RESUMEN
Recently we have shown that protein disulfide isomerase (PDI or PDIA1) is involved in mediating chemically-induced, glutathione (GSH) depletion-associated ferroptotic cell death through NOS activation (dimerization) and NO accumulation. The present study aims to determine the role of PDI in mediating chemically-induced hepatocyte injury in vitro and in vivo and whether PDI inhibitors can effectively protect against chemically-induced hepatocyte injury. We show that during the development of erastin-induced ferroptotic cell death, accumulation of cellular NO, ROS and lipid-ROS follows a sequential order, i.e., cellular NO accumulation first, followed by accumulation of cellular ROS, and lastly cellular lipid-ROS. Cellular NO, ROS and lipid-ROS each play a crucial role in mediating erastin-induced ferroptosis in cultured hepatocytes. In addition, it is shown that PDI is an important upstream mediator of erastin-induced ferroptosis through PDI-mediated conversion of NOS monomer to its dimer, which then leads to accumulation of cellular NO, ROS and lipid-ROS, and ultimately ferroptotic cell death. Genetic manipulation of PDI expression or pharmacological inhibition of PDI function each can effectively abrogate erastin-induced ferroptosis. Lastly, evidence is presented to show that PDI is also involved in mediating acetaminophen-induced liver injury in vivo using both wild-type C57BL/6J mice and hepatocyte-specific PDI conditional knockout (PDIfl/fl Alb-cre) mice. Together, our work demonstrates that PDI is an important upstream mediator of chemically-induced, GSH depletion-associated hepatocyte ferroptosis, and inhibition of PDI can effectively prevent this injury.
Asunto(s)
Glutatión , Hepatocitos , Proteína Disulfuro Isomerasas , Especies Reactivas de Oxígeno , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Animales , Glutatión/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ratones , Ratones Endogámicos C57BL , Piperazinas/farmacología , Ferroptosis/efectos de los fármacos , Óxido Nítrico/metabolismo , Masculino , HumanosRESUMEN
PDIA5 is responsible for modification of disulfide bonds of proteins. However, its impact on the malignant progression of glioblastoma multiforme (GBM) remains unknown. We analyzed the expression and prognostic significance of PDIA5 in cohorts of GBM and clinical samples. The PDIA5 protein was significantly overexpressed in GBM tissues, and higher expression of PDIA5 was statistically associated with a worse prognosis in patients with GBM. Transcriptional data from PDIA5 knockdown GBM cells revealed that downstream regulatory genes of PDIA5 were enriched in malignant regulatory pathways and PDIA5 enhanced the proliferative and invasive abilities of GBM cells. By constructing a PDIA5 CXXC motif mutant plasmid, we found CCAR1 was the vital downstream factor of PDIA5 in regulating GBM malignancy in vitro and in vivo. Additionally, RUNX1 bound to the promoter region of PDIA5 and regulated gene transcription, leading to activation of the PDIA5/CCAR1 regulatory axis in GBM. The RUNX1/PDIA5/CCAR1 axis significantly influenced the malignant behavior of GBM cells. In conclusion, this study comprehensively elucidates the crucial role of PDIA5 in the malignant progression of GBM. Downregulating PDIA5 can mitigate the malignant biological behavior of GBM both in vitro and in vivo, potentially improving the efficacy of treatment for clinical patients with GBM.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Neoplasias Encefálicas , Proteínas de Ciclo Celular , Glioblastoma , Proteína Disulfuro Isomerasas , Animales , Femenino , Humanos , Masculino , Ratones , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patología , Ratones Desnudos , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
Protein disulfide isomerases (PDIs) and PDI-like proteins catalyze the oxidation and reduction in protein disulfide bonds, inhibit aggregation of misfolded proteins, and participate in isomerization and abiotic stress responses. The wild type 'duli' pear (Pyrus betulaefolia) is an important rootstock commonly used for commercial pear tree grafting in northern China. In this study, we identified 24 PDI genes, named PbPDIs, from the genome of 'duli' pear. With 12 homologous gene pairs, these 24 PbPDIs distribute on 12 of its 17 chromosomes. Phylogenetic analysis placed the 24 PbPDIs into four clades and eleven groups. Collinearity analysis of the PDIs between P. betulaefolia, Arabidopsis thaliana, and Oryza sativa revealed that the PbPDIs of 'duli' pear show a strong collinear relationship with those from Arabidopsis, a dicot; but a weak collinear relationship with those from rice, a monocot. Quantitative RT-PCR analysis showed that most of the PbPDIs were upregulated by salt stress. Identification and expression analysis of 'duli' pear PbPDIs under salt stress conditions could provide useful information for further research in order to generate salt-resistant rootstock for pear grafting in the future.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Filogenia , Proteínas de Plantas , Proteína Disulfuro Isomerasas , Pyrus , Estrés Salino , Pyrus/genética , Pyrus/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrés Salino/genética , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
