65 research outputs found

    High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

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    BACKGROUND: Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. RESULTS: Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further region in each proinsulin domain (A- and B-chain, C-peptide). Among the latter, peptides covering most of the B-chain region B11-23 exhibited strongest binding, which was in the range of known high-affinity DnaK ligands, dissociation equilibrium constant (K'd) of 2.2 ± 0.4 μM. The B-chain region B11-23 is located at the interface between two insulin molecules and not accessible in insulin oligomers. Indeed, native insulin oligomers showed very low DnaK affinity (K'd 67.8 ± 20.8 μM) whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM). CONCLUSIONS: Intact insulin only weakly interacts with the hsp70 chaperone DnaK whereas monomeric proinsulin and peptides from 3 distinct proinsulin regions show substantial chaperone binding. Strongest binding was seen for the B-chain peptide B 11-23. Interestingly, peptide B11-23 represents a dominant autoantigen in type 1 diabetes

    Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition

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    Vascular smooth muscle cells (VSMCs) are central players in carotid atherosclerosis plaque development. Although the precise mechanisms involved in plaque destabilization are not completely understood, it is known that VSMC proliferation and migration participate in plaque stabilization. In this study, we analyzed expression patterns of genes involved in carotid atherosclerosis development (e.g., transcription factors of regulation of SMC genes) of VSMCs located inside or outside the plaque lesion that may give clues about changes in phenotypic plasticity during atherosclerosis. VSMCs were isolated from 39 carotid plaques extracted from symptomatic and asymptomatic patients by endarterectomy. Specific biomarker expression, related with VSMC phenotype, was analyzed by qPCR, western immunoblot, and confocal microscopy. MYH11, CNN1, SRF, MKL2, and CALD1 were significantly underexpressed in VSMCs from plaques compared with VSMCs from a macroscopically intact (MIT) region, while SPP1, KLF4, MAPLC3B, CD68, and LGALS3 were found significantly upregulated in plaque VSMCs versus MIT VSMCs. The gene expression pattern of arterial VSMCs from a healthy donor treated with 7-ketocholesterol showed high similarity with the expression pattern of carotid plaque VSMCs. Our results indicate that VSMCs isolated from plaque show a typical SMC dedifferentiated phenotype with macrophage-like features compared with VSMCs isolated from a MIT region of the carotid artery. Additionally, MYH11, KLF5, and SPP1 expression patterns were found to be associated with symptomatology of human carotid atherosclerosis.This work was supported by Spanish Institute for Health Carlos III, RETICS program (Grant Number RD16/0019/0007) and by Basque Government, Education Department, Consolidated Groups program (Grant Number IT512-10)

    Pathophysiology of Atherosclerosis

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    Atherosclerosis is the main risk factor for cardiovascular disease (CVD), which is the leading cause of mortality worldwide. Atherosclerosis is initiated by endothelium activation and, followed by a cascade of events (accumulation of lipids, fibrous elements, and calcification), triggers the vessel narrowing and activation of inflammatory pathways. The resultant atheroma plaque, along with these processes, results in cardiovascular complications. This review focuses on the different stages of atherosclerosis development, ranging from endothelial dysfunction to plaque rupture. In addition, the post-transcriptional regulation and modulation of atheroma plaque by microRNAs and lncRNAs, the role of microbiota, and the importance of sex as a crucial risk factor in atherosclerosis are covered here in order to provide a global view of the disease.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019/2020; U.G-G. was supported by Margarita Salas Grant; and S.J. and A.L-S were supported by a grant PIF (2017–2018) and PIF (2019–2020) Gobierno Vasco, respectively

    Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

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    The human IL22RA2 gene co-produces three protein isoforms in dendritic cells [IL-22 binding protein isoform-1 (IL-22BPi1), IL-22BPi2, and IL-22BPi3]. Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. Using transfected human cell lines, we demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170, and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and IL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 and IL-22BPi2 are bona fide clients of the ER chaperones GRP78 and GRP94. However, only IL-22BPi1 activates an unfolded protein response (UPR) resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. We also found that CD14++/CD16+ intermediate monocytes produced a higher level of IL22RA2 mRNA than classical and non-classical monocytes, but this difference disappeared in immature dendritic cells (moDC) derived thereof. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating GRP78 levels in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response

    Cholesterol Efflux Efficiency of Reconstituted HDL Is Affected by Nanoparticle Lipid Composition

