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1.
Small ; 18(13): e2105915, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35156292

RESUMEN

Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances toward clinical application. New nanotechnology-based therapeutic opportunities have emerged with apoA-I nanoparticles (recombinant/reconstituted high-density lipoproteins, rHDL) as ideal carriers to deliver molecules and the discovery that microRNAs participate in atherosclerosis establishment and progression. Here, a therapeutic strategy to improve cholesterol efflux is developed based on a two-step administration of rHDL consisting of a first dose of antagomiR-33a-loaded rHDLs to induce adenosine triphosphate-binding cassette transporters A1 overexpression, followed by a second dose of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine rHDLs, which efficiently remove cholesterol from foam cells. A triple-cell 2D-atheroma plaque model reflecting the cellular complexity of atherosclerosis is used to improve efficiency of the nanoparticles in promoting cholesterol efflux. The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach may be used in vivo to more efficiently target atherosclerotic lesions and improve prognosis of the disease.


Asunto(s)
Aterosclerosis , MicroARNs , Aterosclerosis/tratamiento farmacológico , Colesterol , Células Espumosas , Humanos , Macrófagos , MicroARNs/uso terapéutico
2.
Int J Mol Sci ; 23(6)2022 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-35328769

RESUMEN

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.


Asunto(s)
Aterosclerosis , Calcinosis , Enfermedades Cardiovasculares , Placa Aterosclerótica , Aterosclerosis/metabolismo , Calcinosis/complicaciones , Enfermedades Cardiovasculares/metabolismo , Humanos , Factores de Riesgo
3.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540814

RESUMEN

Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet can also propagate cardiovascular pathologies through proliferative and pro-inflammatory activities. Such phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. However, lncRNA characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. We aimed to improve lncRNA representation in such contexts by assembling non-reference transcripts in RNA sequencing datasets describing VSMCs stimulated in vitro with cytokines, growth factors, or mechanical stress, as well as those isolated from atherosclerotic plaques. All transcripts were then subjected to a rigorous lncRNA prediction pipeline. We substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21-32% for each dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, and transcription from enhancer sites, suggesting particular relevance to VSMC processes, and the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33-46% of these lncRNAs would remain hidden. Our comprehensive VSMC lncRNA repertoire allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states.


Asunto(s)
Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Placa Aterosclerótica/metabolismo , ARN Largo no Codificante/análisis , Aorta/citología , Vasos Coronarios/citología , Citocinas/farmacología , Conjuntos de Datos como Asunto , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , ARN Largo no Codificante/aislamiento & purificación , RNA-Seq , Estrés Mecánico , Transcripción Genética/efectos de los fármacos , Transcriptoma
4.
Int J Mol Sci ; 21(24)2020 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-33317170

RESUMEN

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.


Asunto(s)
Arterias Carótidas/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Placa Aterosclerótica/genética , Polimorfismo de Nucleótido Simple , Arterias Carótidas/patología , Humanos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología
5.
Int J Mol Sci ; 20(10)2019 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-31108847

RESUMEN

Of the three interleukin-22 binding protein (IL-22BP) isoforms produced by the human IL22RA2 gene, IL-22BPi2 and IL-22BPi3 are capable of neutralizing IL-22. The longest isoform, IL-22BPi1, does not bind IL-22, is poorly secreted, and its retention within the endoplasmic reticulum (ER) is associated with induction of an unfolded protein response (UPR). Therapeutic modulation of IL-22BPi2 and IL-22BPi3 production may be beneficial in IL-22-dependent disorders. Recently, we identified the ER chaperones GRP94 and cyclophilin B in the interactomes of both IL-22BPi1 and IL-22BPi2. In this study, we investigated whether secretion of the IL-22BP isoforms could be modulated by pharmacological targeting of GRP94 and cyclophilin B, either by means of geldanamycin, that binds to the ADP/ATP pocket shared by HSP90 paralogs, or by cyclosporin A, which causes depletion of ER cyclophilin B levels through secretion. We found that geldanamycin and its analogs did not influence secretion of IL-22BPi2 or IL-22BPi3, but significantly enhanced intracellular and secreted levels of IL-22BPi1. The secreted protein was heterogeneously glycosylated, with both high-mannose and complex-type glycoforms present. In addition, cyclosporine A augmented the secretion of IL-22BPi1 and reduced that of IL-22BPi2 and IL-22BPi3. Our data indicate that the ATPase activity of GRP94 and cyclophilin B are instrumental in ER sequestration and degradation of IL-22BPi1, and that blocking these factors mobilizes IL-22BPi1 toward the secretory route.