Protein disulfide isomerase, containing thioredoxin (Trx) domains, serves as a vital enzyme responsible for oxidative protein folding (the formation, reduction, and isomerization of disulfide bonds in newly synthesized proteins) in the endoplasmic reticulum (ER). However, the role of ER-localized PDI proteins in parasite growth and their interaction with secretory proteins remain poorly understood. In this study, we identified two ER-localized PDI proteins, TgPDI8 and TgPDI6, in Toxoplasma gondii. Conditional knockdown of TgPDI8 resulted in a significant reduction in intracellular proliferation and invasion abilities, leading to a complete block in plaque formation on human foreskin fibroblast monolayers, whereas parasites lacking TgPDI6 did not exhibit any apparent fitness defects. The complementation of TgPDI8 with mutant variants highlighted the critical role of the CXXC active site cysteines within its Trx domains for its enzymatic activity. By utilizing TurboID-based proximity labeling, we uncovered a close association between PDI proteins and canonical secretory proteins. Furthermore, parasites lacking TgPDI8 showed a significant reduction in the expression of secretory proteins, especially those from micronemes and dense granules. In summary, our study elucidates the roles of TgPDI8 and sets the stage for future drug discovery studies. IMPORTANCE: Apicomplexans, a phylum of intracellular parasites, encompass various zoonotic pathogens, including Plasmodium, Cryptosporidium, Toxoplasma, and Babesia, causing a significant economic burden on human populations. These parasites exhibit hypersensitivity to disruptions in endoplasmic reticulum (ER) redox homeostasis, necessitating the presence of ER-localized thioredoxin (Trx) superfamily proteins, particularly protein disulfide isomerase (PDI), for proper oxidative folding. However, the functional characteristics of ER-localized PDI proteins in Toxoplasma gondii remain largely unexplored. In this study, we identified two ER-localized proteins, namely, TgPDI8 and TgPDI6, and demonstrated the indispensable role of TgPDI8 in parasite survival. Through a comprehensive multi-omics analysis, we elucidated the crucial role of TgPDI8 in the processing of secretory proteins in T. gondii. Additionally, we introduced a novel ER-anchored TurboID method to label and identify canonical secretory proteins in T. gondii. This research opens up new avenues for understanding oxidative folding and the secretory pathway in apicomplexan parasites, laying the groundwork for future advancements in antiparasitic drug development.
Asunto(s)
Retículo Endoplásmico , Proteína Disulfuro Isomerasas , Proteínas Protozoarias , Toxoplasma , Toxoplasma/genética , Toxoplasma/enzimología , Toxoplasma/metabolismo , Toxoplasma/crecimiento & desarrollo , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Humanos , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Retículo Endoplásmico/metabolismo , Fibroblastos/parasitología , Fibroblastos/metabolismoRESUMEN
Protein disulfide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER) protein. It has different functions including glycoprotein folding in the ER. The unfavorable prognosis of cancer patients was related to the abnormal PDIA3 expression level. However, it is unclear how PDIA3 correlates with the malignant characteristics of different tumors and its impact on tumor immunity. Pan-cancer data were downloaded from several databases for large-scale bioinformatics analysis. The immunological functions of PDIA3 were systematically explored at the single-cell sequencing level, including cell communication, cell metabolism, cell evolution and epigenetic modification. We performed immunofluorescence staining to visualize PDIA3 expression and infiltration of macrophages in pan-cancer samples. Further, we performed a loss-of-function assay of PDIA3 in vitro. The CCK8 assay, clone formation assay, and transwell assay were performed. M2 macrophages were co-cultured with different cell lines before the transwell assay was performed. The immunofluorescence staining of pan-cancer samples presented a higher expression of PDIA3 than those of the paired normal tissues. According to single-cell sequencing analysis, expression of PDIA3 was closely associated with cell communication, cell metabolism, cell evolution and epigenetic modification. The knockdown of PDIA3 in tumor cells inhibited cell proliferation and invasion, and restrained cocultured M2 macrophage migration. Furthermore, PDIA3 displayed predictive value in immunotherapy response in human cancer cohorts, indicating a potential therapeutic target. Our study showed that PDIA3 was associated with tumor malignant characteristics and could mediate the migration of M2 macrophages in various tumor types. PDIA3 could be a promising target to achieve tumor control and improve the immune response on a pan-cancer scale.