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    Cardiovascular disease (CVD), the leading cause of mortality worldwide is primarily caused by atherosclerosis, which is promoted by the accumulation of low-density lipoproteins into the intima of large arteries. Multiple nanoparticles mimicking natural HDL (rHDL) have been designed to remove cholesterol excess in CVD therapy. The goal of this investigation was to assess the cholesterol efflux efficiency of rHDLs with different lipid compositions, mimicking different maturation stages of high-density lipoproteins (HDLs) occurring in vivo. Methods: the cholesterol efflux activity of soybean PC (Soy-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC:Chol:1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPC) and DPPC:18:2 cholesteryl ester (CE):LysoPC rHDLs was determined in several cell models to investigate the contribution of lipid composition to the effectiveness of cholesterol removal. Results: DPPC rHDLs are the most efficient particles, inducing cholesterol efflux in all cellular models and in all conditions the effect was potentiated when the ABCA1 transporter was upregulated. Conclusions: DPPC rHDLs, which resemble nascent HDL, are the most effective particles in inducing cholesterol efflux due to the higher physical binding affinity of cholesterol to the saturated long-chain-length phospholipids and the favored cholesterol transfer from a highly positively curved bilayer, to an accepting planar bilayer such as DPPC rHDLs. The physicochemical characteristics of rHDLs should be taken into consideration to design more efficient nanoparticles to promote cholesterol efflux.This work was supported by the Basque Government (Grupos Consolidados IT-1264-19). U.G.-G. was supported by Fundación Biofísica Bizkaia. A.B.-V. was supported by Programa de especialización de Personal Investigador Doctor en la UPV/EHU (2019) 2019–2020. S.J.-B. and A.L.-S. were supported by a grant PIF (2017–2018) and (2019–2020), Gobierno Vasco, respectively. A.L.-S. was partially supported by Fundación Biofísica Bizkaia

    Genomic Multiple Sclerosis Risk Variants Modulate the Expression of the ANKRD55–IL6ST Gene Region in Immature Dendritic Cells

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    Autoimmune; Multiple sclerosisAutoinmune; Esclerosis múltipleAutoimmune; Esclerosi múltipleIntronic single-nucleotide polymorphisms (SNPs) in the ANKRD55 gene are associated with the risk for multiple sclerosis (MS) and rheumatoid arthritis by genome-wide association studies (GWAS). The risk alleles have been linked to higher expression levels of ANKRD55 and the neighboring IL6ST (gp130) gene in CD4+ T lymphocytes of healthy controls. The biological function of ANKRD55, its role in the immune system, and cellular sources of expression other than lymphocytes remain uncharacterized. Here, we show that monocytes gain capacity to express ANKRD55 during differentiation in immature monocyte-derived dendritic cells (moDCs) in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). ANKRD55 expression levels are further enhanced by retinoic acid agonist AM580 but downregulated following maturation with interferon (IFN)-γ and lipopolysaccharide (LPS). ANKRD55 was detected in the nucleus of moDC in nuclear speckles. We also analyzed the adjacent IL6ST, IL31RA, and SLC38A9 genes. Of note, in healthy controls, MS risk SNP genotype influenced ANKRD55 and IL6ST expression in immature moDC in opposite directions to that in CD4+ T cells. This effect was stronger for a partially correlated SNP, rs13186299, that is located, similar to the main MS risk SNPs, in an ANKRD55 intron. Upon analysis in MS patients, the main GWAS MS risk SNP rs7731626 was associated with ANKRD55 expression levels in CD4+ T cells. MoDC-specific ANKRD55 and IL6ST mRNA levels showed significant differences according to the clinical form of the disease, but, in contrast to healthy controls, were not influenced by genotype. We also measured serum sgp130 levels, which were found to be higher in homozygotes of the protective allele of rs7731626. Our study characterizes ANKRD55 expression in moDC and indicates monocyte-to-dendritic cell (Mo–DC) differentiation as a process potentially influenced by MS risk SNPs.This research was supported by grants to KV from MINECO (SAF2016-74891-R), Instituto de Salud Carlos III (FIS-PI20/00123), Gobierno Vasco RIS3 (Ref. 2019222043), and Red Española de Esclerosis Múltiple (REEM; RD16/0015/0005). NV and LMV were supported by ISCIII (FIS-PI18/00572) and REEM (RD16/0015/0001). RTN is a recipient of a fellowship from the Secretaría Nacional de Ciencia y Tecnología e Innovación (SENACYT; Convocatoria Doctorado de Investigación Ronda III, 2018; Ref. BIDP-III-2018-12) of the Gobierno Nacional, República de Panamá

