Interplay between the Chaperone System and Gut Microbiota Dysbiosis in Systemic Lupus Erythematosus Pathogenesis: Is Molecular Mimicry the Missing Link between Those Two Factors?

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by self-immune tolerance breakdown and the production of autoantibodies, causing the deposition of immune complexes and triggering inflammation and immune-mediated damage. SLE pathogenesis involves genetic predisposition and a combination of environmental factors. Clinical manifestations are variable, making an early diagnosis challenging. Heat shock proteins (Hsps), belonging to the chaperone system, interact with the immune system, acting as pro-inflammatory factors, autoantigens, as well as immune tolerance promoters. Increased levels of some Hsps and the production of autoantibodies against them are correlated with SLE onset and progression. The production of these autoantibodies has been attributed to molecular mimicry, occurring upon viral and bacterial infections, since they are evolutionary highly conserved. Gut microbiota dysbiosis has been associated with the occurrence and severity of SLE. Numerous findings suggest that proteins and metabolites of commensal bacteria can mimic autoantigens, inducing autoimmunity, because of molecular mimicry. Here, we propose that shared epitopes between human Hsps and those of gut commensal bacteria cause the production of anti-Hsp autoantibodies that cross-react with human molecules, contributing to SLE pathogenesis. Thus, the involvement of the chaperone system, gut microbiota dysbiosis, and molecular mimicry in SLE ought to be coordinately studied.

Expression of scFv-anti-CHIKV-E2 in Escherichia coli with chaperones Co-expression, and its functional assay by electrochemical immunosensor.

Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express anti-CHIKV E2 scFv which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of anti-CHIKV-E2 scFv was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 µg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2,24388 ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.

Chaperone-mediated MHC-I peptide exchange in antigen presentation.

This work focuses on molecules that are encoded by the major histocompatibility complex (MHC) and that bind self-, foreign- or tumor-derived peptides and display these at the cell surface for recognition by receptors on T lymphocytes (T cell receptors, TCR) and natural killer (NK) cells. The past few decades have accumulated a vast knowledge base of the structures of MHC molecules and the complexes of MHC/TCR with specificity for many different peptides. In recent years, the structures of MHC-I molecules complexed with chaperones that assist in peptide loading have been revealed by X-ray crystallography and cryogenic electron microscopy. These structures have been further studied using mutagenesis, molecular dynamics and NMR approaches. This review summarizes the current structures and dynamic principles that govern peptide exchange as these relate to the process of antigen presentation.

HtpG-A Major Virulence Factor and a Promising Vaccine Antigen against Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading global cause of death f rom an infectious bacterial agent. Therefore, limiting its epidemic spread is a pressing global health priority. The chaperone-like protein HtpG of M. tuberculosis (Mtb) is a large dimeric and multi-domain protein with a key role in Mtb pathogenesis and promising antigenic properties. This dual role, likely associated with the ability of Heat Shock proteins to act both intra- and extra-cellularly, makes HtpG highly exploitable both for drug and vaccine development. This review aims to gather the latest updates in HtpG structure and biological function, with HtpG operating in conjunction with a large number of chaperone molecules of Mtb. Altogether, these molecules help Mtb recovery after exposure to host-like stress by assisting the whole path of protein folding rescue, from the solubilisation of aggregated proteins to their refolding. Also, we highlight the role of structural biology in the development of safer and more effective subunit antigens. The larger availability of structural information on Mtb antigens and a better understanding of the host immune response to TB infection will aid the acceleration of TB vaccine development.

The aryl hydrocarbon receptor-interacting protein in cancer and immunity: Beyond a chaperone protein for the dioxin receptor.

The aryl hydrocarbon receptor (AhR)-interacting protein (AIP) is a ubiquitously expressed, immunophilin-like protein best known for its role as a co-chaperone in the AhR-AIP-Hsp90 cytoplasmic complex. In addition to regulating AhR and the xenobiotic response, AIP has been linked to various aspects of cancer and immunity that will be the focus of this review article. Loss-of-function AIP mutations are associated with pituitary adenomas, suggesting that AIP acts as a tumor suppressor in the pituitary gland. However, the tumor suppressor mechanisms of AIP remain unclear, and AIP can exert oncogenic functions in other tissues. While global deletion of AIP in mice yields embryonically lethal cardiac malformations, heterozygote, and tissue-specific conditional AIP knockout mice have revealed various physiological roles of AIP. Emerging studies have established the regulatory roles of AIP in both innate and adaptive immunity. AIP interacts with and inhibits the nuclear translocation of the transcription factor IRF7 to inhibit type I interferon production. AIP also interacts with the CARMA1-BCL10-MALT1 complex in T cells to enhance IKK/NF-κB signaling and T cell activation. Taken together, AIP has diverse functions that vary considerably depending on the client protein, the tissue, and the species.

