(176aa) Modulating Lipid Droplet Breakdown in Mammalian Cells through a Split Mediated Interaction

Obesity is in large part a consequence of an excessive cellular fraction of neutral lipids, which are sequestered in organelles called lipid droplets (LDs). The droplet(s) take up most of the volume of adipocytes, the main component of fat tissue. On the cellular level, cells convert fatty acids sourced from a high fat diet into neutral lipids - mostly triacylglycerol (TAG) - in the endoplasmic reticulum (ER). When local TAG levels in the ER membrane are high, an organelle called a lipid droplet forms. Droplets are easily observed in adipocytes and hepatocytes from obese mammals. In such cases, it is also common to see LDs form in other tissues such as skeletal muscle, which can impair the health and function of the tissue. They are also observed in vitro when eukaryotic cells are incubated in media containing fatty acids. Once formed, lipid droplets have a core of neutral lipids, a surrounding phospholipid monolayer, and dozens of bound proteins.

Of equal importance to droplet formation is droplet breakdown (lipolysis). This occurs when cells need to use stored neutral lipids for energy or components for phospholipids. Lipolysis occurs through multiple pathways, here we are interested in the links between lipolysis and chaperone-mediated autophagy (CMA). During CMA, the heat shock cognate protein of 70 kDa (Hsc70) binds proteins with a pentapeptide sequence/motif. CMA can only breakdown LDs in times of cellular stress. This is because PLIN2 has to be modified by AMP activated protein Kinase (AMPK). Mouse perilipin 2 (mPLIN2) has a known pentapeptide sequence. It is hypothesized that hPLIN2 also contains a pentapeptide sequence. However, the location of the pentapeptide sequence in hPLIN2 is not known. After binding, Hsc70 guides the murine PLIN2 bound LD to the lysosome surface where they form a complex with murine Lysosomal Associated Membrane Protein 2 variant A (LAMP2A). This is the rate-limiting step of CMA. To increase the rate of CMA driven LD degradation, we have developed a molecular system based on a split intein to increase the affinity of LDs to lysosomes via an engineered PLIN2- LAMP2A interactions. This system has a high affinity between intein segments (NpuC and NpuN, K_d = 1.2 nM).

We first validated that split intein could be used to target molecular bodies to the lysosomal surface by expressing both NpuC-mCherry and LAMP2A-NpuN-eGFP from plasmids in NIH/3T3 cells. The signals from the two proteins strongly overlapped, signifying mCherry was bound to LAMP2A rich lysosomes. We then moved to see if the 2 organelles, LDs and lysosomes, could be targeted together with a split intein. To do this, we expressed NpuC-mCherry-PLIN2 with LAMP2A-NpuN-GFP with oleic acid supplemented media to simulate a high fat diet and promote the formation of LDs. I saw close overlap between the two fluorescent proteins and LDs. Nearly all lipid droplets appear near with the lysosome whereas non-treated cells appear to have evenly distributed LDs.

To test the intein's ability to increase LD degradation, the intein system was expressed in LD synthesizing conditions and then the cells were exposed to a 24 hr starving condition. Imaging LDs droplets after starving showed a decrease from 30-50 LDs for control to >10 LDs for treated cells.