Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Development of Clarstatin, a Novel Drug Lead for the Therapy of Autoimmune Uveitis.

We describe the design, synthesis, and activity of a potent thiourea-bridged backbone cyclic peptidomimetic known as Clarstatin, comprising a 5-amino acid sequence (Q/D)1-(R/K)2-X3-X4-A5-(Gln/Asp)1-(Arg/Lys)2-AA3-AA4-Ala5-based on a motif called "shared epitope (SE)", specifically present in specific alleles of the HLA-DRB1 gene. This SE binds to a particular site within the proline reach domain (P-domain) of the cell surface-calreticulin (CS-CRT). CS-CRT is a multifunctional endoplasmic reticulum (ER) calcium-binding protein that is located on the cell surface of T cells and triggers innate immune signaling, leading to the development of inflammatory autoimmune diseases. The development of Clarstatin was based on the parent peptide W-G-D1-K2-S3-G4-A5- derived from the active region of the SE. Following the design based on the cycloscan method, the synthesis of Clarstatin was performed by the Fmoc solid phase peptide synthesis (SPPS) method, purified by HPLC to 96% homogeneity, and its structure was confirmed by LC-MS. Clarstatin reduced calcium levels in Jurkat lymphocyte cultures, ameliorated uveitis in vivo in the experimental autoimmune uveitis (EAU) mice model, and was safe upon acute toxicity evaluation. These findings identify Clarstatin as a promising lead compound for future drug development as a novel class of therapeutic agents in the therapy of uveitis.

Proteasome inhibitors FHND6091 enhance the ability of NK cells to kill tumor cells through multiple mechanisms.

The immune system has a strong connection to tumors. When a tumor cell is recognized as an abnormal cell by the immune system, the immune system may initiate an immune response to kill the tumor cell. In this study, RNA sequencing was performed on multiple myeloma (MM) cells treated with the proteasome inhibitor FHND6091. The transcriptional changes induced by FHND6091 in RPMI8226 cells aligned notably with immune response activation and results indicated upregulation of cGAS-STING pathway-related genes in the FHND6091-treated group. In vivo and in vitro experiments had demonstrated that FHND6091 stimulated the immunoreaction of MM cells via activation of the cyclic guanosine monophosphate-adenosine synthase/stimulator of interferon genes (cGAS-STING) pathway. This activation resulted in the generation of type-I interferons and the mobilization of natural killer (NK) cells. Notably, FHND6091 upregulated the levels of calreticulin and the protein ligands UL16-binding protein 2/5/6, MHC class I chain-related A (MICA), and MICB on the surface of MM cells. Subsequently, upon engaging with the surface activation receptors of NK cells, these ligands triggered NK cell activation, leading to the subsequent elimination of tumor cells. Thus, our findings elucidated the mechanism whereby FHND6091 exerted its immunotherapeutic activity as a STING agonist, enhancing the killing ability of NK cells against tumor cells.

Detection of anti-calreticulin antibody in the sera of Chinese patients with primary Sjögren syndrome.

