Pyroptosis is an identified programmed cell death that has been highly linked to endoplasmic reticulum (ER) dynamics. However, the crucial proteins for modulating dynamic ER membrane curvature change that trigger pyroptosis are currently not well understood. In this study, a biotin-labeled chemical probe of potent pyroptosis inducer α-mangostin (α-MG) was synthesized. Through protein microarray analysis, reticulon-4 (RTN4/Nogo), a crucial regulator of ER membrane curvature, was identified as a target of α-MG. We observed that chemically induced proteasome degradation of RTN4 by α-MG through recruiting E3 ligase UBR5 significantly enhances the pyroptosis phenotype in cancer cells. Interestingly, the downregulation of RTN4 expression significantly facilitated a dynamic remodeling of ER membrane curvature through a transition from tubules to sheets, consequently leading to rapid fusion of the ER with the cell plasma membrane. In particular, the ER-to-plasma membrane fusion process is supported by the observed translocation of several crucial ER markers to the "bubble" structures of pyroptotic cells. Furthermore, α-MG-induced RTN4 knockdown leads to pyruvate kinase M2 (PKM2)-dependent conventional caspase-3/gasdermin E (GSDME) cleavages for pyroptosis progression. In vivo, we observed that chemical or genetic RTN4 knockdown significantly inhibited cancer cells growth, which further exhibited an antitumor immune response with anti-programmed death-1 (anti-PD-1). In translational research, RTN4 high expression was closely correlated with the tumor metastasis and death of patients. Taken together, RTN4 plays a fundamental role in inducing pyroptosis through the modulation of ER membrane curvature remodeling, thus representing a prospective druggable target for anticancer immunotherapy.
Pyroptosis/immunology* ; Humans ; Endoplasmic Reticulum/immunology* ; Animals ; Nogo Proteins/antagonists & inhibitors* ; Mice ; Cell Line, Tumor ; Xanthones/pharmacology* ; Neoplasms/pathology* ; Mice, Nude

Interleukin 24 (IL-24) is a member of the IL-10 cytokine family and is primarily synthesized by lymphocytes and activated monocytes. IL-24 exerts its immunological functions by interacting with membrane receptors or intracellular proteins, leading to the activation of Janus protein tyrosine kinase/signal transducer and activator of transcription (JAK/STAT), p38 mitogen-activated protein kinase (p38 MAPK), and endoplasmic reticulum stress pathways in target cells. This versatile cytokine has specific abilities to inhibit tumor proliferation and invasion, expedite wound healing, and contribute to cardiovascular protection. IL-24 is involved in the pathogenesis of various autoimmune and inflammatory disorders, presenting itself as a prospective therapeutic target for the treatment of such conditions. This article primarily delves into the role and mechanisms of IL-24 in physiological processes, aiming to provide novel insights and avenues for disease treatment.
Humans ; Animals ; Interleukins/physiology* ; Signal Transduction ; Endoplasmic Reticulum Stress ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Neoplasms/metabolism* ; Autoimmune Diseases/metabolism* ; Inflammation/immunology* ; STAT Transcription Factors/metabolism* ; Janus Kinases/metabolism*

OBJECTIVES: To explore a causal relationship between ferroptosis-related gene heat shock protein A5 (HSPA5) and hepatocellular carcinoma (HCC). METHODS: A two-sample Mendelian randomization (MR) design was employed to evaluate the causal relationships among HSPA5, regulatory T cells (Tregs), and HCC. Single nucleotide polymorphisms (SNPs) associated with HSPA5, Tregs and HCC were selected as instrumental variables through publicly available genome-wide association studies (GWAS) databases. MR analysis was used to assess the direct effect of HSPA5 on HCC, followed by two-step MR to analyze the potential mediating role of Tregs. Reverse MR analysis was conducted with HCC as the exposure and HSPA5 as the outcome. Inverse variance weighting was the primary method for testing causal associations in all MR analyses. Robustness of the results was confirmed through MR-Egger, weighted median, weighted mode, and simple mode methods. Heterogeneity of instrumental variables was evaluated using Cochrane's Q statistic, while pleiotropy was tested by MR-Egger intercept and MR-PRESSO, with leave-one-out sensitivity analysis performed for robustness. Data from The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) were utilized to verify the expression levels of HSPA5 in HCC tissues and its correlation with Tregs to reveal the interaction mechanisms between HSPA5 and Tregs in HCC progression and their relationship with patient prognosis. RESULTS: MR analysis showed a positive correlation between elevated HSPA5 expression and HCC risk (all P<0.01), while reverse MR analysis found no statistically significant association between HCC and HSPA5 (P>0.05). HSPA5 expression was significantly correlated with Tregs function (all P<0.05), and the enrichment of Tregs in HCC microenvironment was positively associated with HCC progression (all P<0.05). Mediation analysis indicated that Tregs accounted for 5.00% and 7.45% of the mediation effect between HSPA5 and HCC. TCGA and HPA database analysis revealed that both HSPA5 mRNA and protein expression levels were higher in HCC tissues compared to normal tissues, and high HSPA5 expression was significantly associated with poor prognosis. Immune infiltration analysis confirmed a significant positive correlation between HSPA5 and Tregs, with high Tregs infiltration closely related to HCC progression. CONCLUSIONS Elevated HSPA5 expression is significantly associated with HCC development and poor prognosis. HSPA5 may promote HCC progression by regulating the function of Tregs in the tumor microenvironment.
Humans ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/genetics* ; Endoplasmic Reticulum Chaperone BiP ; Mendelian Randomization Analysis ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; Heat-Shock Proteins/genetics* ; Ferroptosis/genetics* ; T-Lymphocytes, Regulatory/immunology*

