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Cullin-RING ligases (CRLs) recognize and interact with substrates for ubiquitination and degradation, and can be targeted for disease treatment when the abnormal expression of substrates involves pathologic processes. Phosphorylation, either of substrates or receptors of CRLs, can alter their interaction. Phosphorylation-dependent ubiquitination and proteasome degradation influence various cellular processes and can contribute to the occurrence of various diseases, most often tumorigenesis. These processes have the potential to be used for tumor intervention through the regulation of the activities of related kinases, along with the regulation of the stability of specific oncoproteins and tumor suppressors. This review describes the mechanisms and biological functions of crosstalk between phosphorylation and ubiquitination, and most importantly its influence on tumorigenesis, to provide new directions and strategies for tumor therapy.
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BACKGROUND: Autoimmune diseases are a class of diseases that cause a strong immune response to the continuous lack of self-tissue-specific antigens in the thymus. Hypothyroidism and unstable expression of tissue-specific antigens in the thymus can limit the therapeutic effect. The thymus is mainly composed of thymic epithelial cells, but the limited number of mature thymic epithelial cells and thymic epithelial progenitor cells in the thymus has greatly limited related research. OBJECTIVE: To detect the expression of autoimmune regulator (AIRE) when mouse embryonic stem cells were transformed into thymic epithelial progenitor cells. METHODS: A two-step differentiation method was used to induce the differentiation of mouse embryonic stem cells into endoderm and then into thymic epithelial progenitor cells. The cells were collected at 0, 3, and 13 days of induced differentiation. Immunofluorescence, flow cytometry, western blot and real-time PCR were used to detect the expression of cell-associated genes and proteins. RESULTS AND CONCLUSION: Positive expression of OCT4 and SSEA1 was detected by immunofluorescence at 0 day of induction. The double positive expression of SOX17 and FoxA2 was measured by immunofluorescence at 3 days of induction. The positive expression of EpCAM, K5 and K8 were analyzed by flow cytometry at 13 days of induction. During the directional differentiation of mouse embryonic stem cells, real-time PCR indicated that the expression of PAX1, PAX9, FOXN1 and PLET1 showed an increasing trend. The expression of AIRE gene increased significantly at 0, 3, and 13 days of induction. At the same time, the expression of INS2 gene and GAD67 gene also increased. Western blot assay showed that the expression of AIRE protein gradually decreased at 0, 3, and 13 days of induction; however, insulin protein and GAD67 protein were not detected. Overall findings indicate that mouse embryonic stem cells can successfully differentiate into thymic epithelial progenitor cells with highly expressed AIRE gene, which promotes the expression of INS2 and GAD67 genes, and provides an evaluation basis for cell transplantation in the treatment of autoimmune diseases.
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Objective@#To analyze the mutation of autoimmune regulator ( AIRE ) gene in a pedigree with autoimmune polyendocrine syndrome type Ⅰ (APS-Ⅰ). @*Methods@#The peripheral blood samples from family members were collected for DNA extraction, and then the mutation sites on AIRE gene were screened by PCR and Sanger sequencing. The mutation sites were further verified in 100 healthy persons by the created restriction site PCR (CRS-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). The effects of mutation on the structure and function of AIRE protein were analyzed with SIFT, PolyPhen-2, Mutation Taster and Antheprot Editor softwares. The effects of mutation on the splicing sites of AIRE mRNA were predicted with Alternative Splice Site Predictor, FruitFly Splice Predictor and SplicePort softwares, and further verified by Sanger sequencing. @*Results@#Two novel heterozygous mutations c.47 C＞G T16R and c.1631-2 A＞T were found in the proband. The c.47 site is highly conserved and homologous in different species. The missense mutation of c.47C＞G changed the secondary structure and hydrophobicity of AIRE protein, and affected its function. The c.1631 -2 A＞T mutation changed the splicing site of AIRE mRNA, and led to the deletion of exon 13. @*Conclusion@#Two novel pathogenic mutations c.47 C＞G T16R and c.1631-2 A＞T are identified in a pedigree with autoimmune polyendocrine syndrome type Ⅰ.
