with different degrees of liver dysfunction (LD) were processed for light (LM) and electron (EM) microscopic examination. The degree of calcification was estimated as mild-moderate or severe. The degree of cell vacuolization, used as a surrogate marker of cell necrosis, was reported as mild-moderate or severe. RESULTS: Two patients with severe LD had obvious calcifications by LM and EM examinations. Both showed significant vacuole formation, suggesting a severe degree of cell necrosis, and both succumbed to the sequelae of their LD. One patient showed evidence of mild calcifications at EM (but not LM) examination, with mild vacuole formation. The remaining two patients displayed no microcalcifications. Both presented only mild vacuole formation. Both patients recovered from LD and are currently alive. CONCLUSION: In this preliminary report, we conclude that the clinically observed degree of LD after orthotopic liver transplant (OLT)correlates well with ultrastructural modifications. These include calcification and vacuole formation. We believe that both findings can be used as surrogate markers of a clinically significant hepatic I/R injury.
Assuntos
Transplante de Fígado/patologia , Traumatismo por Reperfusão/patologia , Biópsia , Calcinose/patologia , Sobrevivência de Enxerto , Humanos , Fígado/ultraestrutura , Necrose , Período Pós-OperatórioRESUMO
Schwann cell-derived peripheral myelin protein-22 (PMP-22) when mutated or overexpressed causes heritable neuropathies with a previously unexplained "gain-of-function" endoplasmic reticulum (ER) retention phenotype. In wild-type sciatic nerves, PMP-22 associates in a specific, transient (t(1/2 ) approximately equal to 11 min), and oligosaccharide processing-dependent manner with the lectin chaperone calnexin (CNX), but not calreticulin nor BiP. In Trembler-J (Tr-J) sciatic nerves, prolonged association of mutant PMP-22 with CNX is found (t(1/2) > 60 min). In 293A cells overexpressing PMP-22(Tr-J), CNX and PMP-22 colocalize in large intracellular structures identified at the electron microscopy level as myelin-like figures with CNX localization in the structures dependent on PMP-22 glucosylation. Similar intracellular myelin-like figures were also present in Schwann cells of sciatic nerves from homozygous Trembler-J mice with no detectable activation of the stress response pathway as deduced from BiP and CHOP expression. Sequestration of CNX in intracellular myelin-like figures may be relevant to the autosomal dominant Charcot-Marie-Tooth-related neuropathies.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Mielina/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Nervo Isquiático/fisiologia , Animais , Sequência de Bases , Células COS , Calnexina , Linhagem Celular , Chlorocebus aethiops , Primers do DNA , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas da Mielina/genética , Doenças do Sistema Nervoso/genética , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The receptor for the type 1 insulin-like growth factor (IGF-I) has been implicated in cellular transformation and the acquisition of an invasive/metastatic phenotype in various tumors. Following ligand binding, the IGF-I receptor is internalized, and the receptor.ligand complex dissociates as the ligand is degraded by endosomal proteinases. In the present study we show that the inhibition of endosomal IGF-I-degrading enzymes in human breast and murine lung carcinoma cells by the cysteine proteinase inhibitors, E-64 and CA074-methyl ester, profoundly altered receptor trafficking and signaling. In treated cells, intracellular ligand degradation was blocked, and although the receptor and two substrates, Shc and Insulin receptor substrate, were hyperphosphorylated on tyrosine, IGF-I-induced DNA synthesis, anchorage-independent growth, and matrix metalloproteinase synthesis were inhibited. The results suggest that ligand processing by endosomal proteinases is a key step in receptor signaling and function and a potential target for therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Inibidores de Cisteína Proteinase/farmacologia , Endossomos/metabolismo , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/metabolismo , Leucina/análogos & derivados , Receptor IGF Tipo 1/metabolismo , Animais , Western Blotting , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , DNA/biossíntese , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Endossomos/enzimologia , Feminino , Citometria de Fluxo , Humanos , Cinética , Leucina/farmacologia , Ligantes , Fígado/metabolismo , Masculino , Camundongos , Modelos Biológicos , Metástase Neoplásica , Fosforilação , Testes de Precipitina , Ligação Proteica/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fatores de Tempo , Células Tumorais Cultivadas , Tirosina/metabolismoAssuntos
Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Lectinas/metabolismo , Chaperonas Moleculares/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Calnexina , Calreticulina , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Lectinas/química , Chaperonas Moleculares/química , Dados de Sequência Molecular , Dobramento de Proteína , Ribonucleoproteínas/químicaRESUMO
The endoplasmic reticulum is the entry point into the secretory pathway. To acquire a correct conformation, secretory proteins encounter the endoplasmic reticulum molecular machines of folding, quality control, signaling and disposal, which function as an integrated mechanism. The creation of such a molecular network, spatially regulated, suggests how the endoplasmic reticulum promotes the release of correctly folded secretory proteins.
Assuntos
Retículo Endoplasmático/fisiologia , Dobramento de Proteína , Proteínas/metabolismo , Modelos Biológicos , Transporte Proteico , Transdução de SinaisRESUMO
A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.