Asunto(s)
Benzoquinonas/farmacología , Ciclofilinas/metabolismo , Ciclosporina/farmacología , Lactamas Macrocíclicas/farmacología , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Sitios de Unión/efectos de los fármacos , Ciclofilinas/química , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Perfilación de la Expresión Génica , Glicosilación , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Monocitos/metabolismo , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteolisis , Receptores de Interleucina/química , Receptores de Interleucina/genética
6.
J Immunol ; 196(11): 4553-65, 2016 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-27183579

RESUMEN

An intronic variant in ANKRD55, rs6859219, is a genetic risk factor for multiple sclerosis, but the biological reasons underlying this association are unknown. We characterized the expression of ANKRD55 in human PBMCs and cell lines. Three ANKRD55 transcript variants (Ensembl isoforms 001, 005, and 007) could be detected in PBMCs and CD4(+) T cells but were virtually absent in CD8(+), CD14(+), CD19(+), and CD56(+) cells. Rs6859219 was significantly associated with ANKRD55 transcript levels in PBMCs and CD4(+) T cells and, thus, coincides with a cis-expression quantitative trait locus. The processed noncoding transcript 007 was the most highly expressed variant in CD4(+) T cells, followed by 001 and 005, respectively, but it was not detected in Jurkat, U937, and SH-SY5Y cell lines. Homozygotes for the risk allele produced more than four times more transcript copies than did those for the protective allele. ANKRD55 protein isoforms 005 and 001 were predominantly located in the nucleus of CD4(+) T cells and Jurkat and U937 cells. ANKRD55 was produced by primary cultures of murine hippocampal neurons and microglia, as well as by the murine microglial cell line BV2, and it was induced by inflammatory stimuli. ANKRD55 protein was increased in the murine mouse model of experimental autoimmune encephalomyelitis. Flow cytometric analysis of CNS-infiltrating mononuclear cells showed that CD4(+) T cells and monocytes expressed ANKRD55 in experimental autoimmune encephalomyelitis mice, with the higher fluorescence intensity found in CD4(+) cells. A low percentage of microglia also expressed ANKRD55. Together, these data support an important role for ANKRD55 in multiple sclerosis and neuroinflammation.


Asunto(s)
Proteínas Portadoras/genética , Esclerosis Múltiple/genética , Animales , Proteínas Portadoras/inmunología , Línea Celular , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Factores de Riesgo
7.
Hum Mol Genet ; 24(19): 5619-27, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26152201

RESUMEN

Several variants in strong linkage disequilibrium (LD) at the SP140 locus have been associated with multiple sclerosis (MS), Crohn's disease (CD) and chronic lymphocytic leukemia (CLL). To determine the causal polymorphism, we have integrated high-density data sets of expression quantitative trait loci (eQTL), using GEUVADIS RNA sequences and 1000 Genomes genotypes, with MS-risk variants of the high-density Immunochip array performed by the International Multiple Sclerosis Genetic Consortium (IMSGC). The variants most associated with MS were also correlated with a decreased expression of the full-length RNA isoform of SP140 and an increase of an isoform lacking exon 7. By exon splicing assay, we have demonstrated that the rs28445040 variant was the causal factor for skipping of exon 7. Western blots of peripheral blood mononuclear cells from MS patients showed a significant allele-dependent reduction of the SP140 protein expression. To confirm the association of this functional variant with MS and to compare it with the best-associated variant previously reported by GWAS (rs10201872), a case-control study including 4384 MS patients and 3197 controls was performed. Both variants, in strong LD (r(2) = 0.93), were found similarly associated with MS [P-values, odds ratios: 1.9E-9, OR = 1.35 (1.22-1.49) and 4.9E-10, OR = 1.37 (1.24-1.51), respectively]. In conclusion, our data uncover the causal variant for the SP140 locus and the molecular mechanism associated with MS risk. In addition, this study and others previously reported strongly suggest that this functional variant may be shared with other immune-mediated diseases as CD and CLL.


