Fatty acids (FAs) are essential for cellular energy storage and as the precursors for synthesis of many lipids. FAs are predominantly stored as triglycerides in organelles called lipid droplets (LDs). LDs form dynamic membrane contacts with other organelles including mitochondria (Mito), a site of FA oxidation. These contacts are proposed to facilitate the trafficking of lipids between organelles. Impaired FA trafficking and metabolism can lead to a variety of serious diseases. Lipodystrophy and cachexia are caused by deficient lipid storage, while the metabolic syndrome, type 2 diabetes, atherosclerosis and fatty liver disease involve excess lipid storage. Therefore, there is a critical need to identify the mechanism and physiological functions of LD- organelle contacts. Perilipin 5 (Plin5) is an LD protein that mediates LD-Mito contacts. The overall objectives are to (i) identify the mechanism by which Plin5 induces LD-Mito contacts (ii), to determine the role of these contacts in FA trafficking, and (iii) to evaluate the effect of Plin5 on lipid metabolism organelles. The central hypothesis is that Plin5 utilizes a unique C-terminal domain to induce LD-Mito contacts that promote ?- oxidization during nutrient starvation by directing FA trafficking from LDs to Mito. The rationale for this project is that identifying the mechanism and physiological role of LD-Mito contacts will result in strategies to treat lipid storage/trafficking diseases. The central hypothesis will be tested by pursuing three specific aims.
In Aim 1, affinity purification mass spectrometry of Plin5 constructs lacking the minimal LD-Mito contact domain will be performed to identify mitochondrial binding partners. Identified binding partners will then be assessed for a role in LD-Mito contact formation.
In Aim 2, fluorescent and radioactive pulse-chase assays will be utilized to identify the physiological effect of Plin5 on FA storage, trafficking and metabolism.
In Aim 3, multispectral imaging will be utilized to evaluate the effect of Plin5 on the morphology and contacts between 5 organelles involved in lipid metabolism. The expected outcomes are to define the mechanism by which Plin5 induces LD- Mito contacts and to demonstrate the physiological function of these contacts. These results will have important positive impact because they will establish a strong basis from which to further study dysregulated lipid trafficking and suggest a framework from which to develop novel therapies for lipid-associated diseases. To enable these research objectives, mentoring and technical/theoretical training will be provided by Dr. Cohen, Dr. Coleman, and Dr. Klett whom are experts in multispectral imaging and lipid trafficking/metabolism respectively. Career development will be facilitated by the Office of Postdoctoral Affairs through seminars and workshops. The proposed research and training will be conducted at UNC Chapel Hill, which is well known for its outstanding cell biology community, including extensive expertise in membrane trafficking and the development of novel techniques in light microscopy.
The proposed research is relevant to public health because an improved understanding of how the storage and mobilization of lipids is regulated at lipid droplet-organelle contact sites will have implications for a variety of human diseases. Once our understanding of lipid trafficking is improved, there is the potential for novel insight into the mechanisms of disease states such as lipodystrophy and cachexia resulting from impaired storage as well as the metabolic syndrome, type 2 diabetes, atherosclerosis, and fatty liver disease caused by excess lipid storage. Thus, the proposed research is relevant to the part of the NIH?s mission that pertains to reducing illness and disability.