RESUMO
BACKGROUND: Cryptochromes (CRYs) are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL1 heterodimer activates transcription of the Per and Cry genes as well as clock-regulated genes. The PER2 proteins interact with CRY and CKIε, and the resulting ternary complexes translocate into the nucleus, where they negatively regulate the transcription of Per and Cry core clock genes and other clock-regulated output genes. Recent studies have indicated that the extended C-termini of the mammalian CRYs, as compared to photolyase proteins, interact with PER proteins. RESULTS: We identified a region on mCRY2 (between residues 493 and 512) responsible for direct physical interaction with mPER2 by mammalian two-hybrid and co-immunoprecipitation assays. Moreover, using oligonucleotide-based degenerate PCR, we discovered that mutation of Arg-501 and Lys-503 of mCRY2 within this C-terminal region totally abolishes interaction with PER2. CONCLUSIONS: Our results identify mCRY2 amino acid residues that interact with the mPER2 binding region and suggest the potential for rational drug design to inhibit CRYs for specific therapeutic approaches.
Assuntos
Criptocromos/genética , Criptocromos/metabolismo , Proteínas Circadianas Period/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Sequência de Aminoácidos , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Linhagem Celular , Ritmo Circadiano/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese , Proteínas Circadianas Period/genética , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Twenty-eight synovial effusions (SE) were obtained from 24 patients, paired samples of peripheral blood (PB) from 10 of these patients, and PB from 36 healthy individuals for analysis of CD146 on T-lymphocytes by flow cytometry. CD146+ or CD146- T-lymphocytes were sorted from three SE to study gene expression profiles and selected genes revalidated using QPCR assays. We found more CD3+CD146+ and CD4+CD146+ T-lymphocytes in PB from patients compared with PB of healthy individuals (4.71% +/- 2.48% vs. 2.53% +/- 1.08%, P = 0.028) and (6.29% +/- 2.74% vs. 2.41% +/- 0.96%, P = 0.0017), respectively, whereas CD8+CD146+ T-lymphocytes were not significantly different (2.55% +/- 1.65% vs. 3.18% +/- 2.59%, P = 0.5008). SE displayed CD146 staining on 16.32% +/- 6.06% of CD3+ cells. This expression was skewed toward CD4+ T-lymphocytes, with CD146 present on 24.06% +/- 8.20% of the CD4+ T-lymphocytes compared with 6.19% +/- 5.22% of the CD8+ T-lymphocytes. CD146 on CD3+, CD4+ and CD8+ T-lymphocytes in SE was significantly higher compared with PB in patients (P < 0.0001, P < 0.0001 and P = 0.0036, respectively). Gene expression profiles of sorted CD146+CD4+CD3+ vs. CD146-CD4+CD3+ T-lymphocytes (n = 2) and CD2+CD146+ vs. CD2+CD 146- (n = 1) from SE, displayed increased CD146, LAIR2, CXCL13, CD109, IL6ST, IL6R, TNFRsf18, and TNFRsf4 genes, whereas decreased CCR7, CCL5, and cytotoxicity-associated genes including granzymes b, h, and k, perforin were found with the CD146- T-lymphocytes. By QPCR higher mRNA expression of CXCL13, CD146 and CD109 was also noted in the CD146+ subset, compared with the CD146- subset, in PB of healthy individuals and in PB and SE from patients. Our study establishes increased CD146+ T-lymphocytes in diseases with joint effusions, and demonstrates pro-inflammatory gene profiles in these cells.
