RESUMEN
The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and ß-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another ß-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications.
Asunto(s)
Calnexina/fisiología , Inflamación/metabolismo , Interleucinas/fisiología , Antígenos de Histocompatibilidad Menor/fisiología , Chaperonas Moleculares/fisiología , Dimerización , Glicoproteínas/química , Humanos , Interleucinas/química , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias/metabolismo , Neoplasias/patología , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Subunidades de Proteína , Receptores de Interleucina/químicaRESUMEN
Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca 2+ homeostasis, and Ca 2+ signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts in vivo, and the in vitro cardiac fibroblasts activation model was established by transforming growth factor ß1 (TGFß1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFß1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.
Asunto(s)
Calnexina , Fibroblastos , Corazón , Chaperonas Moleculares , Animales , Calnexina/fisiología , Estrés del Retículo Endoplásmico , Fibroblastos/fisiología , Corazón/fisiología , RatonesRESUMEN
Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G1M9GN2-proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae, which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G1M9GN2-ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G1M9GN2-RNase was chemoenzymatically synthesized from G1M9GN-oxazoline and GN-RNase. Denatured G1M9GN2-RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae, TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX.
Asunto(s)
Aspergillus oryzae/metabolismo , Calnexina/fisiología , Proteínas Fúngicas/fisiología , Glicoproteínas/fisiología , Proteínas de Choque Térmico/fisiología , Secuencia de Carbohidratos , Proteínas Fúngicas/química , Glicoproteínas/química , Glicosilación , Proteínas de Choque Térmico/química , Cinética , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Procesamiento Proteico-PostraduccionalRESUMEN
Calnexin (Cnx) is an endoplasmic reticulum membrane-bound lectin chaperone that comprises a dedicated maturation system with another lectin chaperone calreticulin (Crt). This maturation system is known as the Cnx/Crt cycle. The main functions of Cnx are Ca(2+) storage, glycoprotein folding, and quality control of synthesis. Recent studies have shown that Cnx is important in phagocytosis and in optimizing dendritic cell immunity. However, the functions of Cnx in invertebrate innate immunity remain unclear. In this research, we characterized Cnx in the kuruma shrimp Marsupenaeus japonicus (designated as MjCnx) and detected its function in shrimp immunity. The expression of MjCnx was upregulated in several tissues challenged with Vibrio anguillarum. Recombinant MjCnx could bind to bacteria by binding polysaccharides. MjCnx protein existed in the cytoplasm and on the membrane of hemocytes and was upregulated by bacterial challenge. The recombinant MjCnx enhanced the clearance of V. anguillarum in vivo, and the clearance effects were impaired after silencing MjCnx with RNA interference assay. Recombinant MjCnx promoted phagocytosis efficiency of hemocytes. These results suggest that MjCnx functions as one of the pattern recognition receptors and has crucial functions in shrimp antibacterial immunity.
Asunto(s)
Proteínas de Artrópodos/fisiología , Calnexina/fisiología , Inmunidad Innata , Penaeidae/inmunología , Animales , Proteínas de Artrópodos/química , Bacillus/inmunología , Calnexina/química , Células Cultivadas , Expresión Génica/inmunología , Hemocitos/inmunología , Hemocitos/microbiología , Micrococcus/inmunología , Penaeidae/metabolismo , Penaeidae/microbiología , Fagocitosis , Filogenia , Polisacáridos Bacterianos/química , Unión Proteica , Transporte de Proteínas , Staphylococcus aureus/inmunología , Vibrio/inmunologíaRESUMEN
Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.
Asunto(s)
Aspergillus fumigatus/metabolismo , Calnexina/química , Calnexina/fisiología , Proteínas Fúngicas/fisiología , Lectinas/química , Animales , Calnexina/genética , Cationes , Medios de Cultivo/farmacología , Cartilla de ADN/genética , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Femenino , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Inmunosupresores/uso terapéutico , Ratones , Reacción en Cadena de la Polimerasa/métodos , Pliegue de Proteína , Temperatura , VirulenciaRESUMEN
Peripheral myelin protein 22 (PMP22) and protein 0 (P0) are major peripheral myelin glycoproteins, and mutations in these two proteins are associated with hereditary demyelinating peripheral neuropathies. Calnexin, calreticulin, and ERp57 are critical components of protein quality control responsible for proper folding of newly synthesized glycoproteins. Here, using confocal microscopy, we show that cell surface targeting of P0 and PMP22 is not affected in the absence of the endoplasmic reticulum chaperones. However, the folding and function (adhesiveness) of PMP22 and P0, measured using the adhesion assay, are affected significantly in the absence of calnexin but not in the absence of calreticulin. Deficiency in oxidoreductase ERp57 results in impaired folding and function of P0, a disulfide bond-containing protein, but does not have any effect on folding or function of PMP22 (a protein that does not contain a disulfide bond). We concluded that calnexin and ERp57, but not calreticulin, play an important role in the biology of peripheral myelin proteins PMP22 and P0, and, consequently, these chaperones may contribute to the pathogenesis of peripheral neuropathies and the diversity of these neurological disorders.