BACKGROUND: Primary Sjögren syndrome (pSjS) is one of the most prevalent systemic autoimmune diseases and characterized with hyperactivation of B cell and the abundant presence of autoantibodies in sera. The salivary gland epithelial cells (SGECs) release autoantigens to evoke autoimmunity through releasing elevated apoptosis or secreting autoantigen-containing exosomes, thus identifying autoantibodies directly to SGECs might provide insights into disease related biomarkers as well as further elucidating pathogenesis mechanisms. The present study was undertaken to identify autoantibodies to SGECs and to evaluate its clinical values in Chinese pSjS. METHODS: Cell-based indirect immunofluorescence and immunostaining, two-dimensional electrophoresis and liquid chromatograph-tandem mass spectrometry were conducted to identify the autoantibodies to human salivary gland cell line A253 in pSjS sera. Enzyme-linked immunosorbent assay (ELISA) was applied to identify autoantibody titer in pSjS cohort and healthy controls. The prevalence and clinical significance of the identified autoantibodies was further assessed in pSjS population. RESULTS: Anti-calreticulin (CALR) antibody was identified as a new autoantibody directly to SGECs in sera from pSjS patients. Anti-CALR antibody were detected in 37 of 120 pSjS patients (30.83 %) and 1 of 54 healthy controls (1.85 %). It was found in 40.85 % pSjS with anti-SSA positive, 53.85 % with anti-SSB positive, and 14.7 % in sero-negative pSjS. Anti-CALR antibody was associated with clinical manifestations including weight loss(p = 0.045), vasculitis (p = 0.031), and laboratory parameters including erythrocyte sedimentation rate (ESR) (r = 0.056, p = 0.021), Krebs von den Lungen-6 (KL-6) (r = 0.121, p = 0.035), IgG (r = 0.097, p < 0.001), IgG2 (r = 0.142, p = 0.022), IgG3 (r = 0.287, p < 0.001), fibrinogen (r = 0.084, p = 0.016), D-Dimer (r = 0.086, p = 0.012) and fibrinogen degradation production (r = 0.150, p = 0.002). The expression of CALR in salivary glands was related to lymphocytes infiltration into salivary glands in pSjS patients (r = 0.7076, p = 0.0034). CONCLUSION: To our knowledge, this was the first study to investigate the prevalence and clinical significance of anti-CALR antibody in Chinses pSjS patients. The present study identified an autoimmune antibody, anti-CALR antibody, as a good autoimmune biomarker for sero-negative pSjS.

Calreticulin regulates hepatic stellate cell activation through modulating TGF-beta-induced Smad signaling.

Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) as a wound healing process. Activated hepatic stellate cells (HpSCs) are the major producer of the ECM and play a central role in liver fibrogenesis. It has been widely accepted that elimination of activated HpSCs or reversion to a quiescent state can be a feasible strategy for resolving the disease, further highlighting the urgent need for novel therapeutic targets. Calreticulin (CRT) is a molecular chaperone that normally resides in the endoplasmic reticulum (ER), important in protein folding and trafficking through the secretory pathway. CRT also plays a critical role in calcium (Ca2+) homeostasis, with its Ca2+ storage capacity. In the current study, we aimed to demonstrate its function in directing HpSC activation. In a mouse liver injury model, CRT was up-regulated in HpSCs. In cellular experiments, we further showed that this activation was through modulating the canonical TGF-ß signaling. As down-regulation of CRT in HpSCs elevated intracellular Ca2+ levels through a form of Ca2+ influx, named store-operated Ca2+ entry (SOCE), we examined whether moderating SOCE affected TGF-ß signaling. Interestingly, blocking SOCE had little effect on TGF-ß-induced gene expression. In contrast, inhibition of ER Ca2+ release using the inositol trisphosphate receptor inhibitor 2-APB increased TGF-ß signaling. Treatment with 2-APB did not alter SOCE but decreased intracellular Ca2+ at the basal level. Indeed, adjusting Ca2+ concentrations by EGTA or BAPTA-AM chelation further enhanced TGF-ß-induced signaling. Our results suggest a crucial role of CRT in the liver fibrogenic process through modulating Ca2+ concentrations and TGF-ß signaling in HpSCs, which may provide new information and help advance the current discoveries for liver fibrosis.

Crystal structure of Trichinella spiralis calreticulin and the structural basis of its complement evasion mechanism involving C1q.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

In vitro immunoregulatory role of recombinant Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestines of humans and animals (dogs and cats), leading to malnutrition and iron-deficiency anemia. Helminth parasites secrete calreticulin (CRT), which regulates or blocks the host's immune response. However, no data on A. ceylanicum calreticulin (Ace-CRT) are available. We investigated the biological function of recombinant Ace-CRT (rAce-CRT). rAce-CRT showed reliable antigenicity and stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells. Quantitative reverse-transcription PCR assays revealed that rAce-CRT primarily promoted the expression of T helper 2 cytokines, particularly IL-13, in canine peripheral blood lymphocytes. rAce-CRT inhibited complement-mediated sheep erythrocyte hemolysis in vitro. Our findings indicate that Ace-CRT plays an immunomodulatory role and may be a promising candidate molecule for a hookworm vaccine.

