RESUMO
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.
Assuntos
Macrófagos , Neoplasias , Isomerases de Dissulfetos de Proteínas , Análise de Célula Única , Humanos , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Macrófagos/metabolismo , Macrófagos/imunologia , Proliferação de Células , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão GênicaRESUMO
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.
Assuntos
Morte Celular Imunogênica , Células Matadoras Naturais , Oxaliplatina , Isomerases de Dissulfetos de Proteínas , Humanos , Animais , Camundongos , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Morte Celular Imunogênica/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/metabolismo , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/terapia , FemininoRESUMO
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.
Assuntos
Cálcio , Proteínas de Neoplasias , Oxirredução , Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas , Molécula 1 de Interação Estromal , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Cálcio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Ligação Proteica , Sinalização do Cálcio , Molécula 2 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genéticaRESUMO
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.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Hepatócitos , Isomerases de Dissulfetos de Proteínas , Transdução de Sinais , Tunicamicina , Chaperona BiP do Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Hepatócitos/metabolismo , Animais , Tunicamicina/farmacologia , Retículo Endoplasmático/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Fator 6 Ativador da Transcrição/genética , Linhagem Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Ácido Palmítico/farmacologia , Ácido Palmítico/metabolismo , Tapsigargina/farmacologia , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Sobrevivência Celular/efeitos dos fármacosRESUMO
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.
Assuntos
Anticorpos Monoclonais , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Ligação Proteica , Humanos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Anticorpos Monoclonais/química , Ligantes , Plaquetas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Conformação Proteica , Regulação Alostérica , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidoresRESUMO
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.
Assuntos
Antineoplásicos , Cobre , Ensaios de Seleção de Medicamentos Antitumorais , Isomerases de Dissulfetos de Proteínas , Solubilidade , Tiossemicarbazonas , Água , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologia , Tiossemicarbazonas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Humanos , Água/química , Animais , Cobre/química , Cobre/farmacologia , Camundongos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/síntese química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Linhagem Celular TumoralRESUMO
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.
Assuntos
Cálcio , Sulfeto de Hidrogênio , Osteoporose , Isomerases de Dissulfetos de Proteínas , Animais , Masculino , Camundongos , Cálcio/metabolismo , Linhagem Celular , Cistationina gama-Liase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Absorção Intestinal/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoporose/metabolismo , Osteoporose/prevenção & controle , Isomerases de Dissulfetos de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Invasividade Neoplásica , Isomerases de Dissulfetos de Proteínas , Humanos , Linhagem Celular Tumoral , Isomerases de Dissulfetos de Proteínas/metabolismo , Feminino , Regulação para Baixo/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Membrana Nuclear/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
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.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Camundongos Knockout , Isomerases de Dissulfetos de Proteínas , Espermatogênese , Testículo , Animais , Masculino , Espermatogênese/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Camundongos , Testículo/metabolismo , Chaperona BiP do Retículo Endoplasmático/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Apoptose , Espermatócitos/metabolismo , Estresse do Retículo Endoplasmático , Oligospermia/genética , Oligospermia/metabolismo , Oligospermia/patologiaRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Corpos de Inclusão , Isomerases de Dissulfetos de Proteínas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/genética , Animais , Camundongos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Superóxido Dismutase-1/genética , MutaçãoRESUMO
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.
Assuntos
Antineoplásicos , Proliferação de Células , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Sondas Moleculares/síntese química , Estrutura Molecular , Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , Relação Estrutura-Atividade , Peptídeos/química , Peptídeos/farmacologiaRESUMO
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.
Assuntos
Isomerases de Dissulfetos de Proteínas , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Oxirredução , Dobramento de Proteína , Motivos de Aminoácidos , Humanos , Glutationa/metabolismo , Glutationa/químicaRESUMO
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.
