RESUMEN
The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.
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Proteínas Fluorescentes Verdes , Factor de Apareamiento , Señales de Clasificación de Proteína , Saccharomycetales , Glicosilación , Saccharomycetales/metabolismo , Saccharomycetales/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Señales de Clasificación de Proteína/genética , Factor de Apareamiento/metabolismo , Factor de Apareamiento/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Calnexina/metabolismo , Calnexina/genética , Pichia/metabolismo , Pichia/genética , Retículo Endoplásmico/metabolismoRESUMEN
In mammalian cells, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are cleared out of the ER to the Golgi via a constitutive and a stress-inducible pathway called RESET. From the Golgi, misfolded GPI-APs transiently access the cell surface prior to rapid internalization for lysosomal degradation. What regulates the release of misfolded GPI-APs for RESET during steady-state conditions and how this release is accelerated during ER stress is unknown. Using mutants of prion protein or CD59 as model misfolded GPI-APs, we demonstrate that inducing calnexin degradation or upregulating calnexin-binding glycoprotein expression triggers the release of misfolded GPI-APs for RESET. Conversely, blocking protein synthesis dramatically inhibits the dissociation of misfolded GPI-APs from calnexin and subsequent turnover. We demonstrate an inverse correlation between newly synthesized calnexin substrates and RESET substrates that coimmunoprecipitate with calnexin. These findings implicate competition by newly synthesized substrates for association with calnexin as a key factor in regulating the release of misfolded GPI-APs from calnexin for turnover via the RESET pathway.
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Calnexina , Proteínas Ligadas a GPI , Priones , Animales , Calnexina/genética , Membrana Celular , Glicosilfosfatidilinositoles , Mamíferos , Chaperonas Moleculares , Retículo Endoplásmico , Aparato de Golgi , Pliegue de ProteínaRESUMEN
BACKGROUND: TP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer. METHODS: We analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-ß1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy. RESULTS: Mutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner. CONCLUSIONS: The mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies.
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Adenocarcinoma , Antineoplásicos , Animales , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Calnexina/genética , Calnexina/metabolismo , Integrina alfa5/metabolismo , Calreticulina/metabolismo , Anticuerpos Bloqueadores/metabolismo , Inhibidores de Glicósido Hidrolasas , Línea Celular Tumoral , Chaperonas Moleculares/metabolismo , Modelos Animales de Enfermedad , Pirofosfatasas/metabolismo , Proteínas Oncogénicas/metabolismoRESUMEN
N-linked glycans are specifically attached to asparagine residues in a N-X-S/T motif of secretory pathway glycoproteins. N-glycosylation of newly synthesized glycoproteins directs their folding via the lectin chaperones calnexin and calreticulin that are associated with protein-folding enzymes and glycosidases of the endoplasmic reticulum (ER). Misfolded glycoproteins are retained in the ER by the same lectin chaperones. The work by Sun et al. (FEBS J 2023, 10.1111/febs.16757) in this issue focusses on hepsin, a serine protease on the surface of liver and other organs. The authors deduce that spatial positioning of N-glycans on one side of a conserved domain of hepsin, known as the scavenger receptor-rich cysteine domain, regulates calnexin selection for hepsin maturation and transport through the secretory pathway. If N-glycosylation is elsewhere on hepsin, then it is misfolded and has a prolonged accumulation with calnexin and BiP. This association coincides with the engagement of stress response pathways that sense glycoprotein misfolding. The topological considerations of N-glycosylation dissected by Sun et al. may help unravel how key sites of N-glycosylation sites required for protein folding and transport have evolved to select the lectin chaperone calnexin pathway for folding and quality control.
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Serina Proteasas , Calnexina/genética , Calnexina/metabolismo , Calreticulina/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Lectinas/genética , Lectinas/metabolismo , Chaperonas Moleculares/metabolismo , Polisacáridos/metabolismo , Pliegue de Proteína , Control de CalidadRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.
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COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , Calnexina/genética , SARS-CoV-2/genética , Replicación ViralRESUMEN
PURPOSE OF THE STUDY: Calreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homolog calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloproliferative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for myeloproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin. PATIENTS AND METHODS: We analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay. RESULTS: Antibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high correlation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin. CONCLUSION: Collectively, these findings indicate that calreticulin mutations may be common and not necessarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloproliferative neoplasm.