Gold standard assessment of immunogenic cell death induced by photodynamic therapy: From in vitro to tumor mouse models and anti-cancer vaccination strategies.

The discovery of the concept of immunogenic cell death (ICD) is a cornerstone in the development of novel anti-cancer immunotherapeutic approaches. Induction of the ICD pathway by specific anti-cancer therapeutic regimens can eliminate cancer cells by directly killing them during therapy and by activation of strong and specific anti-cancer immunity, leading to a long-lasting immunological memory that prevents cancer recurrence. ICD encompasses different forms of regulated cell death and can be triggered by many anti-cancer treatment modalities, including photodynamic therapy (PDT). PDT is a multistep procedure involving the accumulation of a light-sensitive dye known as a photosensitizer (PS) in tumor cells, followed by its activation by irradiation with a light of an appropriate wavelength. In the presence of molecular oxygen, the irradiated PS leads to the generation of cytotoxic reactive oxygen species, which can lead to ICD induction in the cancer cells. Here, we first describe in vitro methods to help optimize the PDT procedure for a specific PS. We also provide a collection of protocols and techniques for assessing ICD in vitro, including analysis of the emission of damage associated molecular patterns (DAMPs), efferocytosis, and the maturation and activation state of antigen presenting cells. Next, we describe in detail protocols for diverse tumor mouse models for assessing and characterizing ICD in vivo, such as murine tumor vaccination models. Finally, as an immunotherapeutic vaccine, we suggest using either PDT-induced dead cancer cells, preferably undergoing ICD, or dendritic cells loaded with lysates of PDT-induced cancer cells in a syngeneic orthotopic glioma model. Overall, this methodological article provides a quantitative, comprehensive set of validated tools that can be successfully used, with some adaptations, to identify, optimize and validate novel PSs in vitro and in vivo for the efficient induction of ICD during photodynamic treatment.

Nano-delivery of Silibinin Potentiate the Induction of Immunogenic Cell Death (ICD) in Melanoma Cells.

BACKGROUND: Induction of immunogenic cell death (ICD) in tumors can enhance antitumor immunity and modulate immunosuppression in the tumor microenvironment (TME). OBJECTIVE: In the current study, we investigated the effect of silibinin, a natural compound with anticancer activity, and its polymer-based nanoformulations on the induction of apoptosis and ICD in cancer cells. METHODS: Free and nanoparticulate silibinin were evaluated for their growth-inhibitory effects using an MTT assay. Annexin V/PI staining was used to analyze apoptosis. Calreticulin (CRT) expression was measured by flow cytometry. Western blotting was conducted to examine the levels of elf2α, which plays a role in the ICD pathway. The HSP90 and ATP levels were determined using specific detection kits. RESULTS: Compared to the free drug, silibinin-loaded nanocarriers significantly increased the induction of apoptosis and ICD in B16F10 cells. ICD induction was characterized by significantly increased levels of ICD biomarkers, including CRT, HSP90, and ATP. We also observed an increased expression of p-elf-2α/ elf-2α in B16F10 cells treated with silibinin-loaded micelles compared to cells that received free silibinin. CONCLUSION: Our findings showed that the encapsulation of silibinin in polymeric nanocarriers can potentiate the effects of this drug on the induction of apoptosis and ICD in B16F10 melanoma cells.

Combining cisplatin with Pinellia pedatisecta Schott lipid-soluble extract induces tumor immunogenic cell death in cervical cancer.

