Regulation of SLC26 proteins by plasma membrane recycling and novel interacting proteins

肠道电中性NaCl的吸收是通过NHE3 （SLC9A3）和DRA（在腺瘤中下调，SLC26A3）介导的Na/H-和Cl/ hco3交换偶联发生的。至于其他SLC26亚型，其转运活性的调节涉及内吞作用/再循环，导致质膜中转运蛋白的数量变化。DRA直接与NHERF家族的PDZ适配器蛋白（Na/H交换调节因子1-4和分选连接蛋白27；SNX27）相互作用。研究表明，DRA的PDZ相互作用对其循环利用具有重要意义。初步研究表明，DRA在早期核内体的逆转录复合物中与SNX27相互作用。这表明其他多蛋白复合物也起着重要作用。我们的目标是更好地了解DRA的细胞内转运，其PDZ相互作用的相关作用以及含有DRA的多蛋白复合物的功能，并将这种方法应用于其他SLC26亚型。我们将利用生物化学和显微技术以及应用工程细胞外标签，以时间和区室分解的方式表征DRA的内吞循环。荧光含dra配合物将通过蓝色天然凝胶电泳（BNE）进行监测。结合BNE和天然质谱的络合物分析，以及靶向相互作用蛋白质组学将揭示涉及dra内化、内体运输和再循环的蛋白质复合物的全面信息。货物分选蛋白将通过免疫分离的含dra的内体部分的深层蛋白质组学和复杂组学来破译。候选蛋白将在HEK293细胞、分化肠Caco2细胞和肠类器官中进行功能表征。我们将进一步向5046研究组的所有成员提供我们的蛋白质组学平台，以研究SLC26复合物及其在蛋白质组重塑中的功能后果。该结果将揭示SLC26亚型的分子病理机制，并为未来的翻译研究奠定基础。

Intestinal electroneutral NaCl absorption occurs by coupled Na/H- and Cl/HCO3-exchange mediated by NHE3 (SLC9A3) and DRA (down regulated in adenoma, SLC26A3). As for other SLC26 isoforms, regulation of their transport activity involves endocytosis/recycling resulting in varying numbers of transporters in the plasma membrane. DRA directly interacts with PDZ adapter proteins of the NHERF family (Na/H exchange regulatory factors 1-4 and sorting nexin 27; SNX27). We have shown that the PDZ interaction of DRA is important for its recycling. Preliminary work indicates that DRA interacts with SNX27 in the retromer complex in early endosomes. This suggests that other multi-protein complexes play an important role as well. Our aim is to better understand the intracellular trafficking of DRA, the related role of its PDZ interaction and the function of DRA-containing multi-protein complexes, and to apply this approach to other SLC26 isoforms. We will characterize DRA’s endocytic recycling in a time- and compartment-resolved manner using biochemical and microscopic techniques and applying an engineered extracellular tag. Fluorescent DRA-containing complexes will be monitored by blue native gel electrophoresis (BNE). Complexome profiling, which combines BNE and native mass spectrometry, and targeted interaction proteomics will reveal comprehensive information on protein complexes involved in DRA-internalization, endosomal trafficking and recycling. Cargo sorting proteins will be deciphered by deep proteomics and complexomics of immunoisolated DRA-containing endosomal fractions. Candidate proteins will be functionally characterized in HEK293 cells, differentiated intestinal Caco2 cells and intestinal organoids. We will further provide our proteomics platform to all members of the Research Unit FOR 5046 to study SLC26 complexes and functional consequences in proteome remodeling. The results will reveal molecular pathomechanisms involving SLC26 isoforms and allow future translational studies.