Transgelin Differentially Modulates the Uptake of LDL in Hepatocytes and Smooth Muscle Cells: A New Gene Involved in LDL Clearance and Atherosclerosis. - Summary Patients with genetic hyperlipidemias are at the highest risk for atherosclerotic cardiovascular disease due to a lifetime exposure to high plasma lipids. Although several key genes in lipoprotein metabolism have already been identified, the causality of hyperlipidemia in many patients is unknown. My long-term goal is to contribute to the diagnosis of genetic hyperlipidemia by characterizing new genes involved in the metabolism of low-density lipoprotein (LDL). The overall objectives in this application are to (1) elucidate how transgelin (TAGLN), an actin binding protein, interacts with LDL endocytic machinery and its impact on LDL clearance from plasma, (2) explore TAGLN's role in atherosclerosis by modulating LDL uptake in vascular smooth muscle cells (VSMC), and (3) investigate TAGLN's interactions with cofilin 1 (CFL1) and hormonally upregulated neu- associated kinase (HUNK) and their joint effect 0n cellular LDL uptake. The central hypothesis is that TAGLN plays a critical role in lipoprotein metabolism by facilitating LDL endocytosis. Furthermore, TAGLN also appears to take part in atherogenesis by modulating LDL uptake in VSMC. The rationale for this project is that the characterization of TAGLN as a novel factor involved in LDL endocytosis will contribute to a better understanding of LDL endocytosis and, ultimately, the diagnosis of genetic hyperlipidemias. Uncovering novel aspects of TAGLN in atherogenesis will also open the door to future potential therapeutics. The central hypothesis will be tested by pursuing three specific aims: 1) identify TAGLN's interactions with LDL/LDL receptor (LDLR) endocytic machinery in vitro and determine TAGLN's role on LDL catabolism in vivo, 2) elucidate TAGLN's role in LDL uptake in VSMC, and 3) investigate the protein-protein interactions between TAGLN, CFL1, and HUNK, and their relevance for LDL uptake. Under the first aim, high-resolution microscopy will be used to assess TAGLN's associations with LDL endocytic machinery and animal studies will reveal TAGLN's role in plasma LDL clearance. For the second aim, in vitro assays, using specific probes and inhibitors, and in vivo models will be used to test TAGLN's role in LDL uptake by fluid-phase pinocytosis in VSMC. In the third aim, co-IP will assess the interactions between TAGLN, CFL1, and HUNK. RNAi and microscopy assays will evaluate their joint role in LDL uptake. The proposed research is innovative because it proposes a new physiologic role for TAGLN in modulating LDL endocytosis in hepatocytes and VSMC. Also, it explores for the first time how the interactions between TAGLN, CFL1, and HUNK affect LDL uptake. The proposed research is significant because it is expected to positively impact the diagnosis of genetic hyperlipidemias by providing a new target for genetic testing. Also, it will uncover TAGLN and associated proteins as potential therapeutic targets to avert atherosclerosis. Most importantly, the execution of this training award will allow the candidate to acquire the needed training in cellular biology, high-resolution microscopy, and murine models, to establish an independent investigator career in the field of lipoprotein metabolism and atherosclerosis.