Assuntos
Complexo de Golgi/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteoma/análise , Sequência de Aminoácidos , Animais , Células Cultivadas , Complexo de Golgi/ultraestrutura , Dados de Sequência Molecular , Neurônios/química , Octoxinol , Polietilenoglicóis/química , Ratos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/químicaRESUMO
The ATPase associated with different cellular activities family member p97, associated p47, and the t-SNARE syntaxin 5 are necessary for the cell-free reconstitution of transitional endoplasmic reticulum (tER) from starting low-density microsomes. Here, we report that membrane-associated tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities regulate tER assembly by stabilizing (PTPase) or destabilizing (tyrosine kinase) p97 association with membranes. Incubation with the PTPase inhibitor bpV(phen) inhibited tER assembly coincident with the enhanced tyrosine phosphorylation of endogenous p97 and its release from membranes. By contrast, the tyrosine kinase inhibitor, genistein, promoted tER formation and prevented p97 dissociation from membranes while increasing p97 association with the t-SNARE syntaxin 5. Purification of the endogenous tyrosine kinase activity from low-density microsomes led to the identification of JAK-2, whereas PTPH1 was identified as the relevant PTPase. The p97 tyrosine phosphorylation state is proposed to coordinate the assembly of the tER as a regulatory step of the early secretory pathway.
Assuntos
Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/ultraestrutura , Proteínas Nucleares/metabolismo , Tirosina/metabolismo , Animais , Sistema Livre de Células , Fosforilação , Testes de Precipitina , RatosRESUMO
Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost alpha-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum. We have isolated two cDNA isoforms of the human alpha subunit (alpha1 and alpha2) differing by a 66 bp stretch, and a cDNA for the corresponding beta subunit. The alpha1 and alpha2 forms have distinct mobilities on SDS-PAGE and are expressed in most of the cell lines we have tested, but were absent from the glucosidase II-deficient cell line PHA(R) 2.7. Using COS7 cells, the coexpression of the beta subunit with the catalytic alpha subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the alpha/beta complex. Transfected cell extracts expressing either alpha1 or alpha2 forms with the beta subunit showed similar activities, while mutating( )the nucleophile (D542N) predicted from the glycoside hydrolase Family 31 active site consensus sequence abolished enzymatic activity. In order to compare the kinetic parameters of both alpha1/beta and alpha2/beta forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human alpha or beta subunit alone led to the formation of active human/insect heteroenzymes, demonstrating functional complementation by the endogenous insect glucosidase II subunits. The activity of both forms of recombinant human glucosidase II was examined with a p-nitrophenyl alpha-D-glucopyranoside substrate, and a two binding site kinetic model for this substrate was shown. The K(M1-2) values and apparent K(i1-2 )for deoxynojirimycin and castanospermine were determined and found to be identical for both isoforms suggesting they have similar catalysis and inhibition characteristics. The substrate specificities of both isoforms using the physiological oligosaccharides were assessed and found to be similar.
Assuntos
alfa-Glucosidases/metabolismo , Animais , Sequência de Bases , Células COS , Clonagem Molecular , Primers do DNA , DNA Complementar , Dimerização , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Relação Estrutura-Atividade , Especificidade por Substrato , alfa-Glucosidases/química , alfa-Glucosidases/genéticaRESUMO
The effects of Fibroblast Growth Factor-2 (FGF-2) on breast cancer cell DNA synthesis are controversial. To elucidate the mechanisms by which FGF-2 stimulates or inhibits DNA synthesis, we analysed FGF-2 signaling pathways in breast cancer MCF-7 and MCF-7 cells overexpressing Ha-Ras (MCF-7ras). We found that FGF-2-induction of DNA synthesis correlates with Ras transient activation, FRS-2 tyrosine phosphorylation and low level of expression of p66Shc. In addition, Nck-associated proteins are highly tyrosine phosphorylated and JNK reaches a higher level of activation when FGF-2 triggers DNA synthesis. Interestingly upon FGF-2 treatment, JNK activation and DNA synthesis are dependent on Rac-1 activity. These results confirm that in MCF-7 cells, induction of DNA synthesis by FGF-2 requires a transient activation of the Ras/MAPK cascade and demonstrates for the first time that intact Rac-1 and Nck signaling networks are required.
Assuntos
Replicação do DNA/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Proteínas Oncogênicas/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Primers do DNA , Humanos , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
The endoplasmic reticulum (ER) is an intracellular compartment devoted to the synthesis, segregation and folding of soluble and membrane secretory proteins. Some mutations in these proteins lead to their incorrect or incomplete folding in the ER. The ER has a quality control system which detects misfolded proteins and then specifies their fate. Some mutated proteins are retained in the ER wherein they accumulate (Russell bodies for misfolded immunoglobulin heavy chains, the PiZZ for alpha 1-antitrypsin), others are retrotranslocated from the ER and degraded by the cytosolic proteasomal system, and yet other proteins are eventually secreted (in AZC-treated cells). In this review we summarize the role of ER resident proteins in quality control of mutated secretory proteins.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Calnexina , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de Aminoácidos , alfa 1-Antitripsina/metabolismoRESUMO
Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER-associated protein kinases. Although the role of the ER luminal domain of calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of calnexin. Analysis by two-dimensional phosphopeptide maps revealed that calnexin was in vitro phosphorylated in isolated microsomes by casein kinase 2 (CK2) and extracellular-signal regulated kinase-1 (ERK-1) at sites corresponding to those for in vivo phosphorylation. In canine pancreatic microsomes, synergistic phosphorylation by CK2 and ERK-1 led to increased association of calnexin with membrane-bound ribosomes. In vivo, calnexin-associated ERK-1 activity was identified by co-immunoprecipitation. This activity was abolished in cells expressing a dominant-negative MEK-1. Activation of ERK-1 in cells by addition of serum led to a 4-fold increase in ribosome-associated calnexin over unstimulated cells. Taken together with studies revealing calnexin association with CK2 and ERK-1, a model is proposed whereby phosphorylation of calnexin leads to a potential increase in glycoprotein folding close to the translocon.