Novel temperature-responsive fusions of clathrin and caveolin to explore TGF-beta mediated fibrosis

项目总结 这项建议研究转化生长因子β受体(TGFBRs)使用新的融合蛋白用于 快速、可逆和精确地抑制胞苷介导的内吞作用(CME)和激活脂质 RAFT/小窝蛋白介导的内吞作用(CAVME)。CME和CAVME是主要的机制 受体内化及其去调控与多种疾病有关，包括肾纤维化。 还有肝脏。转化生长因子β1(TGF-β1)是肝纤维化的主要调节因子，因此， 将使用原代小鼠肝星状细胞以及转化的细胞系来评估信号转导。转化生长因子- B1的纤维化信号是通过其两个细胞表面受体TGFBR1和TGFBR2介导的。内吞作用 在TGFBR介导的信号转导中起主要调节作用，因为转化生长因子-β1/TGFBR通过CME导联内化 促纤维化的SMAD信号转导，而通过CAVME的内化则下调该信号转导。尽管 内吞作用在纤维化和正常生理中的重要性，区分货物(例如，受体、药物、 病原体)通过CME或CAVME选择性地内化仍然具有挑战性，因为科学家不能 一次操纵一条路径。这项提议旨在克服现有就业人员的局限性。 利用温度敏感型重组蛋白的胞内抑制物/激活剂 链(CLC)或小窝蛋白1(CAV1)和热响应性弹性蛋白样多肽(ELP)。我假设 ELP介导的CLC组装可抑制CME并下调转化生长因子-β1介导的纤维化信号，而 CAV1-ELP组装将激活CAVME，但也下调该信号。总而言之，这项提案 利用转化生长因子-β1/TGFBR途径和原代肝星状细胞进一步鉴定CAV1-ELP和 ClC-ELP融合蛋白。该项目的完成将为科学界提供：1)新的 快速和可逆地操纵内吞作用的工具；2)更好地理解TGFBR是如何 人口贩运导致纤维化的细胞表型；3)关于如何 内吞作用调节肝星状细胞的激活；以及4)新的实验分析，可能确定靶点 抗纤维化治疗药物。

PROJECT SUMMARY This proposal studies Transforming Growth Factor Beta Receptors (TGFBRs) using novel fusion proteins for rapid, reversible, and precise inhibition of Clathrin mediated endocytosis (CME) and activation of lipid raft/Caveolin mediated endocytosis (CAVME). CME and CAVME are the predominant mechanisms for receptor internalization, and their deregulation is implicated in myriad diseases, including fibrosis of the kidney and liver. Transforming Growth Factor Beta 1 (TGF-B1) is a master regulator of liver fibrosis; therefore, signaling will be evaluated using primary mouse hepatic stellate cells, along with transformed cell lines. TGF- B1 fibrotic signaling is mediated through its two cell surface receptors, TGFBR1 and TGFBR2. Endocytosis plays a major regulatory role in TGFBR mediated signaling, as TGF-B1/TGFBR internalization via CME leads to pro-fibrotic SMAD signaling while internalization via CAVME downregulates this signaling. Despite the importance of endocytosis in fibrosis and normal physiology, distinguishing if cargo (e.g. a receptor, a drug, a pathogen) is selectively internalized via CME or CAVME remains challenging, as scientists cannot exclusively manipulate one pathway at a time. This proposal aims to overcome the limitations of currently employed endocytic inhibitors/activators by using temperature sensitive recombinant proteins composed of Clathrin light chain (CLC) or Caveolin 1 (CAV1) and a thermally responsive Elastin-like polypeptide (ELP). I hypothesize that ELP-mediated assembly of CLC will inhibit CME and down regulate TGF-B1 mediated fibrotic signaling, while CAV1-ELP assembly will activate CAVME but also down regulate this signaling. In summary, this proposal employs the TGF-B1/TGFBR pathway and primary hepatic stellate cells to further characterize CAV1-ELP and CLC-ELP fusion proteins. Completion of this project will provide the scientific community with: 1) new tools to rapidly and reversibly manipulate endocytosis; 2) a better understanding of how TGFBR trafficking contributes to a fibrotic cellular phenotype; 3) higher-fidelity knowledge about how endocytosis regulates HSC activation; and 4) new experimental assays that may identify targets for anti-fibrotic therapeutics.