The Prognostic and Immunotherapeutic Significance of AHSA1 in Pan-Cancer, and Its Relationship With the Proliferation and Metastasis of Hepatocellular Carcinoma.

The AHSA1 is a main activator of ATPase of Hsp90. Hsp90 is involved in various metabolic and developmental processes of tumor cells. Although, the role of AHSA1 in tumor cells is still unrecognized. In the current research, the RNA-seq of 33 tumors were downloaded using The Cancer Genome Atlas (TCGA) database for the analysis of AHSA1 expression in tumors. The Kaplan-Meier method was used for the evaluation of the prognostic significance of AHSA1 in patients with pan-cancer. Additionally, the correlation between AHSA1 and immune cell infiltration, immune checkpoint, pyroptosis-related molecules, epithelial cell transformation-related molecules, and autophagy-related molecules were analyzed by co-expression. Furthermore, we examined the effect of AHSA1 knockdown on cell function in Huh7 and HCCLM3 cells of hepatocellular carcinoma (HCC) cell lines. According to the finding of this study, up-regulation of AHSA1 expression was observed in numerous tumor tissues, and its over-expression in liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), and esophageal carcinoma (ESCA) could affect the overall survival and disease-specific survival of the patients. Meanwhile, as per the correlation analysis the expression of AHSA1 was greatly correlated with the expression of various immune cell infiltrates, immune checkpoint inhibitors, tumor mutation load, and microsatellite instability. Moreover, this study focused on analyzing the association of AHSA1 expression with multiple pathological stages in HCC, and confirmed that AHSA1 was an independent prognostic factor of HCC by univariate and multivariate COX regression in TCGA and The International Cancer Genome Consortium (ICGC) cohorts. At the same time, cellular experiments proved that the AHSA1 knockdown could decrease the proliferation activity, cell migration and invasion ability of HCC cells. Therefore, the results of this study indicated that AHSA1 can be used as a potential prognostic biomarker of tumors and it may have a significant role in the proliferation as well as migration of HCC cells.

Partnering for the major histocompatibility complex class II and antigenic determinant requires flexibility and chaperons.

Cytotoxic, or helper T cells recognize antigen via T cell receptors (TCRs) that can see their target antigen as short sequences of peptides bound to the groove of proteins of major histocompatibility complex (MHC) class I, and class II respectively. For MHC class II epitope selection from exogenous pathogens or self-antigens, participation of several accessory proteins, molecular chaperons, processing enzymes within multiple vesicular compartments is necessary. A major contributing factor is the MHC class II structure itself that uniquely offers a dynamic and flexible groove essential for epitope selection. In this review, I have taken a historical perspective focusing on the flexibility of the MHC II molecules as the driving force in determinant selection and interactions with the accessory molecules in antigen processing, HLA-DM and HLA-DO.

Are IgG antibodies to heat shock proteins HSP27 and HSP60 useful markers in endometrial cancer and cervical cancer?

OBJECTIVES: Heat shock proteins are overexpressed in many human malignancies. The role of heat shock proteins as a therapeutic target in cancer as well as their association with drug resistance were widely documented. The aim of this study was to evaluate the concentration of IgG class HSP27 and HSP60 antibodies in serum of patients with endometrial and cervical cancer, as well as to analyse the variability of concentrations of the examined antibodies depending on the cancer stage. MATERIAL AND METHODS: The study included 59 women with adenocarcinoma of the endometrium and 36 women with cervical cancer, the control group consisted of 54 healthy women. The concentrations of IgG class antibodies against the tested heat shock proteins were determined by an immunoenzymatic assay (ELISA) using commercial assays. RESULTS: In both endometrial and cervical cancer, the serum concentration of IgG anti-HSP27 antibody was significantly higher than in the healthy control group. The concentration of IgG anti-HSP60 antibody in endometrial cancer, cervical cancer and healthy control was similar. The median IgG anti-HSP27 antibody serum concentration of endometrial cancer patients was not correlated with FIGO-stage. In cervical cancer inverse correlation between concentration of this antibody and FIGO stage was observed. The median IgG anti-HSP60 antibody concentration in serum of endometrial cancer patients was lower in FIGO stage I and II compared to FIGO stage IV and in FIGO stage IA compared to FIGO stage IB. Concentrations of examined antibodies correlated positively with each other, both in the group of women with cancer and in the group of healthy women. The strongest correlations were found in the group of patients with endometrial cancer. CONCLUSIONS: Concentration of anti-HSP27 antibody could help in detection of cervical and endometrial cancer. We need to look for the cut-off point in large cohort studies. Anti-HSP27 and anti-HSP60 antibodies should be further evaluated for their potential usage as biomarkers in cervical and endometrial cancer as they shown some correlation with stage of disease.

