S3-12 (11mer repeats): Impact on Triacylglycerol Storage in Lipid Droplets

Michelle Mo, Nathan E. Wolins, James R. Skinner, Christin M. Lepus, Benjamin K. Quaynor, and Perry E. Bickel, Department of Medicine, Division of Endocrinology, Metabolism, and Lipid Research, and the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO

The structure and function of lipid droplet proteins and the method by which triacylglycerols (TAGs) are packaged in adipocytes is still not fully understood. S3-12, a member of the PAT (Perilipin, Adipophilin, TIP47) family of lipid droplet proteins, has only recently emerged as a potentially important protein in the processes of lipid trafficking and coating. First discovered during a test for adipocyte-specific proteins, S3-12 was found to have an amino acid sequence very similar to that of the PAT protein family ⁽¹⁾. These similarities between the sequences have led to further research that has ended with the prospective conclusion that S3-12 is involved in adipocyte lipid storage ⁽¹⁾. Such an understanding is potentially significant, considering that failure to effectively partition lipids into storage is implicated in cardiomyopathy and Type II Diabetes ⁽²⁾.

The S3-12 protein has been linked with particular lipid droplets that only emerge during TAG storage driven by the addition of the long chain fatty acid, oleate, to adipocytes. These particular lipid droplets are smaller than the average droplet and are more peripherally located in the adipocyte. Further experimentation has also suggested that the S3-12 coated lipid droplets are imbued with newly synthesized TAG, thus causing a large accumulation of TAG in the presence of S3-12 ⁽³⁾.

In order to truly understand the function and purpose of S3-12, one must first comprehend the molecular structure of the protein. Approximately two-thirds of the S3-12 amino acid sequence is tandem repeats of an 11mer motif ⁽¹⁾; about 105 x 11mer repeats are in one S3-12 protein. These repeats resemble the repeats of the a-Synuclein lipid binding protein, a synaptic vesicle-associated protein that amasses into amyloid fibrils of Lewy body deposits found in the neural regions of Parkinson’s disease victims ⁽⁴⁾. a-Synuclein consists of a hydrophilic side and a hydrophobic side that is fashioned of 11mer repeats like that of S3-12. Because of the similar structure between S3-12 and a-Synuclein and the fact that they both coat lipids, it is probable that S3-12 also consists of hydrophobic and hydrophilic sides.

Keeping with the fact that S3-12 and a-Synuclein are structurally similar, I hypothesized that S3-12 is involved with the transport of TAG to the lipid droplet by using its hydrophobic side to bind directly onto the lipid droplet and its hydrophilic side to recruit proteins for lipid droplet upkeep and for transportation of lipid droplets to the perinuclear area.

To test my hypothesis, I created a construct using two vectors: pEGFP-C1 and pCR 2.1 TOPO. I excised the region encoding the 18 x 11mer repeats from S3-12, and ligated this fragment after DNA encoding EGFP (Enhanced Green Fluorescent Protein). This resulted in a construct encoding a fusion protein with eighteen repeats tagged on the N-terminus with EGFP.

In order to assess the newly created construct’s ability to associate with lipid droplets, I transfected the following five constructs into COS-7 cells: ¹⁾ EGFP fused to S3-12 18 x 11mer repeats (EGFP S3-12 18 x 11mer repeats), ²⁾ EGFP fused to full-length S3-12 (EGFP S3-12), ³⁾ EGFP S3-12 18 x 11mer repeats & Untagged S3-12 (pCAGGS/S3-12), ⁴⁾ EGFP S3-12 with only 6 x 11mer repeats (S3-12 6 x 11mer repeats), and ⁵⁾ EGFP fused to Perilipin (EGFP-Perilipin). To observe non-EGFP tagged proteins within my transfected COS-7 cells, I labeled all my cells with red fluorescence by incubating the newly transfected COS-7 cells with oleate and Red Fatty Acid.

Under direct and indirect fluorescence microscopy, it was observed that the construct with 18 x 11mer repeats was sufficient to bind onto lipid droplets - supporting the theory of hydrophilic/hydrophobic binding for transport of TAG. Additional examination uncovered the fact that the repeats also tended to coalesce toward the stress fibers of adipocytes - suggesting and sustaining the supposition that S3-12 may be involved with the recruitment of proteins for lipid droplet transport. Despite the drastic difference in the number of repeats, my newly created construct with 18 x 11mer repeats associated with the lipid droplets just as well as construct EGFP S3-12. It was also observed that the construct, S3-12 6 x 11mer repeats, did not associate with lipid droplets as well as EGFP S3-12 18 x 11mer repeats. When comparing the constructs with entire S3-12 repeat sequences, it was discovered that the construct without the EGFP, pCAGGS/S3-12, associated much better with the lipid droplet than did the construct with the EGFP, EGFP S3-12.

Future experimentation will include further examination into the coalescence of S3-12 repeats onto stress fibers and a more meticulous assessment of the method and purpose in which S3-12 repeats coat the lipid droplet. I also plan on uncovering if and how S3-12 recruits proteins, as well as the type of proteins the repeats are engaging onto the lipid droplet. These upcoming experiments will hopefully provide our laboratory with a better understanding of S3-12, which will then lead us, in the long run, to an improved comprehension of the aforementioned diseases - cardiomyopathy and Type II Diabetes.

REFERENCES

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3. Wolins, N.E., Quaynor B.K., Skinner, J.R., Schoenfish M.J., Tzekov A., & Bickel P.E. (2005). S3-12, Adipophilin, and TIP47 Package Lipid in Adipocytes. J. Biol Chem. 280, 19146-19155.

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