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Calreticulina , Neoplasias , Humanos , Calreticulina/genética , Calnexina/genética , Calnexina/química , Calnexina/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.
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Proteínas de Arabidopsis , Arabidopsis , Calnexina/genética , Calnexina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Chaperonas Moleculares/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/genética , Fosfatos/metabolismo , Glicoproteínas/metabolismo , Adenosina Trifosfatasas/metabolismoRESUMEN
Type A γ-aminobutyric acid receptors (GABAARs) represent a family of pentameric GABA-gated Cl-/HCO3- ion channels which mediate inhibitory transmission in the central nervous system. Cell surface expression of GABAARs, a prerequisite for their function, is dependent on the appropriate assembly of the receptor subunits and their transient interactions with molecular chaperones within the endoplasmic reticulum (ER) and Golgi apparatus. Here, we describe a highly conserved amino acid sequence within the extracellular N-terminal domain of the receptor subunits adjoining the first transmembrane domain as a region important for GABAAR processing within the ER. Modifications of this region in the α1, ß3, and γ2 subunits using insertion or site-directed mutagenesis impaired GABAAR trafficking to the cell surface in heterologous cell systems although they had no effect on the subunit assembly. We found that mutated receptors accumulated in the ER where they were shown to associate with chaperones calnexin, BiP, and Grp94. However, their surface expression was increased when ER-associated degradation or proteosome function was inhibited, while modulation of ER calcium stores had little effect. When compared to the wt, mutated receptors showed decreased interaction with calnexin, similar binding to BiP, and increased association with Grp94. Structural modeling of calnexin interaction with the wt or mutated GABAAR revealed that disruption in structure caused by mutations in the conserved region adjoining the first transmembrane domain may impair calnexin binding. Thus, this previously uncharacterized region plays an important role in intracellular processing of GABAARs at least in part by stabilizing their interaction with calnexin.
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Proteínas Portadoras , Receptores de GABA-A , Animales , Ratones , Calnexina/genética , Calnexina/metabolismo , Espacio Extracelular/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Chaperonas Moleculares/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Subunidades de Proteína/metabolismoRESUMEN
Functional and epidemiological data suggest that N-linked glycans on the SARS-CoV-2 Spike protein may contribute to viral infectivity. To investigate this, we created a panel of N-to-Q mutations at N-glycosylation sites proximal to the Spike S1-S2 (N61, N603, N657, and N616) and S2' (N603 and N801) proteolysis sites. Some of these mutations, particularly N61Q and N801Q, reduced Spike incorporation into Spike-pseudotyped lentivirus and authentic SARS-CoV-2 virus-like particles (VLPs). These mutations also reduced pseudovirus and VLP entry into ACE2-expressing cells by 80 to 90%. In contrast, glycan mutations had a relatively minor effect on cell surface expression of Spike, ACE2 binding, and syncytia formation. A similar dichotomy in function was observed when virus was produced in host cells lacking ER chaperones, calnexin and calreticulin. Here, while both chaperones regulated pseudovirus function, only VLPs produced in calnexin KOs were less infectious. Overall, Spike N-glycans are likely critical for SARS-CoV-2 function and could serve as drug targets for COVID-19.
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COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Calnexina/genética , Calnexina/metabolismo , Calreticulina , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Polisacáridos/metabolismo , Glicoproteína de la Espiga del CoronavirusRESUMEN
Objective To investigate the killing effect and molecular mechanism of aberrant expression of calnexin (CNX) in the colorectal cancer (CRC) on the CD8+ T immune cells. Methods Immunohistochemistry was used to detect CNX protein level in 102 pairs of CRC cancer and adjacent non-cancerous tissues. Western blotting was employed to examine the protein expression of MHC I in the HCT-15 cells overexpressed with CNX or in the SW480 cells whose CNX expressions were knockdown by siRNA. Murine CD8+ T cells isolated from the spleen were cocultured with CT-26 murine CRC cells infected with lentivirus-mediated CNX overexpression. The killing effect of CD8+ T cells on CT-26 cells was determined by cytotoxicity kit. The secretion of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) in the culture medium were examined by ELISA. Results The protein level of CNX in colorectal cancer tissues were significantly lower than that in non-cancerous tissues. CNX overexpressed in HCT-15 cells was upregulated and CNX knockdown in SW480 cells downregulated the MHC I expression in these cells. Furthermore, the overexpression of CNX could not only enhance the killing effect of CD8+ T cells on CT-26 cells, but also promote the secretion of IFN-γ and TNF-α from these cells. Conclusion CNX can enhance the killing potential of CD8+ T cells on tumor cells through upregulating the MHC I expression in colorectal cancer cells.