Conformational sensing of major histocompatibility complex (MHC) class I molecules by immune receptors and intracellular assembly factors.

Major histocompatibility complex class I (MHC-I) molecules play a critical role in both innate and adaptive immune responses. The heterodimeric complex of a polymorphic MHC-I heavy chain and a conserved light chain binds to a diverse set of peptides which are presented at the cell surface. Peptide-free (empty) versions of MHC-I molecules are typically retained intracellularly due to their low stability and bound by endoplasmic reticulum chaperones and assembly factors. However, emerging evidence suggests that at least some MHC-I allotypes are relatively stable and detectable at the cell-surface as peptide-deficient conformers, under some conditions. Such MHC-I conformers interact with multiple immune receptors to mediate various immunological functions. Furthermore, conformational sensing of MHC-I molecules by intracellular assembly factors and endoplasmic reticulum chaperones influences the peptide repertoire, with profound consequences for immunity. In this review, we discuss recent advances relating to MHC-I conformational variations and their pathophysiological implications.

HSP25 Vaccination Attenuates Atherogenesis via Upregulation of LDLR Expression, Lowering of PCSK9 Levels and Curbing of Inflammation.

[Figure: see text].

Regulation of cardiomyocyte DNA damage and cell death by the type 2A protein phosphatase regulatory protein alpha4.

The type 2A protein phosphatase regulatory protein alpha4 (α4) constitutes an anti-apoptotic protein in non-cardiac tissue, however it's anti-apoptotic properties in the heart are poorly defined. To this end, we knocked down α4 protein expression (α4 KD) using siRNA in cultured H9c2 cardiomyocytes and confirmed the lack of DNA damage/cell death by TUNEL staining and MTT assay. However, α4 KD did increase the phosphorylation of p53 and ATM/ATR substrates, decreased the expression of poly ADP-ribose polymerase and associated fragments. Expression of anti-apoptotic proteins Bcl-2 and Bcl-xL was reduced, whereas expression of pro-apoptotic BAX protein did not change. Alpha4 KD reduced basal H2AX Ser139 phosphorylation, whereas adenoviral-mediated re-expression of α4 protein following α4 KD, restored basal H2AX phosphorylation at Ser139. The sensitivity of H9c2 cardiomyocytes to doxorubicin-induced DNA damage and cytotoxicity was augmented by α4 KD. Adenoviral-mediated overexpression of α4 protein in ARVM increased PP2AC expression and augmented H2AX Ser139 phosphorylation in response to doxorubicin. Furthermore, pressure overload-induced heart failure was associated with reduced α4 protein expression, increased ATM/ATR protein kinase activity, increased H2AX expression and Ser139 phosphorylation. Hence, this study describes the significance of altered α4 protein expression in the regulation of DNA damage, cardiomyocyte cell death and heart failure.

MHC I assembly and peptide editing - chaperones, clients, and molecular plasticity in immunity.

Peptides presented on MHC I molecules allow the immune system to detect diseased cells. The displayed peptides typically stem from proteasomal degradation of cytoplasmic proteins and are translocated into the ER lumen where they are trimmed and loaded onto MHC I. Peptide translocation is carried out by the transporter associated with antigen processing, which forms the central building block of a dynamic assembly called the peptide-loading complex (PLC). By coordinating peptide transfer with MHC I loading and peptide optimization, the PLC is a linchpin in the adaptive immune system. Peptide loading and optimization is catalyzed by the PLC component tapasin and the PLC-independent TAPBPR, two MHC I-dedicated enzymes chaperoning empty or suboptimally loaded MHC I and selecting stable peptide-MHC I complexes in a process called peptide editing or proofreading. Recent structural and functional studies of peptide editing have dramatically improved our understanding of this pivotal event in antigen processing/presentation. This review is dedicated to Vincenzo Cerundolo (1959-2020) for his pioneering work in the field of antigen processing/presentation.

A mutant α1antitrypsin in complex with heat shock proteins as the primary antigen in type 1 diabetes in silico investigation.