Asunto(s)
Macrófagos , Neoplasias , Proteína Disulfuro Isomerasas , Análisis de la Célula Individual , Humanos , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Macrófagos/metabolismo , Macrófagos/inmunología , Proliferación Celular , Línea Celular Tumoral , Epigénesis Genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
Protein disulfide isomerase-A1 (PDIA1) is a master regulator of oxidative protein folding and proteostasis in the endoplasmic reticulum (ER). However, PDIA1 can reach the extracellular space, impacting thrombosis and other pathophysiological phenomena. Whether PDIA1 is externalized via passive release or active secretion is not known. To investigate how PDIA1 negotiates its export, we generated a tagged variant that undergoes N-glycosylation in the ER (Glyco-PDIA1). Addition of N-glycans does not alter its enzymatic functions. Upon either deletion of its KDEL ER-localization motif or silencing of KDEL receptors, Glyco-PDIA1 acquires complex glycans in the Golgi and is secreted. In control cells, however, Glyco-PDIA1 is released with endoglycosidase-H sensitive glycans, implying that it does not follow the classical ER-Golgi route nor does it encounter glycanases in the cytosol. Extracellular Glyco-PDIA1 is more abundant than actin, lactate dehydrogenase, or other proteins released by damaged or dead cells, suggesting active transport through a Golgi-independent route. The strategy we describe herein can be extended to dissect how select ER-residents reach the extracellular space.
Asunto(s)
Retículo Endoplásmico , Aparato de Golgi , Proteína Disulfuro Isomerasas , Transporte de Proteínas , Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Aparato de Golgi/metabolismo , Humanos , Glicosilación , Espacio Extracelular/metabolismo , Células HeLa , Receptores de Péptidos/metabolismo , Receptores de Péptidos/genética , Polisacáridos/metabolismo , Animales , Células HEK293RESUMEN
The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Retículo Endoplásmico , Hepatocitos , Proteína Disulfuro Isomerasas , Transducción de Señal , Tunicamicina , Chaperón BiP del Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Hepatocitos/metabolismo , Animales , Tunicamicina/farmacología , Retículo Endoplásmico/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción Activador 6/metabolismo , Factor de Transcripción Activador 6/genética , Línea Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Ácido Palmítico/farmacología , Ácido Palmítico/metabolismo , Tapsigargina/farmacología , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Supervivencia Celular/efectos de los fármacosRESUMEN
Copper complexes have shown promising anticancer properties, but they are often poorly soluble in aqueous solutions, thus limiting their possible medical developments and applications. We have recently isolated some copper(II) complexes with salicylaldehyde thiosemicarbazone ligands exhibiting remarkable nanomolar cytotoxic activity, but in vivo tests evidenced several difficulties related to their poor solubility. To overcome these limitations and increase solubility in aqueous solution, herein we report the synthetic strategy that led to the introduction of the sulfonic group on the ligands, then separated as salts (NaH2L1 - NaH2L5), as well as the synthesis and characterization of the related copper(II) complexes. The characterization of the complexes is completed by the analysis of the structures obtained by X-rays diffraction on single crystals on the species [Cu(HL5)(H2O)]2.2H2O, [Cu(HL2)(H2O)2].2H2O, and [Cu(HL1)(H2O]2.2H2O. While the uncoordinated ligands do not affect cancer cell viability, copper(II) complexes exhibit low to sub-micromolar cytotoxic activity, which is maintained in 3D (HCT-15 and 2008) spheroidal models of cancer cell. Notably, copper(II) complexes were selective towards cancer cells, showing high selectivity indexes. Investigations focused on elucidating the mechanism of action evidenced the protein disulfide-isomerase as an innovative molecular target for this class of water-soluble copper(II) complexes. Finally, preliminary in vivo experiments performed with the most representative derivative in the murine Lewis Lung Carcinoma, highlight its significant antitumor efficacy and better tolerability profile with respect to the reference metallodrug, suggesting for this sulfonated Cu(II) complex a potential clinical relevance.
