Functional characterization of SAMD9, a protein deficient in normophosphatemic familial tumoral calcinosis.
Functional characterization of SAMD9, a protein deficient in normophosphatemic familial tumoral calcinosis.
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DOI：
10.1038/jid.2010.387
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发表时间：
2011-03
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营养不良性皮肤钙质沉着症与自身免疫性疾病和癌症等常见疾病有关。为了深入了解这一鲜为人知的过程的发病机制，我们研究了SAMD9的功能，这是一种功能未知的蛋白质，最近被证明在一种遗传性皮肤营养不良钙化形式中缺乏，称为正常磷血症性家族性肿瘤钙质沉着症(NFTC)。与NFTC严重炎症表现总是先于皮肤钙化的事实一致，我们发现SAMD9受到干扰素-γ(干扰素-γ)的严格调控。此外，在荧光素酶报告实验中还发现SAMD9启动子对干扰素-γ有很强的反应。有趣的是，我们在SAMD9转录起始点上游发现了一个关键的30bp片段，负责驱动大部分基因的表达。生物信息学分析表明，SAMD9的功能涉及与额外蛋白质的相互作用(S)。利用RAS募集系统实验和确证免疫沉淀，我们证明了SAMD9与RGL2相互作用。为了研究这种相互作用的生物学重要性，我们评估了RNA干扰介导的这对蛋白质在不同细胞系中下调的效果。我们发现，下调两个蛋白质伙伴中的任何一个都会导致Egr1表达增加，Egr1是一种转录因子，已知在组织钙化、炎症和细胞迁移的调节中发挥作用。支持这些数据的生理学相关性，Egr1水平也在来自NFTC患者的成纤维细胞系中上调。总之，我们的数据表明，SAMD9，一个干扰素-γ反应蛋白，与RGL2相互作用，减少Egr1的表达，这是一种与异位钙化和炎症的发病机制直接相关的蛋白质。
Dystrophic cutaneous calcinosis is associated with disorders as common as autoimmune diseases and cancer. To get insight into the pathogenesis of this poorly understood process, we studied the function of SAMD9, a protein of unknown function, recently shown to be deficient in a hereditary form of dystrophic calcification in the skin, known as normophosphatemic familial tumoral calcinosis (NFTC). Consistent with the fact that in NFTC severe inflammatory manifestations always precede cutaneous calcinosis, we found out that SAMD9 is tightly regulated by interferon-γ (IFN-γ). In addition, the SAMD9 promoter was also found to respond strongly to IFN-γ in a luciferase reporter assay. Of interest, we identified a critical 30-bp fragment upstream to the SAMD9 transcription initiation site responsible for driving most of the gene expression. Bioinformatic analysis suggested that SAMD9 function involves interaction with additional protein(s). Using the Ras recruitment system assay and confirmatory immunoprecipitation, we demonstrated that SAMD9 interacts with RGL2. To study the biological importance of this interaction, we assessed the effect of RNA interference-mediated downregulation of this pair of proteins in various cell lines. We found out that downregulation of any of the two protein partners caused increased expression of EGR1, a transcription factor with a known role in the regulation of tissue calcification, inflammation, and cell migration. Supporting the physiological relevance of these data, EGR1 levels were also upregulated in a fibroblast cell line derived from an NFTC patient. In conclusion, our data indicate that SAMD9, an IFN-γ-responsive protein, interacts with RGL2 to diminish the expression of EGR1, a protein of direct relevance to the pathogenesis of ectopic calcification and inflammation.
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