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Linfocitos T CD8-positivos , Neoplasias Colorrectales , Animales , Linfocitos T CD8-positivos/metabolismo , Calnexina/química , Calnexina/genética , Calnexina/metabolismo , Neoplasias Colorrectales/genética , Interferón gamma/metabolismo , Ratones , Unión ProteicaRESUMEN
Calnexin (Cnx) is a membrane-bound lectin chaperone of the endoplasmic reticulum. In this study, a novel Cnx homologue from the obscure puffer Takifugu obscurus was characterized, tentatively named ToCnx. The cDNA of ToCnx was 1803 bp, and it contained an open reading frame encoding a polypeptide of 600 amino acid residues with a calculated molecular weight of 67.5 kDa. Multiple alignment of the deduced amino acid sequences of ToCnx and other related fish Cnxs revealed that ToCnx had typical characteristics of fish Cnxs. Sequence comparison and phylogenetic tree analysis showed that ToCnx had the closest relationship with Cnxs from Takifugu flavidus and Takifugu rubripes. ToCnx transcripts were detected in all the tissues examined, and they were mainly expressed in the liver, kidney, and intestine. Upon Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila infection, ToCnx transcripts were all significantly upregulated in the kidneys. The recombinant calreticulin domain of ToCnx (rToCnx) was prepared by prokaryotic expression. In the absence of calcium, rToCnx was able to bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and two bacterial saccharides, such as lipopolysaccharide and peptidoglycan. In the presence of calcium, rToCnx could agglutinate all the detected microorganisms. In addition, rToCnx possessed the effect of inhibiting the growth of three microbe strains. These observations suggested that ToCnx is an important participant in host immune defense against bacteria.
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Enfermedades de los Peces , Infecciones por Bacterias Gramnegativas , Animales , Antibacterianos , Calnexina/genética , Proteínas de Peces , Regulación de la Expresión Génica , Humanos , Inmunidad/genética , Filogenia , Takifugu/genéticaRESUMEN
MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.
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Factor de Transcripción Activador 4/genética , Neoplasias Colorrectales/genética , MicroARNs/genética , Ribonucleasa III/genética , Antagomirs/genética , Proteínas Argonautas/genética , Calnexina/genética , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/patología , ARN Helicasas DEAD-box/genética , Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/genética , Factor 2 Eucariótico de Iniciación/genética , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Transducción de Señal/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Nonresponse, or acquired resistance to immune checkpoint inhibitors in colorectal cancer (CRC) highlight the importance of finding potential tolerance mechanisms. Low expression of major histocompatibility complex, class I (MHC-I) on the cell surface of the tumor is one of the main mechanisms of tumor escape from T-cell recognition and destruction. In this study, we demonstrated that a high level of calnexin (CANX) in the tumors is positively correlated with the overall survival in colorectal cancer patients. CANX is a chaperone protein involved in the folding and assembly of MHC-I molecules. Using miRNA target prediction databases and luciferase assays, we identified miR-148a-3p as a potential regulator of CANX. Inhibition of miR-148a-3p restores surface levels of MHC-I and significantly enhanced the effects of CD8+ T-cell-mediated immune attack in vitro and in vivo by promoting CANX expression. These results reveal that miR-148a-3p can function as a tumor promotor in CRC by targeting the CANX/MHC-I axis, which provides a rationale for immunotherapy through targeting the miR-148a-3p/CANX/MHC-I pathway in patients with CRC.