BACKGROUND: Pinellia pedatisecta Schott extract (PE) is extracted from Pinellia pedatisecta Schott (PPS), a traditional Chinese medicinal plant with the potential for direct anticancer effects or eliciting an anti-tumor response by activating the immune system. PURPOSE: To explore PE's ability and mechanism to reconstruct cisplatin's immunogenicity. METHODS: Cervical cancer cells were treated with cisplatin (CDDP) and/or PE. The exposure of calreticulin (CRT) on cell membrane was investigated by flow cytometry. The extracellular of ATP and HMGB1 was investigated by Western blot analysis, immunofluorescence and ELISA assay. Changes in immune profiles were using flow cytometry in vaccination and anti-tumor assays in vivo. Lastly, the mechanism of PE influenced the ROS/ERS pathway was examined by ROS assay kit, flow cytometry and Western blotting. RESULTS: PE treatment induced translocation of CRT from the endoplasmic reticulum to the cell membrane of tumor cells, concomitantly triggering immunogenic cell death (ICD). In terms of mechanisms, endoplasmic reticulum (ER) stress relievers could impede the ability of PE to induce immunogenicity. This indicates that PE is activated by ER stress, leading to subsequent induction of ICD. Upon analyzing RNA-seq data, it was observed that PE primarily induces programmed cell death in tumors by impeding upstream antioxidant mechanisms. Additionally, it transforms dying tumor cells into vaccines, activating a series of immune responses. CONCLUSIONS: This study observed for the first time that PE-induced CRT exposure on the membrane of cervical cancer cells compensates for the defect of nonimmunogenic cell death inducer CDDP thereby stimulating potent ICD. This ability restores the immunogenicity of CDDP through ER stress induced by the ROS signal. ROS played a role in PE's ability to induce ICD, leading to increased expression of ER stress-related proteins, including ATF3 and IRE-1α. PE exerted anti-cancer effects by increasing the ROS levels, and ROS/ERS signaling may be a potential avenue for cervical cancer treatment. Hence, the synergistic use of PE and CDDP holds potential for enhancing immunochemotherapy in cancer treatment.

Calreticulin (CALR) promotes ionophore-induced microneme secretion in Toxoplasma gondii.

The flow of calcium ions (Ca2+) is involved in numerous vital activities of Toxoplasma gondii. Calreticulin is a type of Ca2+-binding protein in the endoplasmic reticulum (ER) that is involved in Ca2+ signaling pathway regulation, Ca2+ storage, and protein folding. In this work, the calreticulin (CALR), a protein predicted to possess a conserved domain of calreticulin in T. gondii, was characterized. The CALR localized in the ER. Using reverse genetics, we discovered that CALR is not necessary for the lytic cycle, including invasion and replication. However, depletion of CALR affected microneme secretion triggered by A23187, which is a Ca2+ ionophore used to increase cytoplasmic Ca2+ concentration. Furthermore, we discovered that CALR influences Ca2+ release. Transcriptomic comparison between Δcalr and Δku80 parasites showed that 226 genes in the Δcalr parasites were significantly downregulated (p < 0.05). The cellular biological functions of the downregulated genes were mainly involved in calmodulin-dependent protein kinase pathways. Furthermore, in the absence of CALR, tachyzoites were still able to cause acute infection in mice. These results imply that by influencing ER Ca2+ release content, CALR may further impair the ionophore-induced secretion of the parasite. However, this protein is not required for the completion of the parasite's lytic cycle or for the acute virulence of the parasite.

DAMPening Tumor Immune Escape: The Role of Endoplasmic Reticulum Chaperones in Immunogenic Chemotherapy.

Significance: Preclinical and clinical research in the past two decades has redefined the mechanism of action of some chemotherapeutics that are able to activate the immune system against cancer when cell death is perceived by the immune cells. This immunogenic cell death (ICD) activates antigen-presenting cells (APCs) and T cells to induce immune-mediated tumor clearance. One of the key requirements to achieve this effect is the externalization of the damage-associated molecular patterns (DAMPs), molecules released or exposed by cancer cells during ICD that increase the visibility of the cancer cells by the immune system. Recent Advances: In this review, we focus on the role of calreticulin (CRT) and other endoplasmic reticulum (ER) chaperones, such as the heat-shock proteins (HSPs) and the protein disulfide isomerases (PDIs), as surface-exposed DAMPs. Once exposed on the cell membrane, these proteins shift their role from that of ER chaperone and regulator of Ca2+ and protein homeostasis to act as an immunogenic signal for APCs, driving dendritic cell (DC)-mediated phagocytosis and T-mediated antitumor response. Critical Issues: However, cancer cells exploit several mechanisms of resistance to immune attack, including subverting the exposure of ER chaperones on their surface to avoid immune recognition. Future Directions: Overcoming these mechanisms of resistance represents a potential therapeutic opportunity to improve cancer treatment effectiveness and patient outcomes.