Asunto(s)
Antineoplásicos , Cobre , Ensayos de Selección de Medicamentos Antitumorales , Proteína Disulfuro Isomerasas , Solubilidad , Tiosemicarbazonas , Agua , Tiosemicarbazonas/química , Tiosemicarbazonas/farmacología , Tiosemicarbazonas/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Humanos , Agua/química , Animales , Cobre/química , Cobre/farmacología , Ratones , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Proliferación Celular/efectos de los fármacos , Línea Celular TumoralRESUMEN
ABSTRACT: Monoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbß3. Protein disulfide isomerase (PDI) has been implicated in αIIbß3 activation and binds to thrombin-activated αIIbß3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor-activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate-induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-ß3 ß-I domain), and immunoblot studies indicated that R21D10 binds to ß3. The dissociation of αIIbß3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbß3-R21D10 Fab complex revealed that R21D10 binds to the ß3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbß3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the ß3 I-EGF2 domain associated with a new interaction between the ß3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the ß3 hybrid domain required for high-affinity ligand binding and protect αIIbß3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbß3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.
Asunto(s)
Anticuerpos Monoclonales , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Unión Proteica , Humanos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Anticuerpos Monoclonales/química , Ligandos , Plaquetas/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Conformación Proteica , Regulación Alostérica , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/antagonistas & inhibidoresRESUMEN
Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca2+), STIM1 mediates a ubiquitous Ca2+ influx process called the store-operated Ca2+ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)-Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)-as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca2+ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca2+ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1's Ca2+ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1's Ca2+ sensitivity was further confirmed by Ca2+ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca2+ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca2+ signaling and redox responses reported herein may have implications for cell physiology and pathology.
Asunto(s)
Calcio , Proteínas de Neoplasias , Oxidación-Reducción , Procolágeno-Prolina Dioxigenasa , Proteína Disulfuro Isomerasas , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 1/genética , Humanos , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Calcio/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Retículo Endoplásmico/metabolismo , Células HEK293 , Unión Proteica , Señalización del Calcio , Molécula de Interacción Estromal 2/metabolismo , Molécula de Interacción Estromal 2/genéticaRESUMEN
BACKGROUND: Chimeric antigen receptor natural killer (CAR-NK) therapy holds great promise for treating hematologic tumors, but its efficacy in solid tumors is limited owing to the lack of suitable targets and poor infiltration of engineered NK cells. Here, we explore whether immunogenic cell death (ICD) marker ERp57 translocated from endoplasmic reticulum to cell surface after drug treatment could be used as a target for CAR-NK therapy. METHODS: To target ERp57, a VHH phage display library was used for screening ERp57-targeted nanobodies (Nbs). A candidate Nb with high binding affinity to both human and mouse ERp57 was used for constructing CAR-NK cells. Various in vitro and in vivo studies were performed to assess the antitumor efficacy of the constructed CAR-NK cells. RESULTS: We demonstrate that the translocation of ERp57 can not only be induced by low-dose oxaliplatin (OXP) treatment but also is spontaneously expressed on the surface of various types of tumor cell lines. Our results show that G6-CAR-NK92 cells can effectively kill various tumor cell lines in vitro on which ERp57 is induced or intrinsically expressed, and also exhibit potent antitumor effects in cancer cell-derived xenograft and patient-derived xenograft mouse models. Additionally, the antitumor activity of G6-CAR-NK92 cells is synergistically enhanced by the low-dose ICD-inducible drug OXP. CONCLUSION: Collectively, our findings suggest that ERp57 can be leveraged as a new tumor antigen for CAR-NK targeting, and the resultant CAR-NK cells have the potential to be applied as a broad-spectrum immune cell therapy for various cancers by combining with ICD inducer drugs.