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Linfocitos T CD8-positivos/fisiología , Calnexina/metabolismo , Neoplasias Colorrectales/terapia , Antígenos de Histocompatibilidad Clase II/metabolismo , MicroARNs/metabolismo , Animales , Calnexina/genética , Línea Celular Tumoral , Neoplasias Colorrectales/inmunología , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Ratones , MicroARNs/genética , Neoplasias Experimentales/terapiaRESUMEN
Long QT syndrome type 2 is caused by a mutation in the humanetheragogorelated gene (HERG) gene encoding the rapidly activating delayed rectifier Kcurrent. HERG is a key cell membrane glycoprotein; however, whether the maturation process of HERG protein involves key molecules derived from the calnexin (CNX)/calreticulin (CRT) cycle and how these molecules work remains unknown. Using western blotting, the present study screened the key molecules CNX/CRT/endoplasmic reticulum protein 57 (ERP57) involved in this cycle, and it was revealed that the protein expression levels of CNX/CRT/ERP57 in wildtype (WT)/A561V cells were increased compared with those in WT cells (n=3; P<0.05). Additionally, a coimmunoprecipitation experiment was used to reveal that the ability of CNX/ERP57/CRT to interact with HERG was significantly increased in A561V and WT/A561V cells (n=3; P<0.05). A plasmid lacking the bb' domain of ERP57 was constructed and it was demonstrated that the key site of ERP57 binding to CRT and immature HERG protein is the bb' domain. The wholecell patchclamp technique detected that the tail current density increased by 46% following overexpression of CRT and by 53% following overexpression of ERP57 in WT/A561V cells. Overexpression of CRT and ERP57 could increased HERG protein levels on the membrane detected by confocal imaging. Furthermore, overexpression of ERP57 and CRT proteins could restore the HERGA561V mutant protein trafficking process and rescue the dominantnegative suppression of WT. Overall, ERP57/CRT served a crucial role in the HERGA561V mutant protein trafficking deficiency and degradation process.
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Calreticulina/genética , Canal de Potasio ERG1/genética , Chaperonas Moleculares/genética , Mutación Missense , Proteína Disulfuro Isomerasas/genética , Calnexina/genética , Calnexina/metabolismo , Calreticulina/metabolismo , Canal de Potasio ERG1/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Potenciales de la Membrana/genética , Microscopía Confocal , Chaperonas Moleculares/metabolismo , Técnicas de Placa-Clamp , Unión Proteica , Proteína Disulfuro Isomerasas/metabolismo , Transporte de Proteínas/genéticaRESUMEN
Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER-associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER-phagy receptors for ER-to-lysosome-associated degradation (ERLAD). Demannosylation of N-linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro-collagen that cycles of de-/re-glucosylation of selected N-glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD-resistant misfolded proteins for FAM134B-driven lysosomal delivery. In summary, we show that mannose and glucose processing of N-glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.
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Degradación Asociada con el Retículo Endoplásmico/fisiología , Lisosomas/metabolismo , Polisacáridos/metabolismo , Animales , Calnexina/genética , Calnexina/metabolismo , Fibroblastos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Oligosacáridos/metabolismo , Procolágeno/genética , Procolágeno/metabolismo , Pliegue de Proteína , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
Infantile nystagmus syndrome (INS) denominates early-onset, involuntary oscillatory eye movements with different etiologies. Nystagmus is also one of the symptoms in oculocutaneus albinism (OCA), a heterogeneous disease mainly caused by defects in melanin synthesis or melanosome biogenesis. Dopachrome tautomerase (DCT, also called TYRP2) together with tyrosinase (TYR) and tyrosin-related protein 1 (TYRP1) is one of the key enzymes in melanin synthesis. Although DCT´s role in pigmentation has been proven in different species, until now only mutations in TYR and TYRP1 have been found in patients with OCA. Detailed ophthalmological and orthoptic investigations identified a consanguineous family with two individuals with isolated infantile nystagmus and one family member with subtle signs of albinism. By whole-exome sequencing and segregation analysis, we identified the missense mutation c.176G > T (p.Gly59Val) in DCT in a homozygous state in all three affected family members. We show that this mutation results in incomplete protein maturation and targeting in vitro compatible with a partial or total loss of function. Subsequent screening of a cohort of patients with OCA (n = 85) and INS (n = 25) revealed two heterozygous truncating mutations, namely c.876C > A (p.Tyr292*) and c.1407G > A (p.Trp469*), in an independent patient with OCA. Taken together, our data suggest that mutations in DCT can cause a phenotypic spectrum ranging from isolated infantile nystagmus to oculocutaneous albinism.