Asunto(s)
Calnexina/fisiología , Retículo Endoplásmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteína P0 de la Mielina/química , Proteínas de la Mielina/química , Proteína Disulfuro Isomerasas/fisiología , Animales , Calnexina/deficiencia , Calreticulina/deficiencia , Células HEK293 , Humanos , Ratones , Proteína Disulfuro Isomerasas/deficiencia , Pliegue de ProteínaRESUMEN
OBJECTIVE: In diabetes, when glucose consumption is restricted, the heart adapts to use fatty acid (FA) exclusively. The majority of FA provided to the heart comes from the breakdown of circulating triglyceride (TG), a process catalyzed by lipoprotein lipase (LPL) located at the vascular lumen. The objective of the current study was to determine the mechanisms behind LPL processing and breakdown after moderate and severe diabetes. RESEARCH DESIGN AND METHODS: To induce acute hyperglycemia, diazoxide, a selective, ATP-sensitive K(+) channel opener was used. For chronic diabetes, streptozotocin, a ß-cell-specific toxin was administered at doses of 55 or 100 mg/kg to generate moderate and severe diabetes, respectively. Cardiac LPL processing into active dimers and breakdown at the vascular lumen was investigated. RESULTS: After acute hyperglycemia and moderate diabetes, more LPL is processed into an active dimeric form, which involves the endoplasmic reticulum chaperone calnexin. Severe diabetes results in increased conversion of LPL into inactive monomers at the vascular lumen, a process mediated by FA-induced expression of angiopoietin-like protein 4 (Angptl-4). CONCLUSIONS: In acute hyperglycemia and moderate diabetes, exaggerated LPL processing to dimeric, catalytically active enzyme increases coronary LPL, delivering more FA to the heart when glucose utilization is compromised. In severe chronic diabetes, to avoid lipid oversupply, FA-induced expression of Angptl-4 leads to conversion of LPL to inactive monomers at the coronary lumen to impede TG hydrolysis. Results from this study advance our understanding of how diabetes changes coronary LPL, which could contribute to cardiovascular complications seen with this disease.
Asunto(s)
Diabetes Mellitus Experimental/enzimología , Ácidos Grasos no Esterificados/metabolismo , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/biosíntesis , Animales , Calnexina/fisiología , Diazóxido , Hiperglucemia/inducido químicamente , Hiperglucemia/metabolismo , Lipoproteína Lipasa/metabolismo , Masculino , Miocardio/metabolismo , Multimerización de Proteína , Ratas , Ratas Wistar , EstreptozocinaRESUMEN
Calnexin is a molecular chaperone and a component of the quality control of the secretory pathway. We have generated calnexin gene-deficient mice (cnx(-/-)) and showed that calnexin deficiency leads to myelinopathy. Calnexin-deficient mice were viable with no discernible effects on other systems, including immune function, and instead they demonstrated dysmyelination as documented by reduced conductive velocity of nerve fibers and electron microscopy analysis of sciatic nerve and spinal cord. Myelin of the peripheral and central nervous systems of cnx(-/-) mice was disorganized and decompacted. There were no abnormalities in neuronal growth, no loss of neuronal fibers, and no change in fictive locomotor pattern in the absence of calnexin. This work reveals a previously unrecognized and important function of calnexin in myelination and provides new insights into the mechanisms responsible for myelin diseases.