The nematode effector calreticulin competes with the high mobility group protein OsHMGB1 for binding to the rice calmodulin-like protein OsCML31 to enhance rice susceptibility to Meloidogyne graminicola.

The root-knot nematode Meloidogyne graminicola secretes effectors into rice tissues to modulate host immunity. Here, we characterised MgCRT1, a calreticulin protein of M. graminicola, and identified its target in the plant. In situ hybridisation showed MgCRT1 mRNA accumulating in the subventral oesophageal gland in J2 nematodes. Immunolocalization indicated MgCRT1 localises in the giant cells during parasitism. Host-induced gene silencing of MgCRT1 reduced the infection ability of M. graminicola, while over-expressing MgCRT1 enhanced rice susceptibility to M. graminicola. A yeast two-hybrid approach identified the calmodulin-like protein OsCML31 as an interactor of MgCRT1. OsCML31 interacts with the high mobility group protein OsHMGB1 which is a conserved DNA binding protein. Knockout of OsCML31 or overexpression of OsHMGB1 in rice results in enhanced susceptibility to M. graminicola. In contrast, overexpression of OsCML31 or knockout of OsHMGB1 in rice decreases susceptibility to M. graminicola. The GST-pulldown and luciferase complementation imaging assay showed that MgCRT1 decreases the interaction of OsCML31 and OsHMGB1 in a competitive manner. In conclusion, when M. graminicola infects rice and secretes MgCRT1 into rice, MgCRT1 interacts with OsCML31 and decreases the association of OsCML31 with OsHMGB1, resulting in the release of OsHMGB1 to enhance rice susceptibility.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

Insights into cucumber (Cucumis sativus) genetics: Genome-wide discovery and computational analysis of the Calreticulin Domain-Encoding gene (CDEG) family.

Cucumber is an essential vegetable crop throughout the world. Cucumber development is vital for accomplishing both quality and productivity requirements. Meanwhile, numerous factors have resulted in substantial cucumber losses. However, the calreticulin domain-encoding genes (CDEGs) in cucumber were not well-characterized and had little function. In the genome-wide association study (GWAS), we recognized and characterized the CDEGs in Cucumis sativus (cucumber). Through a comprehensive study of C. sativus, our research has unveiled the presence of three unique genes, denoted as CsCRTb, CsCRT3, and CsCNX1, unevenly distributed on three chromosomes in the genome of C. sativus. In accordance to the phylogenetic investigation, these genes may be categorized into three subfamilies. Based on the resemblance with AtCDE genes, we reorganized the all CsCDE genes in accordance with international nomenclature. The expression analysis and cis-acting components revealed that each of CsCDE gene promoter region enclosed number of cis-elements connected with hormone and stress response. According to subcellular localization studies demonstrated that, they were found in deferent locations of the cell such as endoplasmic reticulum, plasma membrane, golgi apparatus, and vacuole, according to subcellular localization studies. Chromosomal distribution analysis and synteny analysis demonstrated the probability of segmental or tandem duplications within the cucumber CDEG gene family. Additionally, miRNAs displayed diverse modes of action, including mRNA cleavage and translational inhibition. We used the RNA seq data to analyze the expression of CDEG genes in response to cold stress and also improved cold tolerance, which was brought on by treating cucumber plants to an exogenous chitosan oligosaccharide spray. Our investigation revealed that these genes responded to this stress in a variety of ways, demonstrating that they may adapt quickly to environmental changes in cucumber plants. This study provides a base for further understanding in reference to CDE gene family and reveals that genes play significant functions in cucumber stress responses.