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Objective:To study whether autoimmune regulator (Aire) affects macrophage polarization. Methods: The mouse mononuclear macrophage cell line RAW264. 7 cells, stable expressing GFP-Aire protein RAW264. 7 cells ( A33-3 ) and stable expressing GFP protein RAW264. 7 cells (C1-6) were stimulated with LPS,IL-4 and LPS combined with immune complex respectively to make the macrophage polarize to M1 (LPS),M2a (IL-4) and M2b (LPS with immune complex). To investigate the effects of Aire on the various types of macrophages polarization,the M1 related molecules (IL-1α,iNOS and IL-6),M2a related molecules (Arg-1) and M2b related molecule (IL-10) were detected by Real-time PCR. Results:The expression of IL-1α,IL-6 and inducible nitric oxide synthase ( iNOS) ,the products of M1 macrophages, were significantly upregulated in RAW264. 7 cells treated with LPS at 0. 5 μg/ml. The expression of Arg-1 and IL-10 mRNA in RAW264. 7 cells increased in a dose-dependent manner after stimulation with IL-4 or LPS combined with immune complexes, respectively. M1 macrophage-related marker iNOS and IL-1 increased, whereas IL-6 levels decreased in A33-3 cells compared with C1-6 cells after treatment with LPS. The expression of Arg1 and IL-10 were downregulated in A33-3 cells compared with C1-6 cells after IL-4 and LPS combined with immune complexes stimulation,respectively. Conclusion:Aire may promote M1 macrophage polarization while inhibit M2a and M2b macrophage polarization.
ABSTRACT
Autoimmune polyendocrinopathy syndrome type Ⅰ( APS-Ⅰ) is a rare autosomal recessive disorder caused by mutations in autoimmune regulator gene( AIRE) . A number of mutations have been described in the AIRE gene of patients with APS-Ⅰ, including nonsense mutation, missense mutation, silent mutation, splice site mutation, insertions and deletions mutation, et al. The mutation characteristics of the APS-Ⅰ pathogenic gene have been reviewed in the article.
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To investigate the effect of autoimmune regulator(AIRE)on phagocytic clearance of apoptotic cells,a recombinant expression vector containing full-length human AIRE cDNA was transfected into 16HBE cells.After incubation with transfected 16HBE cells,engulfment of apoptotic HL-60 cells induced by camptothecin was detected by myeloperoxidase(MPO)staining.The change in the expression of Rac 1 in transfected 16HBE cells was determined by RT-PCR and Western blotting.The results showed that the phagocytosis percentage of the experimental group,the mock trans-fection group and the negative control group(non-apoptotic cells)was(25.50±3.67)%,(6.25±1.58)%and(1.0±0.67)%,respectively.Moreover,the expressions of Rac 1 mRNA and protein were up-regulated in AIRE-transfected 16HBE cells,suggesting that A1RE may function as a regulator in the phagocytic clearance of apoptotic cells by promoting the expression of Rac 1.
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Objective:To clarify the role of Aire on the production of regulatory T cells on mice.Methods:The distribution of CD4~+CD25~+T cells and the expression of Foxp3 mRNA from Aire~ -/- thymus and spleen were separately analyzed using FACS and real-time PCR.Results:Compared with control mice, the total numbers of thymocytes/splenocytes and T cells from Aire~ -/- mice did not show any significant changes; The total CD4~+CD25~+ regulatory T-cell number and Foxp3 mRNA expression did not exhibit any statistically difference; The ratios of CD4~+CD25~+T cells out of CD4~+T cells were similar based on adult /day3/day7 mice analysis.Conclusion:Aire gene does not affect the production of CD4~+CD25~+ regulatory T cells.