Asunto(s)
Calnexina/genética , Calnexina/fisiología , Enfermedades Desmielinizantes/metabolismo , Vaina de Mielina/metabolismo , Animales , Animales Recién Nacidos , Calnexina/metabolismo , Membrana Celular/metabolismo , Electrofisiología/métodos , Retículo Endoplásmico/metabolismo , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pliegue de Proteína , Nervio Ciático/metabolismo , Nervio Ciático/ultraestructura , Médula Espinal/metabolismo , Médula Espinal/ultraestructuraRESUMEN
Inositol is a precursor of numerous phospholipids and signalling molecules essential for the cell. Schizosaccharomyces pombe is naturally auxotroph for inositol as its genome does not have a homologue of the INO1 gene encoding inositol-1-phosphate synthase, the enzyme responsible for inositol biosynthesis. In this work, we demonstrate that inositol starvation in S. pombe causes cell death with apoptotic features. This apoptotic death is dependent on the metacaspase Pca1p and is affected by the UPR transducer Ire1p. Previously, we demonstrated that calnexin is involved in apoptosis induced by ER stress. Here, we show that cells expressing a lumenal version of calnexin exhibit a 2-fold increase in the levels of apoptosis provoked by inositol starvation. This increase is reversed by co-expression of a calnexin mutant spanning the transmembrane domain and C-terminal cytosolic tail. Coherently, calnexin is physiologically cleaved at the end of its lumenal domain, under normal growth conditions when cells approach stationary phase. This cleavage suggests that the two naturally produced calnexin fragments are needed to continue growth into stationary phase and to prevent cell death. Collectively, our observations indicate that calnexin takes part in at least two apoptotic pathways in S. pombe, and suggest that the cleavage of calnexin has regulatory roles in apoptotic processes involving calnexin.
Asunto(s)
Apoptosis/fisiología , Calnexina/fisiología , Inositol/metabolismo , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Calnexina/química , Calnexina/genética , Cromatografía en Gel , Hidrólisis , Espectrometría de Masas , Datos de Secuencia Molecular , Mutación , Schizosaccharomyces/citologíaRESUMEN
Stress conditions affecting the functions of the endoplasmic reticulum (ER) cause the accumulation of unfolded proteins. ER stress is counteracted by the unfolded-protein response (UPR). However, under prolonged stress the UPR initiates a proapoptotic response. Mounting evidence indicate that the ER chaperone calnexin is involved in apoptosis caused by ER stress. Here, we report that overexpression of calnexin in Schizosaccharomyces pombe induces cell death with apoptosis markers. Cell death was partially dependent on the Ire1p ER-stress transducer. Apoptotic death caused by calnexin overexpression required its transmembrane domain (TM), and involved sequences on either side of the ER membrane. Apoptotic death caused by tunicamycin was dramatically reduced in a strain expressing endogenous levels of calnexin lacking its TM and cytosolic tail. This demonstrates the involvement of calnexin in apoptosis triggered by ER stress. A genetic screen identified the S. pombe homologue of the human antiapoptotic protein HMGB1 as a suppressor of apoptotic death due to calnexin overexpression. Remarkably, overexpression of human calnexin in S. pombe also provoked apoptotic death. Our results argue for the conservation of the role of calnexin in apoptosis triggered by ER stress, and validate S. pombe as a model to elucidate the mechanisms of calnexin-mediated cell death.
Asunto(s)
Apoptosis , Calnexina/fisiología , Retículo Endoplásmico/metabolismo , Regulación Fúngica de la Expresión Génica , Muerte Celular , Supervivencia Celular , Citosol/metabolismo , Proteína HMGB1/metabolismo , Humanos , Modelos Biológicos , Mutación , Plásmidos , Desnaturalización Proteica , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Cystic fibrosis (CF) is caused by the mutation in CF transmembrane conductance regulator (CFTR), a cAMP-dependent Cl(-) channel at the plasma membrane of epithelium. The most common mutant, DeltaF508 CFTR, has competent Cl(-) channel function, but fails to express at the plasma membrane since it is retained in the endoplasmic reticulum (ER) by the ER quality control system. Here, we show that calnexin (CNX) is not necessary for the ER retention of DeltaF508 CFTR. Our data show that CNX knockout (KO) does not affect the biosynthetic processing, cellular localization or the Cl(-) channel function of DeltaF508 CFTR. Importantly, cAMP-induced Cl(-) current in colonic epithelium from CNX KO/DeltaF508 CFTR mice was comparable with that of DeltaF508 CFTR mice, indicating that CNX KO failed to rescue the ER retention of DeltaF508 CFTR in vivo. Moreover, we show that CNX assures the efficient expression of WT CFTR, but not DeltaF508 CFTR, by inhibiting the proteasomal degradation, indicating that CNX might stimulate the productive folding of WT CFTR, but not DeltaF508 CFTR, which has folding defects.