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by abnormal proliferation of synoviocytes, leukocyte infiltration, and angiogenesis. The endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded proteins in the ER leads to a condition known as ER stress. Failure of the ER's adaptive capacity results in abnormal activation of the unfolded protein response. Recently, we have demonstrated that ER stress-associated gene signatures are highly expressed in RA synovium and synovial cells. Mice with Grp78 haploinsufficiency exhibit the suppression of experimentally induced arthritis, suggesting that the ER chaperone GRP78 is crucial for RA pathogenesis. Moreover, increasing evidence has suggested that GRP78 participates in antibody generation, T cell proliferation, and pro-inflammatory cytokine production, and is therefore one of the potential therapeutic targets for RA. In this review, we discuss the putative, pathophysiological roles of ER stress and GRP78 in RA pathogenesis.
Animals ; Arthritis, Rheumatoid/genetics/*pathology ; Autoantibodies/immunology ; Cell Proliferation ; Cytokines/biosynthesis/immunology ; Endoplasmic Reticulum/immunology/pathology ; Endoplasmic Reticulum Stress/*immunology ; Haploinsufficiency/genetics ; Heat-Shock Proteins/*genetics/*immunology ; Humans ; Lymphocyte Activation ; Mice ; Neovascularization, Pathologic/genetics ; Protein Folding ; Synovial Membrane/cytology ; T-Lymphocytes/immunology ; Unfolded Protein Response/*immunology

The endoplasmic reticulum quality control (ER-QC) is a conserved mechanism in surveillance of secreted signaling factors during cell-to-cell communication in eukaryotes. Recent data show that the ER-QC plays important roles in diverse cell-to-cell signaling processes during immune response, vegetative and reproductive development in plants. Pollen tube guidance is a precisely guided cell-cell communication process between the male and female gametophytes during plant reproduction. Recently, the female signal has been identified as small secreted peptides, but how the pollen tube responds to this signal is still unclear. In this review, we intend to summarize the role of ER-QC in plants and discuss the recent advances regarding our understanding of the mechanism of pollen tube response to the female signals.
Animals ; Endoplasmic Reticulum ; metabolism ; Humans ; Plant Cells ; metabolism ; Plant Development ; Plant Proteins ; genetics ; metabolism ; Plants ; immunology ; Pollen Tube ; cytology ; growth & development ; immunology ; metabolism ; Signal Transduction

As a member of the HSP90 family, heat shock protein (HSP) Gp96 is one of the most abundant proteins in the endoplasmic reticulum (ER), which displayed important molecular chaperones function in cells. Gp96 can stimulate the production of cytokines by activating the antigen presentation cells (such as dendritic cell, et al) in innate immunity. It is capable of eliciting an antigen-specific cytotoxic T lymphocyte (CTL) immune response to eliminate pathogens and tumors by facilitating antigen cross-presentation in adaptive immunity. Gp96 is also an ideal adjuvant in many recent researches. Here, we review the progress that addresses the role of biological characteristics, immunogenic mechanism that may be involved in the induction of anti-infection immune response and antitumor immunity, which may guide the new vaccine strategies with the knowledge of Gp96-antigen complexes.
Adjuvants, Immunologic ; genetics ; metabolism ; Antigen-Presenting Cells ; physiology ; Communicable Diseases ; immunology ; Dendritic Cells ; immunology ; Endoplasmic Reticulum ; immunology ; Humans ; Membrane Glycoproteins ; immunology ; Neoplasms ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

BACKGROUNDAlthough CD4(+) T cell apoptosis and CD8(+) T cell responses have been extensively studied during HIV infection, how apoptosis signals being initiated in CD4(+) T cells still need to be elucidated. The present study was designed to characterize the function-unknown gene, C6orf120, and elucidates its primary role in tunicamycin-induced CD4(+) T apoptosis.

METHODSThe C6orf120 coding sequence was amplified from peripheral blood mononuclear cells (PBMCs) total RNA of AIDS patients. The DNA fragment was inserted into the pET-32a expression system, transformed into Escherichia coli, and preparation of C6ORF120 recombinant protein. The magnetic cell separation technology was used to prepare primary CD4(+) T cells and CD8(+) T cells. The primary T cells were cultured at 1 × 10(6) cells/ml, treated with 0, 0.1, 1, 10, 100, and 200 ng/ml of C6orf120 recombinant protein for 48 hours, then harvested for cell cycle and apoptosis analysis. Tunicamycin (0.5 µmol/L) was used to induce endoplasmic reticulum stress in Jurkat cells. The biomarker 78 KDa glucose-regulated protein (GRp78) and growth arrest and DNA damage (GADD) were used to evaluate endoplasmic reticulum stress of Jurkat cells.