Asunto(s)
Muerte Celular Inmunogénica , Células Asesinas Naturales , Oxaliplatino , Proteína Disulfuro Isomerasas , Humanos , Animales , Ratones , Oxaliplatino/farmacología , Oxaliplatino/uso terapéutico , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/efectos de los fármacos , Muerte Celular Inmunogénica/efectos de los fármacos , Proteína Disulfuro Isomerasas/metabolismo , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , FemeninoRESUMEN
Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Ratones Noqueados , Proteína Disulfuro Isomerasas , Espermatogénesis , Testículo , Animales , Masculino , Espermatogénesis/genética , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Ratones , Testículo/metabolismo , Chaperón BiP del Retículo Endoplásmico/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Apoptosis , Espermatocitos/metabolismo , Estrés del Retículo Endoplásmico , Oligospermia/genética , Oligospermia/metabolismo , Oligospermia/patologíaRESUMEN
Dysregulated T cell activation underpins the immunopathology of rheumatoid arthritis (RA), yet the machineries that orchestrate T cell effector program remain incompletely understood. Herein, we leveraged bulk and single-cell RNA sequencing data from RA patients and validated protein disulfide isomerase family A member 3 (PDIA3) as a potential therapeutic target. PDIA3 is remarkably upregulated in pathogenic CD4 T cells derived from RA patients and positively correlates with C-reactive protein level and disease activity score 28. Pharmacological inhibition or genetic ablation of PDIA3 alleviates RA-associated articular pathology and autoimmune responses. Mechanistically, T cell receptor signaling triggers intracellular calcium flux to activate NFAT1, a process that is further potentiated by Wnt5a under RA settings. Activated NFAT1 then directly binds to the Pdia3 promoter to enhance the expression of PDIA3, which complexes with STAT1 or PKM2 to facilitate their nuclear import for transcribing T helper 1 (Th1) and Th17 lineage-related genes, respectively. This non-canonical regulatory mechanism likely occurs under pathological conditions, as PDIA3 could only be highly induced following aberrant external stimuli. Together, our data support that targeting PDIA3 is a vital strategy to mitigate autoimmune diseases, such as RA, in clinical settings.
Asunto(s)
Artritis Reumatoide , Proteína Disulfuro Isomerasas , Factor de Transcripción STAT1 , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Humanos , Artritis Reumatoide/metabolismo , Ratones , Animales , Factor de Transcripción STAT1/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Transporte Activo de Núcleo Celular , Proteínas Portadoras/metabolismo , Transducción de Señal , Proteínas de Unión a Hormona Tiroide , Factores de Transcripción NFATC/metabolismo , Activación de Linfocitos , Hormonas Tiroideas/metabolismo , Regulación de la Expresión Génica , Células Th17/metabolismo , Células Th17/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Modelos Animales de Enfermedad , Piruvato QuinasaRESUMEN
Compounds harboring high acidity and oxidizability of thiol groups permit tuning the redox equilibrium constants of CxxC sites of members of the protein disulphide isomerase (PDI) family and thus can be used to accelerate folding processes and increase the production of native proteins by minimal loading in comparison to glutathione.
Asunto(s)
Proteína Disulfuro Isomerasas , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/química , Oxidación-Reducción , Pliegue de Proteína , Secuencias de Aminoácidos , Humanos , Glutatión/metabolismo , Glutatión/químicaRESUMEN
Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.
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Invasividad Neoplásica , Proteína Disulfuro Isomerasas , Humanos , Línea Celular Tumoral , Proteína Disulfuro Isomerasas/metabolismo , Femenino , Regulación hacia Abajo/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Membrana Nuclear/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Péptidos y Proteínas de Señalización IntracelularRESUMEN
The overexpression of PDIA1 in cancer has spurred the quest for effective inhibitors. However, existing inhibitors often bind to only one active site, limiting their efficacy. In our study, we developed a PROTAC-mimetic probe dPA by combining PACMA31 (PA) analogs with cereblon-directed pomalidomide. Through protein profiling and analysis, we confirmed dPA's specific interaction with PDIA1's active site cysteines. We further synthesized PROTAC variants with a thiophene ring and various linkers to enhance degradation efficiency. Notably, H4, featuring a PEG linker, induced significant PDIA1 degradation and inhibited cancer cell proliferation similarly to PA. The biosafety profile of H4 is comparable to that of PA, highlighting its potential for further development in cancer therapy. Our findings highlight a novel strategy for PDIA1 inhibition via targeted degradation, offering promising prospects in cancer therapeutics. This approach may overcome limitations of conventional inhibitors, presenting new avenues for advancing anti-cancer interventions.