Based on previous results demonstrating that complexes of a mutant α1-antitrypsin with the heat shock proteins (HSP)70 and glucose-regulated protein94 (Grp94) circulate in the blood of patients with type 1 diabetes, we raised the hypothesis that these complexes could represent the primary antigen capable of triggering the autoimmune reactions leading to overt diabetes. As a first approach to this issue, we searched whether A1AT and HSPs had a sequence similarity to major islet antigen proteins so as to identify among the similar sequences those with potential relevance for the pathogenesis of diabetes. A thorough in silico analysis was performed to establish the score of similarity of the human proteins: A1AT, pro-insulin (INS), GAD65, IAPP, IA-2, ICA69, Grp94, HSP70 and HSP60. The sequences of A1AT and HSPs with the highest score of similarity to the islet peptides reported in the literature as the main autoantigens in human diabetes were recorded. At variance with other HSPs, also including HSP90 and Grp78, Grp94 contained the highest number and the longest sequences with structural similarity to A1AT and to well-known immunogenic peptides/epitopes of INS, GAD65, and IA-2. The similarity of A1AT with Grp94 and that of Grp94 with INS also suggested a functional relationship among the proteins. Specific sequences were identified in A1AT, Grp94 and HSP70, with the highest score of cross-similarity to a pattern of eight different islet protein epitopes. The similarity also involved recently discovered autoantigens in type 1 diabetes such as a hybrid peptides of insulin and the defective ribosomal insulin gene product. The significant similarity displayed by specific sequences of Grp94 and A1AT to the islet peptides considered main antigens in human diabetes, is a strong indication for testing these sequences as new peptides of immunogenic relevance in diabetes.

High-resolution Crystal Structure of Human pERp1, A Saposin-like Protein Involved in IgA, IgM and Integrin Maturation in the Endoplasmic Reticulum.

The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.

Oral co-administration of bivalent protein r-BL with U-Omp19 elicits mucosal immune responses and reduces S. Typhimurium shedding in BALB/c mice.

The increase in international food trade and travel has dramatically increased the global incidences of Salmonellosis. In the light of widespread resistance to frontline antibiotics, oral vaccines remain the most reliable alternative. In this study, the fusion protein, r-BL was rationally constructed by splicing the Salmonella Typhimurium sseB and ompL genes through G4S linker by over-lap extension PCR. The oral coadministration of r-BL with B. abortus U-Omp19 protein with known protease inhibitor activity resulted in significant increase of mucosal IgA titres to antilog 4.5051 (p < 0.0001) and 4.806 (p < 0.0001) in the fecal samples and intestinal washes respectively. Antibody isotyping of the intestinal washes demonstrated increase in mucosal IgM, IgG1 and IgG2a isotypes also and demonstrated a significant reduction in fecal shedding of S. Typhimurium in challenge study. The r-BL + U-Omp19 treated mice demonstrated a complete termination of Salmonella fecal shedding by the 12th day of challenge as compared to other study groups. In summary, the bivalent protein r-BL when administered with the mucosal adjuvant U-Omp19 was successful in triggering mucosal arm of the immune system which forms the first line of defence in combating the infections caused by the enteric pathogen like Salmonella.

Identification of BAG5 from orange-spotted grouper (Epinephelus coioides) involved in viral infection.

Bcl-2-associated athanogene 5 (BAG5) is a kind of molecular chaperone that can bind to the Bcl-2 and modulate cell survival. However, little is known about the functions of fish BAG5. In this study, we characterized a BAG5 homolog from orange-spotted grouper (Epinephelus coioides) gene (Ec-BAG5) and investigated its roles during viral infection. The Ec-BAG5 protein encoded 468 amino acids with four BAG domains, which shared high identities with reported BAG5. The highest transcriptional level of Ec-BAG5 was found in the peripheral blood lymphocyte (PBL). And the Ec-BAG5 expression were significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) or Lipopolysaccharide (LPS) stimulation in vitro. Furthermore, Ec-BAG5 overexpression could inhibited viral replication and the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)). Also, overexpression of Ec-BAG5 significantly increased the expression of interferon pathway-related factors including interferon regulatory factor 3 (IRF3), interferon-stimulated gene 15 (ISG15), interferon-induced protein 35 (IFP35), myxovirus resistance gene 1 (Mx1) and inflammatory-related factors including tumor necrosis factor receptor-associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1ß), as well as the activities of NF-κB, ISRE and IFN-1. These data indicate that Ec-BAG5 can affect viral infection through regulating the expression of IFN- and inflammation-related factors, which provide useful information to better understand the immune response against viral infection.

Serum levels of anti-heat shock protein 27 antibodies in patients with chronic liver disease.

