Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non‐classical proline/tyrosine‐nuclear localization signal motif (aa98‐117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.

中文翻译：

---

使用增强的膜蛋白制备和亲和蛋白质组学解决人类巨噬细胞免疫代谢调节剂 (MACIR) 的相互作用组。

巨噬细胞免疫代谢调节基因 (MACIR) 的表达与自身免疫性疾病病理学的严重程度以及通过未知机制对巨噬细胞生物学的调节有关。编码的 206 个氨基酸的蛋白质与任何表征的蛋白质序列缺乏同源性，并且根据结构预测算法是无序蛋白质。为了鉴定 MACIR 与来自所有亚细胞区室的蛋白质的相互作用，采用了膜增溶缓冲液，结合高亲和力 EF 手下拉法，提高了定量质谱分析的分辨率，显着富集了膜结合核和线粒体室与用放射免疫沉淀测定缓冲液制备的样品进行比较。共鉴定了 63 种重要的相互作用蛋白，并通过免疫沉淀验证了与核转运受体 TNPO1 和贩运蛋白 UNC119 同源物 A 和 B 的相互作用。MACIR 氨基酸序列中两个候选核定位信号基序的突变分析表明，与 TNPO1 的相互作用可能是通过非经典的脯氨酸/酪氨酸核定位信号基序 (aa98-117)。结果表明，采用在这种富含甘油和去污剂的缓冲液中高效执行的高度特异性和高亲和力的下拉方法是分析未表征蛋白质相互作用组的有力方法。MACIR 氨基酸序列中两个候选核定位信号基序的突变分析表明，与 TNPO1 的相互作用可能是通过非经典的脯氨酸/酪氨酸核定位信号基序 (aa98-117)。结果表明，采用在这种富含甘油和去污剂的缓冲液中高效执行的高度特异性和高亲和力的下拉方法是分析未表征蛋白质相互作用组的有力方法。MACIR 氨基酸序列中两个候选核定位信号基序的突变分析表明，与 TNPO1 的相互作用可能是通过非经典的脯氨酸/酪氨酸核定位信号基序 (aa98-117)。结果表明，采用在这种富含甘油和去污剂的缓冲液中高效执行的高度特异性和高亲和力的下拉方法是分析未表征蛋白质相互作用组的有力方法。