Asunto(s)
Antígenos Nucleares/sangre , Antígenos Nucleares/genética , Esclerosis Múltiple/genética , Polimorfismo de Nucleótido Simple , Factores de Transcripción/sangre , Factores de Transcripción/genética , Estudios de Casos y Controles , Exones , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Desequilibrio de Ligamiento , Esclerosis Múltiple/sangre , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ARN
8.
J Med Genet ; 52(12): 848-55, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26475045

RESUMEN

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.


Asunto(s)
Esclerosis Múltiple/genética , Estudios de Casos y Controles , Frecuencia de los Genes , Sitios Genéticos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido Simple , Factores de Riesgo
9.
Mult Scler ; 21(12): 1498-512, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25623250

RESUMEN

BACKGROUND: Single nucleotide polymorphisms (SNPs) near SOCS1 are associated with multiple sclerosis (MS), but the most important SNPs in the area and mechanisms by which they influence the disease are unknown. METHODS: A haplotype-tagging association study was performed covering 60.5kbp around SOCS1, and the index SNP was validated in a total of 2292 individuals. mRNA expression of SOCS1 and nearby genes was measured in MS patients with different disease courses and healthy controls. SOCS1 protein expression was studied by flow cytometry in a separate cohort of patients and controls. Differentiation and maturation of monocyte-derived dendritic cells (moDCs) were also studied. RESULTS: One SNP, rs423674, reached genome-wide significance. No genotype-specific mRNA expression differences were seen, but, by flow cytometry, significant interactions were observed between genotypes for rs423674 and disease activity (relapse or remission) in B cells and regulatory T cells. Furthermore, homozygotes for the risk allele (GG) showed higher levels of CD1a and CD86 than carriers of the protective allele (GT) in immature moDCs and a greater increase of HLA-DR+ cell percentage than GT heterozygotes upon maturation. CONCLUSIONS: rs423674, or its genetic proxies, may influence MS risk by modulating SOCS1 expression in a cell-specific manner and by influencing dendritic cell function.


Asunto(s)
Células Dendríticas/metabolismo , Expresión Génica , Esclerosis Múltiple Recurrente-Remitente , Proteínas Supresoras de la Señalización de Citocinas , Adulto , Antígenos CD1 , Linfocitos B/metabolismo , Antígeno B7-2 , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Esclerosis Múltiple Crónica Progresiva/genética , Esclerosis Múltiple Recurrente-Remitente/genética , Esclerosis Múltiple Recurrente-Remitente/inmunología , Polimorfismo de Nucleótido Simple , ARN Mensajero/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Proteínas Supresoras de la Señalización de Citocinas/genética , Linfocitos T/metabolismo
10.
Brain ; 136(Pt 6): 1778-82, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23739915

RESUMEN

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.


Asunto(s)
Esclerosis Múltiple/genética , Receptores CXCR5/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Factores de Transcripción SOXE/genética , Factores de Transcripción/genética , alfa-Manosidasa/genética , Estudios de Casos y Controles , Bases de Datos Genéticas , Femenino , Sitios Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Esclerosis Múltiple/diagnóstico , Polimorfismo de Nucleótido Simple/genética
11.
J Med Genet ; 50(1): 25-33, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23160276

RESUMEN

BACKGROUND AND AIM: Several studies have highlighted the association of the 12q13.3-12q14.1 region with coeliac disease, type 1 diabetes, rheumatoid arthritis and multiple sclerosis (MS); however, the causal variants underlying diseases are still unclear. The authors sought to identify the functional variant of this region associated with MS. METHODS: Tag-single nucleotide polymorphism (SNP) analysis of the associated region encoding 15 genes was performed in 2876 MS patients and 2910 healthy Caucasian controls together with expression regulation analyses. RESULTS: rs6581155, which tagged 18 variants within a region where 9 genes map, was sufficient to model the association. This SNP was in total linkage disequilibrium (LD) with other polymorphisms that associated with the expression levels of FAM119B, AVIL, TSFM, TSPAN31 and CYP27B1 genes in different expression quantitative trait loci studies. Functional annotations from Encyclopedia of DNA Elements (ENCODE) showed that six out of these rs6581155-tagged-SNPs were located in regions with regulatory potential and only one of them, rs10877013, exhibited allele-dependent (ratio A/G=9.5-fold) and orientation-dependent (forward/reverse=2.7-fold) enhancer activity as determined by luciferase reporter assays. This enhancer is located in a region where a long-range chromatin interaction among the promoters and promoter-enhancer of several genes has been described, possibly affecting their expression simultaneously. CONCLUSIONS: This study determines a functional variant which alters the enhancer activity of a regulatory element in the locus affecting the expression of several genes and explains the association of the 12q13.3-12q14.1 region with MS.


Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Predisposición Genética a la Enfermedad , Cinesinas/genética , Metiltransferasas/genética , Esclerosis Múltiple/genética , Sitios de Carácter Cuantitativo , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Elementos de Facilitación Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Esclerosis Múltiple/metabolismo , Polimorfismo de Nucleótido Simple , Transcripción Genética
13.
Mult Scler ; 18(7): 959-65, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22194214

RESUMEN

BACKGROUND AND OBJECTIVES: Ten genes previously showing different evidence of association with multiple sclerosis have been selected to validate. METHODS: Eleven polymorphisms were genotyped with the iPLEX™ Sequenom in a well-powered collection of Spanish origin including 2863 multiple sclerosis cases and 2930 controls. RESULTS: Replication extended to the following polymorphisms: PKN2 (rs305217), GTF2B (rs7538427), EPHA4 (rs1517440), YTHDF3 (rs12115114), ANKFN1 (rs17758761) and PTPRM (rs4798571), which did not reach the threshold of significance in a follow-up of the first genome-wide association study (GWAS) conducted in multiple sclerosis; TMEM39A (rs1132200), which appeared as a newly identified susceptibility gene in the same study; a gene previously reaching GWAS significance in Italy, CBLB (rs9657904); IL12B (rs6887695, rs10045431), a susceptibility gene shared by diverse autoimmune diseases and, finally, another gene showing inconclusive association with multiple sclerosis, CNR1 (rs1049353). CONCLUSIONS: Pooled analysis corroborated the effect on MS predisposition of three genes: TMEM39A [rs1132200: p(M-H)=0.001; OR(M-H) (95% CI)= 0.84 (0.75-0.93)], IL12B [rs6887695: p(M-H)=0.03; OR(M-H) (95% CI)= 1.09 (1.01-1.17)] and CBLB [rs9657904: p(M-H)=0.01; OR(M-H) (95% CI)= 0.89 (0.81-0.97)].


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Predisposición Genética a la Enfermedad/genética , Subunidad p40 de la Interleucina-12/genética , Proteínas de la Membrana/genética , Esclerosis Múltiple/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Adulto , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido Simple
14.
J Biol Chem ; 285(10): 6960-9, 2010 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-20054003

RESUMEN

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RT-PCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate inappropriate inflammation in autoimmune conditions.


Asunto(s)
Inhibidores de la Ciclooxigenasa 2 , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Proteínas de la Membrana/metabolismo , Pirazoles , Sulfonamidas , Animales , Celecoxib , Línea Celular , Cicloheximida/metabolismo , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/metabolismo , Ecdisterona/análogos & derivados , Ecdisterona/metabolismo , Chaperón BiP del Retículo Endoplásmico , Regulación de la Expresión Génica , Proteínas de Choque Térmico/genética , Humanos , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Pirazoles/química , Pirazoles/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo
15.
Front Immunol ; 12: 816930, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35111166

RESUMEN

Intronic 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.


Asunto(s)
Proteínas Portadoras/genética , Receptor gp130 de Citocinas/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Variación Genética , Esclerosis Múltiple/etiología , Esclerosis Múltiple/metabolismo , Alelos , Autoinmunidad/genética , Benzoatos/farmacología , Biomarcadores , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Humanos , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tetrahidronaftalenos/farmacología
16.
BMC Biochem ; 11: 44, 2010 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-21059249

RESUMEN

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.


Asunto(s)
Autoantígenos/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Proinsulina/química , Proinsulina/metabolismo , Secuencia de Aminoácidos , Autoantígenos/inmunología , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Evolución Molecular , Humanos , Insulina/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proinsulina/inmunología , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína
17.
Biomedicines ; 8(10)2020 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-32977626

RESUMEN

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.