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Albinismo Oculocutáneo/genética , Oxidorreductasas Intramoleculares/genética , Melaninas/biosíntesis , Mutación Missense , Nistagmo Congénito/genética , Adolescente , Albinismo Oculocutáneo/diagnóstico , Albinismo Oculocutáneo/enzimología , Albinismo Oculocutáneo/patología , Secuencia de Bases , Calnexina/genética , Calnexina/metabolismo , Niño , Estudios de Cohortes , Consanguinidad , Femenino , Regulación de la Expresión Génica , Células HEK293 , Homocigoto , Humanos , Oxidorreductasas Intramoleculares/deficiencia , Masculino , Melaninas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Nistagmo Congénito/diagnóstico , Nistagmo Congénito/enzimología , Nistagmo Congénito/patología , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Linaje , Secuenciación del Exoma , Adulto JovenRESUMEN
Molecular chaperones assist the folding of nascent chains in the cell. Chaperones also aid in quality control decisions as persistent chaperone binding can help to sort terminal misfolded proteins for degradation. There are two major molecular chaperone families in the endoplasmic reticulum (ER) that assist proteins in reaching their native structure and evaluating the fidelity of the maturation process. The ER Hsp70 chaperone, BiP, supports adenine nucleotide-regulated binding to non-native proteins that possess exposed hydrophobic regions. In contrast, the carbohydrate-dependent chaperone system involving the membrane protein calnexin and its soluble paralogue calreticulin recognize a specific glycoform of an exposed hydrophilic protein modification for which the composition is controlled by a series of glycosidases and transferases. Here, we compare and contrast the properties, mechanisms of action and functions of these different chaperones systems that work in parallel, as well as together, to assist a large variety of substrates that traverse the eukaryotic secretory pathway.
Asunto(s)
Chaperonas Moleculares , Pliegue de Proteína , Calnexina/genética , Calnexina/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Control de CalidadRESUMEN
The endoplasmic reticulum (ER) is an organelle that mediates the proper folding and assembly of proteins destined for the cell surface, the extracellular space and subcellular compartments such as the lysosomes. The ER contains a wide range of molecular chaperones to handle the folding requirements of a diverse set of proteins that traffic through this compartment. The lectin-like chaperones calreticulin and calnexin are an important class of structurally-related chaperones relevant for the folding and assembly of many N-linked glycoproteins. Despite the conserved mechanism of action of these two chaperones in nascent protein recognition and folding, calreticulin has unique functions in cellular calcium signaling and in the immune response. The ER-related functions of calreticulin in the assembly of major histocompatibility complex (MHC) class I molecules are well-studied and provide many insights into the modes of substrate and co-chaperone recognition by calreticulin. Calreticulin is also detectable on the cell surface under some conditions, where it induces the phagocytosis of apoptotic cells. Furthermore, mutations of calreticulin induce cell transformation in myeloproliferative neoplasms (MPN). Studies of the functions of the mutant calreticulin in cell transformation and immunity have provided many insights into the normal biology of calreticulin, which are discussed.
Asunto(s)
Señalización del Calcio , Señalización del Calcio/genética , Calnexina/genética , Calnexina/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Sistema Inmunológico , Pliegue de ProteínaRESUMEN
The lectin chaperones calreticulin (CALR) and calnexin (CANX), together with their co-chaperone PDIA3, are increasingly implicated in studies of human cancers in roles that extend beyond their primary function as quality control facilitators of protein folding within the endoplasmic reticulum (ER). Led by the discovery that cell surface CALR functions as an immunogen that promotes anti-tumour immunity, studies have now expanded to include their potential uses as prognostic markers for cancers, and in regulation of oncogenic signaling that regulate such diverse processes including integrin-dependent cell adhesion and migration, proliferation, cell death and chemotherapeutic resistance. The diversity stems from the increasing recognition that these proteins have an equally diverse spectrum of subcellular and extracellular localization, and which are aberrantly expressed in tumour cells. This review describes key foundational discoveries and highlight recent findings that further our understanding of the plethora of activities mediated by CALR, CANX and PDIA3.