    BIRC6 Is Associated with Vulnerability of Carotid Atherosclerotic Plaque

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    Carotid atherosclerotic plaque rupture can lead to cerebrovascular accident (CVA). By comparing RNA-Seq data from vascular smooth muscle cells (VSMC) extracted from carotid atheroma surgically excised from a group of asymptomatic and symptomatic subjects, we identified more than 700 genomic variants associated with symptomatology (p < 0.05). From these, twelve single nucleotide polymorphisms (SNPs) were selected for further validation. Comparing genotypes of a hospital-based cohort of asymptomatic with symptomatic patients, an exonic SNP in the BIRC6 (BRUCE/Apollon) gene, rs35286811, emerged as significantly associated with CVA symptomatology (p = 0.002; OR = 2.24). Moreover, BIRC6 mRNA levels were significantly higher in symptomatic than asymptomatic subjects upon measurement by qPCR in excised carotid atherosclerotic tissue (p < 0.0001), and significantly higher in carriers of the rs35286811 risk allele (p < 0.0001). rs35286811 is a proxy of a GWAS SNP reported to be associated with red cell distribution width (RDW); RDW was increased in symptomatic patients (p < 0.03), but was not influenced by the rs35286811 genotype in our cohort. BIRC6 is a negative regulator of both apoptosis and autophagy. This work introduces BIRC6 as a novel genetic risk factor for stroke, and identifies autophagy as a genetically regulated mechanism of carotid plaque vulnerability.This work was financially supported by grants from the Departments of Education (Ref. PIBA2018-67) and Health (Ref. RIS3-2019222038) of the Basque Government, Vitoria-Gasteiz, Spain; by the Spanish Neurovascular Network (INVICTUSplus) (Ref. RD16/0019/0007) funded by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Madrid, Spain; and by the research project grant (IKERIKTUS) funded by the RefbioII Trans-Pyrenean Cooperation Network for Biomedical Research financed by Horizon 2020. I.A. is supported by the Maratón EiTB 2017 for Funding of Research into Stroke, Bilbao, Spain (Ref. BIO18/IC/005); R.T.N. is the recipient of a fellowship from the Secretaría Nacional de Ciencia y Tecnología e Innovación (SENACYT; Convocatoria Doctorado de Investigación Ronda III, 2018; Ref. BIDP-III-2018-12) of the Gobierno Nacional, República de Panamá

    The Rare IL22RA2 Signal Peptide Coding Variant rs28385692 Decreases Secretion of IL-22BP Isoform-1, -2 and -3 and Is Associated with Risk for Multiple Sclerosis.

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    The IL22RA2 locus is associated with risk for multiple sclerosis (MS) but causative variants are yet to be determined. In a single nucleotide polymorphism (SNP) screen of this locus in a Basque population, rs28385692, a rare coding variant substituting Leu for Pro at position 16 emerged significantly (p = 0.02). This variant is located in the signal peptide (SP) shared by the three secreted protein isoforms produced by IL22RA2 (IL-22 binding protein-1(IL-22BPi1), IL-22BPi2 and IL-22BPi3). Genotyping was extended to a Europe-wide case-control dataset and yielded high significance in the full dataset (p = 3.17 × 10-4). Importantly, logistic regression analyses conditioning on the main known MS-associated SNP at this locus, rs17066096, revealed that this association was independent from the primary association signal in the full case-control dataset. In silico analysis predicted both disruption of the alpha helix of the H-region of the SP and decreased hydrophobicity of this region, ultimately affecting the SP cleavage site. We tested the effect of the p.Leu16Pro variant on the secretion of IL-22BPi1, IL-22BPi2 and IL-22BPi3 and observed that the Pro16 risk allele significantly lowers secretion levels of each of the isoforms to around 50%-60% in comparison to the Leu16 reference allele. Thus, our study suggests that genetically coded decreased levels of IL-22BP isoforms are associated with augmented risk for MS

    Genome-wide significant association with seven novel multiple sclerosis risk loci

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    Objective: A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. Methods: The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. Results: Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10−8) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10−12), CD28 (rs6435203, p=1.35×10−9), LPP (rs4686953, p=3.35×10−8), ETS1 (rs3809006, p=7.74×10−9), DLEU1 (rs806349, p=8.14×10−12), LPIN3 (rs6072343, p=7.16×10−12) and IFNGR2 (rs9808753, p=4.40×10−10). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. Conclusions: This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases

    Genome-wide significant association with seven novel multiple sclerosis risk loci

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    Objective: A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. Methods: The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. Results: Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10−8) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10−12), CD28 (rs6435203, p=1.35×10−9), LPP (rs4686953, p=3.35×10−8), ETS1 (rs3809006, p=7.74×10−9), DLEU1 (rs806349, p=8.14×10−12), LPIN3 (rs6072343, p=7.16×10−12) and IFNGR2 (rs9808753, p=4.40×10−10). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. Conclusions: This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases
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