RESUMO
The HLA-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with AS, a chronic inflammatory spondyloarthropathy. This study examined the effect of ERAP2 in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones, and the peptides were identified using high-throughput mass spectrometry analyses. The relative abundance of a thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in the presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of the antigen-binding site of the HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and PΩ positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.
Assuntos
Aminopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Antígeno HLA-B27/metabolismo , Peptídeos/metabolismo , Espondilite Anquilosante/metabolismo , Alelos , Sequência de Aminoácidos , Aminopeptidases/genética , Campylobacter jejuni/genética , Linhagem Celular , Biologia Computacional , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Técnicas de Inativação de Genes , Antígeno HLA-B27/química , Antígeno HLA-B27/genética , Ensaios de Triagem em Larga Escala , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Proteoma/metabolismo , Alinhamento de Sequência , Espondilite Anquilosante/enzimologia , Espondilite Anquilosante/genética , Espectrometria de Massas em TandemRESUMO
The immune regulatory receptor CD69 is expressed upon activation in all types of leukocytes and is strongly regulated at the transcriptional level. We previously described that, in addition to the CD69 promoter, there are four conserved noncoding regions (CNS1-4) upstream of the CD69 promoter. Furthermore, we proposed that CNS2 is the main enhancer of CD69 transcription. In the present study, we mapped the transcription factor (TF) binding sites (TFBS) from ChIP-seq databases within CNS2. Through luciferase reporter assays, we defined a ~60 bp sequence that acts as the minimum enhancer core of mouse CNS2, which includes the Oct1 TFBS. This enhancer core establishes cooperative interactions with the 3' and 5' flanking regions, which contain RUNX1 BS. In agreement with the luciferase reporter data, the inhibition of RUNX1 and Oct1 TF expression by siRNA suggests that they synergistically enhance endogenous CD69 gene transcription. In summary, we describe an enhancer core containing RUNX1 and Oct1 BS that is important for the activity of the most potent CD69 gene transcription enhancer.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Elementos Facilitadores Genéticos , Lectinas Tipo C/genética , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Sequência Conservada , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Células Jurkat , Lectinas Tipo C/metabolismo , Camundongos , Fator 1 de Transcrição de Octâmero/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The CD69 type II C-type lectin is one of the earliest indicators of leukocyte activation acting in lymphocyte migration and cytokine secretion. CD69 expression in hematopoietic lineage undergoes rapid changes depending on the cell-lineage, the activation state or the localization of the cell where it is expressed, suggesting a complex and tightly controlled regulation. Here we provide new insights on the transcriptional regulation of CD69 gene in mammal species. Through in silico studies, we analyzed several regulatory features of the 4 upstream conserved non-coding sequences (CNS 1-4) previously described, confirming a major function of CNS2 in the transcriptional regulation of CD69. In addition, multiple transcription binding sites are identified in the CNS2 region by DNA cross-species conservation analysis. By functional approaches we defined a core region of 226bp located within CNS2 as the main enhancer element of CD69 transcription in the hematopoietic cells analyzed. By chromatin immunoprecipitation, binding of RUNX1 to the core-CNS2 was shown in a T cell line. In addition, we found an activating but not essential role of RUNX1 in CD69 gene transcription by site-directed mutagenesis and RNA silencing, probably through the interaction with this potent enhancer specifically in the hematopoietic lineage. In summary, in this study we contribute with new evidences to the landscape of the transcriptional regulation of the CD69 gene.