Heat shock protein 27 (HSP27), an intracellular molecular chaperone, is involved in the pathogenesis of cancer by promoting both tumor cell proliferation and resistance to therapy. HSP27 is also present in the circulation and circulating HSP27 (sHSP27) can elicit an autoimmune response with production of antibodies. Levels of sHSP27 are enhanced in patients with hepatocellular carcinoma (HCC); it is, however, unknown whether changes in HSP27 antibody levels occur in patients with HCC and can be exploited as a circulating biomarker of HCC. Our aim was to assess the potential association between newly diagnosed HCC and serum anti-HSP27 antibody levels. In this cross-sectional study, anti-HSP27 antibody levels were measured in serum samples from 71 HCC patients, 80 subjects with chronic liver disease, and 38 control subjects by immunoenzymatic assay. Anti-HSP27 antibody levels did not differ significantly among groups. However, in patients with chronic active hepatitis/cirrhosis, anti-HSP27 levels were significantly higher in subjects with a positive history of alcoholism (p = 0.03). Our data do not support the hypothesis that anti-HSP27 antibody levels may help identify patients with HCC among subjects with chronic liver disease. However, our finding that alcohol-related liver disease is associated with higher anti-HSP27 levels is novel and deserves further investigations.

MZB1 enables efficient interferon α secretion in stimulated plasmacytoid dendritic cells.

MZB1 is an endoplasmic reticulum (ER)-resident protein that plays an important role in the humoral immune response by enhancing the interaction of the µ immunoglobulin (Ig) heavy chain with the chaperone GRP94 and by augmenting the secretion of IgM. Here, we show that MZB1 is also expressed in plasmacytoid dendritic cells (pDCs). Mzb1-/- pDCs have a defect in the secretion of interferon (IFN) α upon Toll-like receptor (TLR) 9 stimulation and a reduced ability to enhance B cell differentiation towards plasma cells. Mzb1-/- pDCs do not properly expand the ER upon TLR9 stimulation, which may be accounted for by an impaired activation of ATF6, a regulator of the unfolded protein response (UPR). Pharmacological inhibition of ATF6 cleavage in stimulated wild type pDCs mimics the diminished IFNα secretion by Mzb1-/- pDCs. Thus, MZB1 enables pDCs to secrete high amounts of IFNα by mitigating ER stress via the ATF6-mediated UPR.

Unlike estrogens that increase PCSK9 levels post-menopause HSP27 vaccination lowers cholesterol levels and atherogenesis due to divergent effects on PCSK9 and LDLR.

AIMS: The estrogen-inducible protein Heat Shock Protein 27 (HSP27) as well as anti-HSP27 antibodies are elevated in healthy subjects compared to cardiovascular disease patients. Vaccination of ApoE-/- mice with recombinant HSP25 (rHSP25, the murine ortholog), boosts anti- HSP25 levels and attenuates atherogenesis. As estrogens promote HSP27 synthesis, cellular release and blood levels, we hypothesize that menopause will result in loss of HSP27 atheroprotection. Hence, the rationale for this study is to compare the efficacy of rHSP25 vaccination vs. estradiol (E2) therapy for the prevention of post-menopausal atherogenesis. METHODS AND RESULTS: ApoE-/- mice subjected to ovariectomy (OVX) showed a 65 % increase atherosclerotic burden compared to sham mice after 5 weeks of a high fat diet. Relative to vaccination with rC1, a truncated HSP27 control peptide, atherogenesis was reduced by 5-weekly rHSP25 vaccinations (-43 %), a subcutaneous E2 slow release pellet (-52 %) or a combination thereof (-82 %). Plasma cholesterol levels declined in parallel with the reductions in atherogenesis, but relative to rC1/OVX mice plasma PCSK9 levels were 52 % higher in E2/OVX and 41 % lower in rHSP25/OVX mice (p < 0.0001 for both). Hepatic LDLR mRNA levels did not change with E2 treatment but increased markedly with rHSP25 vaccination. Conversely, hepatic PCSK9 mRNA increased 148 % with E2 treatment vs. rC1/OVX but did not change with rHSP25 vaccination. In human HepG2 hepatocytes E2 increased PCSK9 promoter activity 303 %, while the combination of [rHSP27 + PAb] decreased PCSK9 promoter activity by 64 %. CONCLUSION: The reduction in post-OVX atherogenesis and cholesterol levels with rHSP25 vaccination is associated with increased LDLR but not PCSK9 expression. Surprisingly, E2 therapy attenuates atherogenesis and cholesterol levels post-OVX without altering LDLR but increases PCSK9 expression and promoter activity. This is the first documentation of increased PCSK9 expression with E2 therapy and raises questions about balancing physiological estrogenic / PCSK9 homeostasis and targeting PCSK9 in women - are there effects beyond cholesterol?

Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.