Asunto(s)
Antineoplásicos , Proliferación Celular , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Sondas Moleculares/química , Sondas Moleculares/farmacología , Sondas Moleculares/síntesis química , Estructura Molecular , Procolágeno-Prolina Dioxigenasa , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/metabolismo , Relación Estructura-Actividad , Péptidos/química , Péptidos/farmacologíaRESUMEN
Endogenous hydrogen sulfide (H2S) plays an important role in bone metabolism. However, the exact role of H2S in intestinal calcium and phosphorus absorption and its potential in preventing and treating primary osteoporosis remains unknown. Therefore, this study aimed to investigate the potential of H2S in promoting intestinal calcium and phosphorus absorption and alleviating primary osteoporosis. We measured the apparent absorptivity of calcium, femoral bone density, expression and sulfhydration of the duodenal endoplasmic reticulum protein of 57 kDa (ERp57), duodenal cystathionine γ-lyase (CSE) expression, and serum H2S content in adult and old CSE-knockout and wild-type mice. We also assessed intracellular reactive oxygen species (ROS) and Ca2+ content in CSE-overexpressing or knockout intestinal epithelial cell (IEC)-6 cells. In senile mice, CSE knockout decreased endogenous H2S, ERp57 sulfhydration, and intestinal calcium absorption and worsened osteoporosis, which were partially reversed by GYY4137, an H2S donor. CSE overexpression in IEC-6 cells increased ERp57 sulfhydration, protein kinase A and C activity, and intracellular Ca2+, whereas CSE knockout exerted the opposite effects. Furthermore, hydrogen peroxide (H2O2) stimulation had similar effects as in CSE knockout, which were reversed by pretreatment with sodium hydrosulfide before H2O2 stimulation and restored by DL-dithiothreitol. These findings suggest that H2S attenuates primary osteoporosis by preventing ROS-induced ERp57 damage in intestinal epithelial cells by enhancing ERp57 activity and promoting intestinal calcium absorption, thereby aiding in developing therapeutic interventions to prevent osteoporosis.
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Calcio , Sulfuro de Hidrógeno , Osteoporosis , Proteína Disulfuro Isomerasas , Animales , Masculino , Ratones , Calcio/metabolismo , Línea Celular , Cistationina gamma-Liasa/metabolismo , Sulfuro de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/farmacología , Absorción Intestinal/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoporosis/metabolismo , Osteoporosis/prevención & control , Proteína Disulfuro Isomerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease affecting motor neurons. Pathological forms of Tar-DNA binding protein-43 (TDP-43), involving its mislocalisation to the cytoplasm and the formation of misfolded inclusions, are present in almost all ALS cases (97%), and ~ 50% cases of the related condition, frontotemporal dementia (FTD), highlighting its importance in neurodegeneration. Previous studies have shown that endoplasmic reticulum protein 57 (ERp57), a member of the protein disulphide isomerase (PDI) family of redox chaperones, is protective against ALS-linked mutant superoxide dismutase (SOD1) in neuronal cells and transgenic SOD1G93A mouse models. However, it remains unclear whether ERp57 is protective against pathological TDP-43 in ALS. Here, we demonstrate that ERp57 is protective against key features of TDP-43 pathology in neuronal cells. ERp57 inhibited the mislocalisation of TDP-43M337V from the nucleus to the cytoplasm. In addition, ERp57 inhibited the number of inclusions formed by ALS-associated variant TDP-43M337V and reduced the size of these inclusions. ERp57 was also protective against ER stress and induction of apoptosis. Furthermore, ERp57 modulated the steady-state expression levels of TDP-43. This study therefore demonstrates a novel mechanism of action of ERp57 in ALS. It also implies that ERp57 may have potential as a novel therapeutic target to prevent the TDP-43 pathology associated with neurodegeneration.
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Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Cuerpos de Inclusión , Proteína Disulfuro Isomerasas , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/genética , Animales , Ratones , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Superóxido Dismutasa-1/genética , MutaciónRESUMEN
Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and "clickable" ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Cetonas , Escherichia coli/enzimología , Escherichia coli/efectos de los fármacos , Cetonas/química , Cetonas/farmacología , Cetonas/síntesis química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Especificidad por Sustrato , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis químicaRESUMEN
Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.