Assuntos
Artrite Reumatoide , Isomerases de Dissulfetos de Proteínas , Fator de Transcrição STAT1 , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Humanos , Artrite Reumatoide/metabolismo , Camundongos , Animais , Fator de Transcrição STAT1/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Transporte Ativo do Núcleo Celular , Proteínas de Transporte/metabolismo , Transdução de Sinais , Proteínas de Ligação a Hormônio da Tireoide , Fatores de Transcrição NFATC/metabolismo , Ativação Linfocitária , Hormônios Tireóideos/metabolismo , Regulação da Expressão Gênica , Células Th17/metabolismo , Células Th17/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Modelos Animais de Doenças , Piruvato QuinaseRESUMO
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.
Assuntos
Hipertensão , Músculo Liso Vascular , NADPH Oxidase 1 , Isomerases de Dissulfetos de Proteínas , Ratos Endogâmicos SHR , Regulação para Cima , Animais , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , NADPH Oxidase 1/metabolismo , NADPH Oxidase 1/genética , Hipertensão/fisiopatologia , Hipertensão/genética , Hipertensão/metabolismo , Ratos , Músculo Liso Vascular/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/genética , Ratos Wistar , Transcrição GênicaRESUMO
Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Escherichia coli , Vitamina K Epóxido Redutases , Varfarina , Varfarina/metabolismo , Varfarina/química , Vitamina K Epóxido Redutases/metabolismo , Vitamina K Epóxido Redutases/química , Vitamina K Epóxido Redutases/genética , Humanos , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dissulfetos/química , Dissulfetos/metabolismo , Cumarínicos/metabolismo , Cumarínicos/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Anticoagulantes/química , Anticoagulantes/metabolismo , Benzoquinonas/metabolismo , Benzoquinonas/química , Relação Estrutura-Atividade , Ligação Proteica , Proteínas de MembranaRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Cetonas , Escherichia coli/enzimologia , Escherichia coli/efeitos dos fármacos , Cetonas/química , Cetonas/farmacologia , Cetonas/síntese química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por Substrato , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese químicaRESUMO
Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.
Assuntos
Escherichia coli , Interleucina-3 , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes de Fusão , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Interleucina-3/metabolismo , Interleucina-3/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Linhagem Celular Tumoral , SolubilidadeRESUMO
Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I , Peptídeos , Apresentação de Antígeno/imunologia , Humanos , Peptídeos/metabolismo , Peptídeos/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Calreticulina/metabolismo , Calreticulina/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Retículo Endoplasmático/metabolismoRESUMO
The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: ⢠The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. ⢠After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. ⢠The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.
Assuntos
Oxigenases de Função Mista , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/química , Saccharomycetales/genética , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Dosagem de Genes , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Expressão Gênica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/químicaRESUMO
OBJECTIVE: This inquiry endeavors to delineate the influence of PDIA3 on tumor-associated macrophages within the realm of colorectal malignancies, whilst elucidating the intrinsic biochemical pathways. METHOD: Leveraging bioinformatics, we scrutinized the symbiosis between PDIA3, STAT3, and CD274. A xenograft model in immunodeficient murine served to assess PDIA3's impact on colorectal carcinogenesis. Further, Western blot analysis quantified the protein expression of PDIA3, p-STAT3, PD-1, XBP-1, assorted enzymes, and IL-6. Moreover, in vitro assays gauged SW480 cellular dynamics inclusive of migration, invasive potential, and proliferation. RESULTS: Bioinformatics exploration exposed PDIA3's elevated presence in diverse cancers, with a marked expression in colorectal cancer, as per TCGA and GEO repositories. Correlative studies showed PDIA3 positively aligning with STAT3 and CD274, the latter also associated with monocyte-derived macrophages. Comparative analysis of colorectal neoplasms and normal colon samples unveiled heightened levels of PDIA3 markers which, when overexpressed in SW480 cells, escalated tumorigenicity and oncogenic behaviors, with a noted decrease upon PD-1 monoclonal antibody intervention. CONCLUSIONS: PDIA3 augments the M2 polarization of tumor-associated macrophages via modulation of the STAT3/PD-1 cascade, thus invigorating the tumorous proliferation and dissemination in colorectal cancer. Such revelations position PDIA3 as an auspicious target for PD-1 blockade therapeutics, offering a promising foundation for rectifying colorectal carcinoma.