A novel CSN5/CRT O-GlcNAc/ER stress regulatory axis in platinum resistance of epithelial ovarian cancer.

Background: High levels of COP9 signalosome subunit 5 (CSN5) in epithelial ovarian cancer (EOC) are associated with poor prognosis and are implicated in mediating platinum resistance in EOC cells. The underlying mechanisms, however, remained undefined. This study aimed to elucidate the molecular process and identify potential therapeutic targets. Methods: RNA-sequencing was used to investigate differentially expressed genes between platinum-resistant EOC cells with CSN5 knockdown and controls. O-GlcNAc proteomics were employed to identify critical modulators downstream of CSN5. The omics findings were confirmed through qRT-PCR and immunoblotting. In vitro and in vivo experiments assessed the sensitivity of resistant EOCs to platinum. Results: We demonstrated an involvement of aberrant O-GlcNAc and endoplasmic reticulum (ER) stress disequilibrium in CSN5-mediated platinum resistance of EOC. Genetic or pharmacologic inhibition of CSN5 led to tumor regression and surmounted the intrinsic EOC resistance to platinum both in vitro and in vivo. Integration of RNA-sequencing and O-GlcNAc proteomics pinpointed calreticulin (CRT) as a potential target of aberrant O-GlcNAc modification. CSN5 upregulated O-GlcNAc-CRT at T346 to inhibit ER stress-induced cell death. Blocking T346 O-GlcNAc-CRT through CSN5 deficiency or T346A mutation resulted in Ca2+ disturbances, followed by ER stress overactivation, mitochondrial dysfunction, and ultimately cell apoptosis. Conclusion: This study reveals that CSN5-mediated aberrant O-GlcNAc-CRT acts as a crucial ER stress checkpoint, governing cell fate response to stress, and emphasizes an unrecognized role for the CSN5/CRT O-GlcNAc/ER stress axis in platinum resistance of EOC.

Membranal Expression of Calreticulin Induced by Unfolded Protein Response in Melanocytes: A Mechanism Underlying Oxidative Stress-Induced Autoimmunity in Vitiligo.

Calreticulin (CRT), a damage-associated molecular pattern molecule, is reported to translocate from the endoplasmic reticulum to the membrane in melanocytes under oxidative stress. To investigate the potential role of CRT in the pathogenesis of vitiligo, we analyzed the correlation between CRT and ROS in serum and lesions of vitiligo, detected CRT and protein kinase RNA-like endoplasmic reticulum kinase (PERK) expression in vitiligo lesions, and studied the production of CRT and mediators of unfolded protein response (UPR) pathway and then tested the chemotactic migration of CD8+ T cells or CD11c+ CD86+ cells. Initially, we verified the overexpression of CRT in perilesional epidermis that was positively correlated with the disease severity of vitiligo. Furthermore, the PERK branch of UPR was confirmed to be responsible for the overexpression and membranal translocation of CRT in melanocytes under oxidative stress. We also found that oxidative stress-induced membranal translocation of CRT promoted the activation and migration of CD8+ T cells in vitiligo. In addition, dendritic cells from patients with vitiligo were also prone to maturation with the coincubation of melanocytes harboring membranal CRT. CRT could be induced on the membrane of melanocytes through UPR and might play a role in oxidative stress-triggered CD8+ T-cell response in vitiligo.

Bioinspired Tumor Calcification-Guided Early Diagnosis and Eradication of Hepatocellular Carcinoma.