RESULTSWe prepared C6ORF120 recombinant protein and its polyclonal antibody. Immunohistochemical analysis showed that C6orf120 mainly expressed in hepatocytes and cells in germinal center of lymph node. At concentration of 0.1, 1, 10, 100, and 200 ng/ml, C6orf120 recombinant protein could induce apoptosis of Jurkat cells and primary CD4(+) T cells, and promoting G2 phase of its cell cycle. Western blotting analysis showed that C6ORF120 recombinant protein increased the expression of GRp78 and GADD in Jurkat cells in vitro.

CONCLUSIONOur results suggested that C6ORF120 could induce apoptosis of CD4(+) T cells, at least in part, mediated with endoplasmic reticulum stress.

Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; CD8-Positive T-Lymphocytes ; Cell Cycle ; Cells, Cultured ; Endoplasmic Reticulum Stress ; Female ; HIV Infections ; immunology ; Humans ; Immunohistochemistry ; Male ; Microscopy, Confocal ; Proteins ; genetics ; metabolism ; Tunicamycin ; pharmacology

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG-modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.
Animals ; Cartilage, Articular/pathology ; Cells, Cultured ; Chondrocytes/drug effects/immunology/*metabolism/pathology ; Cyclooxygenase 2/genetics/*metabolism ; Deoxyglucose/*pharmacology ; Down-Regulation ; Endoplasmic Reticulum/drug effects/*metabolism/pathology ; Glycosylation/drug effects ; Inflammation ; Rabbits ; Signal Transduction/drug effects ; Stress, Physiological/drug effects/immunology ; src-Family Kinases/*metabolism

The N-terminal segment (FR-1) of the heavy chain (VH) of antibodies may have a great impact on IgG secretion in Escherichia coli and other hosts. Decrease in secretion may be caused by a single amino acid change in the framework region. To investigate the high antibody expression in mammalian cells, we designed the site-directed mutagenesis of the FR-I of the pCMV-RV/VH gene,which expressed the immunoglobulin heavy chain of human anti-Rabies virus antibody. Mutating Glu (H6) to Gln could improve both antibody secretion and affinity. The immunofluorescence assay indicated that both the secretion-deficient antibodies and the secretion- efficient antibodies could be transcribed and translated intracellularly, and led into ER,then transferred to Golgi apparatus,and the difference in secretion may relate to the contribution of the FR-I to the folding and assembly of the antibody. In this study, we have confirmed experimentally that the nature of residues H6 in antibody heavy chains indeed determines the antibody secretion in mammalian cells. These results also provide the basis for antibody production.
Animals ; Antibodies ; genetics ; immunology ; metabolism ; Antibodies, Viral ; genetics ; immunology ; metabolism ; Antibody Affinity ; Biological Transport ; COS Cells ; Cercopithecus aethiops ; Cytomegalovirus ; genetics ; Endoplasmic Reticulum ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Glycoproteins ; genetics ; immunology ; Golgi Apparatus ; metabolism ; Humans ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Heavy Chains ; chemistry ; genetics ; immunology ; Immunoglobulin Variable Region ; chemistry ; genetics ; immunology ; Mutagenesis, Site-Directed ; Plasmids ; genetics ; Rabies virus ; genetics ; immunology ; metabolism

AIMTo investigate the expression and subcellular localization of chemokine-like factor superfamily 2 (CKLFSF2) in human testis and its potential role in spermatogenesis.

METHODSA specific polyclonal antibody against CKLFSF2 was raised. The expression and cellular localization of CKLFSF2 in the seminiferous tubules was checked by immunohistochemistry method. Also, in situ hybridization was applied to localize the mRNA distribution. The EGFP-CKLFSF2 fusion protein was expressed in COS-7 cells to localize its subcellular location in vitro. In addition, the abnormal expression of CKLFSF2 in testes of patients with male infertility was assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry methods.

RESULTSHaving a close correlation with spermatogenesis defects, CKLFSF2 was specifically expressed in meiotic and post-meiotic germ cells, which were localized to the endoplasmic reticulum (ER) near the Golgi apparatus.

CONCLUSIONCKLFSF2 could play important roles in the process of meiosis and spermiogenesis, and might be involved in the vesicular transport or membrane apposition events in the endoplasmic reticulum.

Animals ; Antibody Specificity ; COS Cells ; Cercopithecus aethiops ; Chemokines ; biosynthesis ; immunology ; Endoplasmic Reticulum ; metabolism ; Germ Cells ; metabolism ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Infertility, Male ; metabolism ; MARVEL Domain-Containing Proteins ; Male ; Meiosis ; Microscopy, Confocal ; Spermatogenesis ; physiology ; Testis ; metabolism