Asunto(s)
Calnexina/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Retículo Endoplásmico/metabolismo , Animales , Calnexina/genética , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Epitelio/fisiología , Ratones , Ratones Noqueados , Pliegue de Proteína , Eliminación de SecuenciaRESUMEN
Calnexin is an endoplasmic reticulum (ER) lectin that mediates protein folding on the rough ER. Calnexin also interacts with ER calcium pumps that localize to the mitochondria-associated membrane (MAM). Depending on ER homeostasis, varying amounts of calnexin target to the plasma membrane. However, no regulated sorting mechanism is so far known for calnexin. Our results now describe how the interaction of calnexin with the cytosolic sorting protein PACS-2 distributes calnexin between the rough ER, the MAM, and the plasma membrane. Under control conditions, more than 80% of calnexin localizes to the ER, with the majority on the MAM. PACS-2 knockdown disrupts the calnexin distribution within the ER and increases its levels on the cell surface. Phosphorylation by protein kinase CK2 of two calnexin cytosolic serines (Ser554/564) reduces calnexin binding to PACS-2. Consistent with this, a Ser554/564 Asp phosphomimic mutation partially reproduces PACS-2 knockdown by increasing the calnexin signal on the cell surface and reducing it on the MAM. PACS-2 knockdown does not reduce retention of other ER markers. Therefore, our results suggest that the phosphorylation state of the calnexin cytosolic domain and its interaction with PACS-2 sort this chaperone between domains of the ER and the plasma membrane.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Calnexina/biosíntesis , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Secuencia de Aminoácidos , Calnexina/química , Calnexina/fisiología , Citosol/química , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Transporte VesicularAsunto(s)
Calnexina/fisiología , Calreticulina/fisiología , Regulación de la Expresión Génica , Animales , Calnexina/química , Calreticulina/química , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/química , Glicoproteínas/química , Humanos , Modelos Biológicos , Polisacáridos/química , Pliegue de ProteínaRESUMEN
We cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37 degrees C, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.
Asunto(s)
Calnexina/genética , Calor , Proteína Disulfuro Isomerasas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Eliminación de Secuencia/genética , Calnexina/deficiencia , Calnexina/fisiología , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Chaperonas Moleculares/genética , Proteína Disulfuro Isomerasas/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The key to successful cancer immunotherapy is to induce an effective anticancer immunity that will overcome the acquired cancer-specific immune tolerance. In this study, we found that dendritic cells (DCs) from multiple myeloma (MM) patients suppressed rather than induced a cancer cell-specific immune response. We demonstrated that CD4(+)CD25(high) T cells from MM patients suppressed the proliferation of activated peripheral blood lymphocytes. Further analysis illustrated that MM cell lysates or MM-specific idiotype immunoglobulins (MM Id-Ig) specifically induced the expansion of peripheral CD4(+)CD25(high)FoxP3(high) T regulatory (Treg) cells in vitro. Supraphysiological expression of calnexin (CNX) using lentiviral (LV) vectors in DCs of MM patients overcame the immune suppression and enhanced MM-specific CD4 and CD8 T-cell responses. However, overexpression of CNX did not affect the peripheral expansion of Treg cells stimulated by MM antigens. Thus, the immune suppression effect of Treg cells in cancer patients may be overcome by improving antigen processing in DCs, which in turn may lower the activation threshold of the immune effector cells. This concept of modulating anticancer immunity by genetically engineering cancer patients' DCs may improve immunotherapeutic regimens in cancer treatment.
Asunto(s)
Calnexina/fisiología , Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Mieloma Múltiple/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Calnexina/genética , Proliferación Celular , Células Cultivadas , Células Dendríticas/metabolismo , Humanos , Idiotipos de Inmunoglobulinas/genética , Idiotipos de Inmunoglobulinas/inmunología , Idiotipos de Inmunoglobulinas/metabolismo , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Lentivirus/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mieloma Múltiple/patología , Alineación de Secuencia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Protein secretion is a complex process that can be modulated by folding factors in the endoplasmic reticulum (ER), such as calnexin, a highly-conserved molecular chaperone involved in quality control. In Schizosaccharomyces pombe, calnexin (Cnx1p) is essential for cell viability. The calnexin/Cnx1p determinants required for viability have been mapped within the last 123 residues of its C-terminus. To better understand the role(s) of calnexin/Cnx1p in secretion, we screened for cnx1 mutants 'super-secreting' cellulase. We identified ss14_cnx1, a mutant secreting 10-fold higher levels of the glycoprotein cellulase than the wild-type strain. While cellulase did not interact with ss14_Cnx1p, the ratio of secreted activity/quantity for this enzyme was not affected, suggesting that the quality control of folding in the ER was adequate in the mutant strain. Surprisingly, the ss14_Cnx1p mutant is composed of the 160 N-terminal amino acids of the mature molecule, thus this mutant defines a novel calnexin/Cnx1p region supporting Sz. pombe viability. Interestingly, like viable mutants spanning the last 52 aa of calnexin/Cnx1p, the 160 N-terminal residues encoded by ss14_cnx1 also forms a complex with the essential BiP chaperone. These results reveal the so far unidentified importance of the N-terminal region of calnexin/Cnx1p.