Tumor calcification is found to be associated with the benign prognostic, and which shows considerable promise as a somewhat predictive index of the tumor response clinically. However, calcification is still a missing area in clinical cancer treatment. A specific strategy is proposed for inducing tumor calcification through the synergy of calcium peroxide (CaO2)-based microspheres and transcatheter arterial embolization for the treatment of hepatocellular carcinoma (HCC). The persistent calcium stress in situ specifically leads to powerful tumor calcioptosis, resulting in diffuse calcification and a high-density shadow on computed tomography that enables clear localization of the in vivo tumor site and partial delineation of tumor margins in an orthotopic HCC rabbit model. This osmotic calcification can facilitate tumor clinical diagnosis, which is of great significance in differentiating tumor response during early follow-up periods. Proteome and phosphoproteome analysis identify that calreticulin (CALR) is a crucial target protein involved in tumor calcioptosis. Further fluorescence molecular imaging analysis also indicates that CALR can be used as a prodromal marker of calcification to predict tumor response at an earlier stage in different preclinical rodent models. These findings suggest that upregulated CALR in association with tumor calcification, which may be broadly useful for quick visualization of tumor response.

Plant extracts modulate cellular stress to inhibit replication of mouse Coronavirus MHV-A59.

The Covid-19 infection outbreak led to a global epidemic, and although several vaccines have been developed, the appearance of mutations has allowed the virus to evade the immune response. Added to this is the existing risk of the appearance of new emerging viruses. Therefore, it is necessary to explore novel antiviral therapies. Here, we investigate the potential in vitro of plant extracts to modulate cellular stress and inhibit murine hepatitis virus (MHV)-A59 replication. L929 cells were treated with P2Et (Caesalpinia spinosa) and Anamu SC (Petiveria alliacea) plant extracts during infection and virus production, ROS (reactive oxygen species), UPR (unfolded protein response), and autophagy were assessed. P2Et inhibited virus replication and attenuated both ROS production and UPR activation induced during infection. In contrast, the sustained presence of Anamu SC during viral adsorption and replication was required to inhibit viral infection, tending to induce pro-oxidant effects, and increasing UPR gene expression. Notably, the loss of the PERK protein resulted in a slight decrease in virus yield, suggesting a potential involvement of this UPR pathway during replication. Intriguingly, both extracts either maintained or increased the calreticulin surface exposure induced during infection. In conclusion, our findings highlight the development of antiviral natural plant extracts that differentially modulate cellular stress.

Pinacidil ameliorates cardiac microvascular ischemia-reperfusion injury by inhibiting chaperone-mediated autophagy of calreticulin.

Calcium overload is the key trigger in cardiac microvascular ischemia-reperfusion (I/R) injury, and calreticulin (CRT) is a calcium buffering protein located in the endoplasmic reticulum (ER). Additionally, the role of pinacidil, an antihypertensive drug, in protecting cardiac microcirculation against I/R injury has not been investigated. Hence, this study aimed to explore the benefits of pinacidil on cardiac microvascular I/R injury with a focus on endothelial calcium homeostasis and CRT signaling. Cardiac vascular perfusion and no-reflow area were assessed using FITC-lectin perfusion assay and Thioflavin-S staining. Endothelial calcium homeostasis, CRT-IP3Rs-MCU signaling expression, and apoptosis were assessed by real-time calcium signal reporter GCaMP8, western blotting, and fluorescence staining. Drug affinity-responsive target stability (DARTS) assay was adopted to detect proteins that directly bind to pinacidil. The present study found pinacidil treatment improved capillary density and perfusion, reduced no-reflow and infraction areas, and improved cardiac function and hemodynamics after I/R injury. These benefits were attributed to the ability of pinacidil to alleviate calcium overload and mitochondria-dependent apoptosis in cardiac microvascular endothelial cells (CMECs). Moreover, the DARTS assay showed that pinacidil directly binds to HSP90, through which it inhibits chaperone-mediated autophagy (CMA) degradation of CRT. CRT overexpression inhibited IP3Rs and MCU expression, reduced mitochondrial calcium inflow and mitochondrial injury, and suppressed endothelial apoptosis. Importantly, endothelial-specific overexpression of CRT shared similar benefits with pinacidil on cardiovascular protection against I/R injury. In conclusion, our data indicate that pinacidil attenuated microvascular I/R injury potentially through improving CRT degradation and endothelial calcium overload.