18.
Cells ; 9(1)2020 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-31936765

RESUMEN

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.


Asunto(s)
Predisposición Genética a la Enfermedad , Esclerosis Múltiple/genética , Polimorfismo de Nucleótido Simple/genética , Señales de Clasificación de Proteína/genética , Receptores de Interleucina/genética , Adulto , Secuencia de Aminoácidos , Simulación por Computador , Bases de Datos Genéticas , Frecuencia de los Genes/genética , Células HEK293 , Humanos , Persona de Mediana Edad , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Interleucina/química , Factores de Riesgo
19.
Front Immunol ; 10: 2067, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31620119

RESUMEN

The ankyrin repeat domain-55 (ANKRD55) gene contains intronic single nucleotide polymorphisms (SNPs) associated with risk to contract multiple sclerosis, rheumatoid arthritis or other autoimmune disorders. Risk alleles of these SNPs are associated with higher levels of ANKRD55 in CD4+ T cells. The biological function of ANKRD55 is unknown, but given that ankyrin repeat domains constitute one of the most common protein-protein interaction platforms in nature, it is likely to function in complex with other proteins. Thus, identification of its protein interactomes may provide clues. We identified ANKRD55 interactomes via recombinant overexpression in HEK293 or HeLa cells and mass spectrometry. One hundred forty-eight specifically interacting proteins were found in total protein extracts and 22 in extracts of sucrose gradient-purified nuclei. Bioinformatic analysis suggested that the ANKRD55-protein partners from total protein extracts were related to nucleotide and ATP binding, enriched in nuclear transport terms and associated with cell cycle and RNA, lipid and amino acid metabolism. The enrichment analysis of the ANKRD55-protein partners from nuclear extracts is related to sumoylation, RNA binding, processes associated with cell cycle, RNA transport, nucleotide and ATP binding. The interaction between overexpressed ANKRD55 isoform 001 and endogenous RPS3, the cohesins SMC1A and SMC3, CLTC, PRKDC, VIM, ß-tubulin isoforms, and 14-3-3 isoforms were validated by western blot, reverse immunoprecipitaton and/or confocal microscopy. We also identified three phosphorylation sites in ANKRD55, with S436 exhibiting the highest score as likely 14-3-3 binding phosphosite. Our study suggests that ANKRD55 may exert function(s) in the formation or architecture of multiple protein complexes, and is regulated by (de)phosphorylation reactions. Based on interactome and subcellular localization analysis, ANKRD55 is likely transported into the nucleus by the classical nuclear import pathway and is involved in mitosis, probably via effects associated with mitotic spindle dynamics.


Asunto(s)
Proteínas Portadoras/inmunología , Núcleo Celular/metabolismo , Mitosis/inmunología , Mapas de Interacción de Proteínas/inmunología , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Proteínas Portadoras/genética , Núcleo Celular/genética , Células HEK293 , Células HeLa , Humanos , Mitosis/genética , Polimorfismo de Nucleótido Simple
20.
Cytokine Growth Factor Rev ; 39: 62-70, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29396056

RESUMEN

Inflammation in carotid atherosclerotic plaque is linked to plaque rupture and cerebrovascular accidents. The balance between pro- and anti-inflammatory mediators governs development of the plaque, and may mediate enhancement of lesion broadening or, on the contrary, delay progression. In addition to macrophages and endothelial cells, smooth muscle cells (SMCs), which are the dominant cell subset in advanced plaques, are crucial players in carotid atherosclerosis development given their ability to differentiate into distinct phenotypes in reponse to specific signals received from the environment of the lesion. Carotid atheroma SMCs actively contribute to the inflammation in the lesion because of their acquired capacity to produce inflammatory mediators. We review the successive stages of carotid atheroma plaque formation via fatty streak early-stage toward more advanced rupture-prone lesions and document involvement of cytokines and chemokines and their cellular sources and targets in plaque progression and rupture.


Asunto(s)
Enfermedades de las Arterias Carótidas/patología , Inflamación/complicaciones , Miocitos del Músculo Liso/citología , Placa Aterosclerótica/patología , Animales , Células Endoteliales/citología , Humanos , Mediadores de Inflamación , Macrófagos/citología , Ratones
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