Asunto(s)
Calnexina/fisiología , Proteínas Fúngicas/fisiología , Schizosaccharomyces/fisiología , Secuencias de Aminoácidos , Aspergillus/enzimología , Aspergillus/genética , Southern Blotting , Calnexina/genética , Celulasa/genética , Celulasa/metabolismo , Celulasa/fisiología , ADN de Hongos/genética , Proteínas Fúngicas/genética , Immunoblotting , Microscopía de Interferencia , Mutagénesis Insercional , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Schizosaccharomyces/enzimología , Schizosaccharomyces/genéticaRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/metabolismo , Calnexina/metabolismo , Carcinoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Tunicamicina/farmacología , Secuencia de Aminoácidos , Apoptosis , Calnexina/genética , Calnexina/fisiología , Caspasa 3/metabolismo , Línea Celular Tumoral , Células Clonales , Resistencia a Antineoplásicos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Activación Enzimática , Humanos , Datos de Secuencia MolecularRESUMEN
Calnexin is a membrane-bound chaperone of the endoplasmic reticulum (ER) that participates in the folding and quality control of newly synthesized glycoproteins. Binding to glycoproteins occurs through a lectin site with specificity for Glc1Man9GlcNAc2 oligosaccharides as well as through a polypeptide binding site that recognizes non-native protein conformations. The latter interaction is somewhat controversial because it is based on observations that calnexin can suppress the aggregation of non-glycosylated substrates at elevated temperature or at low calcium concentrations, conditions that may affect the structural integrity of calnexin. Here, we examine the ability of calnexin to interact with a non-glycosylated substrate under physiological conditions of the ER lumen. We show that the soluble ER luminal domain of calnexin can indeed suppress the aggregation of non-glycosylated firefly luciferase at 37 degrees C and at the normal resting ER calcium concentration of 0.4 mM. However, gradual reduction of calcium below the resting level was accompanied by a progressive loss of native calnexin structure as assessed by thermal stability, protease sensitivity, intrinsic fluorescence, and bis-ANS binding. These assays permitted the characterization of a single calcium binding site on calnexin with a Kd = 0.15 +/- 0.05 mM. We also show that the suppression of firefly luciferase aggregation by calnexin is strongly enhanced in the presence of millimolar concentrations of ATP and that the Kd for ATP binding to calnexin in the presence of 0.4 mM calcium is 0.7 mM. ATP did not alter the overall stability of calnexin but instead triggered the localized exposure of a hydrophobic site on the chaperone. These findings demonstrate that calnexin is a potent molecular chaperone that is capable of suppressing the aggregation of substrates through polypeptide-based interactions under conditions that exist within the ER lumen.
Asunto(s)
Calnexina/fisiología , Retículo Endoplásmico/fisiología , Animales , Calcio/metabolismo , Secuencia de Carbohidratos , Luciérnagas , Glutatión Transferasa/metabolismo , Cinética , Lectinas , Luciferasas/metabolismo , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Péptido Hidrolasas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Espectrofotometría Ultravioleta , Termodinámica , TripsinaRESUMEN
MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.
Asunto(s)
Retículo Endoplásmico/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Animales , Calnexina/fisiología , Calreticulina/fisiología , Proteínas de Choque Térmico/fisiología , Humanos , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Ratones , Conformación Proteica , Proteína Disulfuro Isomerasas/fisiologíaRESUMEN
The quality control (QC) system of the endoplasmic reticulum (ER) is an important monitoring mechanism in the protein maturation process, which ensures export of properly folded proteins from the ER. Incorrectly or incompletely folded proteins are retained in the ER for refolding or degradation by the ER-residing proteasome. The calnexin/calreticulin cycle and ER-associated degradation are the key elements in QC. These two mechanisms work together to allow incorrectly folded proteins have additional opportunities to achieve their native conformations. The QC dysfunction is involved in many diseases caused by mutant proteins, many of which are causes of neurodegenerative disorders. A better understanding of molecular regulation in the QC system will uncover the molecular pathogenic mechanisms of many diseases caused by protein misfolding and help discover novel strategies for preventing or treating these diseases.