How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins (ASPPs) are protein phosphatase 1 catalytic subunit (PP1c) binding partners associated with cardiocutaneous diseases. ASPPs localize PP1c to cell-cell junctions, but how ASPPs localize and whether they regulate PP1c activity in vivo is unclear. Through a C. elegans genetic screen, we find that loss of the ASPP homolog, APE-1, suppresses a pathology called ‘jowls,’ providing us with an in vivo assay for APE-1 activity. Using immunoprecipitations and mass spectrometry, we find that APE-1 binds the PP1c called GSP-2. Through structure-function analysis, we discover that APE-1’s N-terminal half directs the APE-1–GSP-2 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1’s ankyrin repeats that suppress jowls yet do not preclude GSP-2 binding, implying APE-1 does more than simply localize GSP-2. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.

Many countries, including the United States, have been afflicted with a group of lipid‐related diseases including type II diabetes, obesity and non‐alcoholic fatty liver disease. The commonality behind these disorders is lipid storage and lipid storage is mediated in part by the perilipin proteins. The perilipin family are a group of five conserved genes that differ with respect to size of the proteins they encode, tissue expression and transcriptional regulation. The only member of the perilipin family to have significant protein structural data collected is perilipin 3, and this is only on the carboxyl terminus. The C‐terminus of perilipin 5 has a high sequence homology to that of perilipin 3. Less is known about the amino terminus. The central focus of this study is to characterize and determine the structure of the N‐terminal domain of perilipin 5. The amino terminal 104 amino acids of perilipin 5 were expressed with a 6‐His tag in E. coli. Optimization of the expression of the protein fragment was ascertained using an immuno‐dot blot that contained cell samples treated with various ITPG concentrations and temperatures. Isolation and purification of the protein will then be performed using nickel affinity chromatography for further structural analysis. Secondary and tertiary structures of the N‐terminal fragment were predicted using Phyre2 and RaptorX. These computer models predicted a predominantly alpha helical structure with the presence of two 4‐helix bundles. Overall, determination of the N‐terminal structure for the perilipin 5 protein will increase our understanding of the perilipin family and their functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bioinformatics research has become increasingly important for fields in Biochemistry and Molecular Biology. An important question considers how to best engage undergraduate students with bioinformatics topics in the classroom. Students in my upper‐level bioinformatics classroom each chose an unknown gene from the RNA Sequencing data generated from my undergraduate research group. This exposed the students in the class to this commonly used Next Gen Sequencing technique that is not typically accessible to undergraduates. It then allowed the students to each have a unique gene/protein to generate novel bioinformatics data with the techniques that they were learning in the classroom. Each student created an annotated portfolio with figures for the various bioinformatics techniques that they applied to their gene of interest. By employing a workshop approach instead of a largely lecture format for the bioinformatics course, the course remained highly interactive. Students were highly invested in the individual gene that they chose, knowing that the techniques they employed would uncover novel information. All twelve students presented their data in class and three of the students presented their projects at a regional scientific conference. The benefits that the students gained from this approach is coupled to the impact on the professor/principal investigator's research program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The United States and other nations are experiencing an epidemic of lipid‐related diseases. Understanding the etiologies of these diseases is an increasingly imperative objective. Finding the function and mechanisms of the molecules involved in these lipid‐related diseases could show new and enhanced treatments for those suffering from these conditions. The perilipins, a family of five conserved lipid droplet proteins, are a chief subject of study in this field. However, an abundance of structural data is absent and would facilitate major strides in clarifying perilipin function. There is three‐dimensional structural data on the carboxyl terminus of one member in the perilipin family, perilipin 3, but not for other family members. The current study is aimed to characterize and determine the structure of the N‐terminal domain of perilipin 5. Perilipin 5 is of interest because of its implications in numerous of the metabolic conditions observed in the population. The N‐terminus is the concentration of the project because the C‐terminus of the protein has a high sequence homology with that of perilipin 3. Therefore, the C‐terminus of perilipin 5 is predicted to take on a similar structure to the C‐terminus of perilipin 3. A cDNA fragment coding for the N‐terminal region of perilipin 5 was PCR amplified from the full‐length Mus musculus perilipin 5 gene. It was then transformed into Escherichia Coli to produce the protein fragment of interest. Isolation of the protein fragment is undergoing via nickel affinity chromatography. Theoretical secondary and tertiary structures of the N‐terminal fragment of perilipin 5 have been obtained using the programs Phyre2, SCRATCH, GOR4, CFSSP, and Jpred. Computer models predict the sequence's secondary structure to contain numerous alpha helices with increasing frequency near the back half of the fragment. In conclusion, determination of the N‐terminal structure of perilipin 5 will further enable research into the functions of the perilipin family.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lipid storage‐related diseases have become prevalent throughout the nation and are responsible for afflictions such as obesity and type II diabetes. Proteins such as the perilipin family serve a key role in the modulation of lipolysis. Although this protein family plays such a major role in biochemical processes, little information has been discovered pertaining to the structure of the Perilipins. Structure is central to understanding the function of a protein, and how that protein will interact will other molecules. There has been data showing the structure of the C‐terminal domain of perilipin 3; however, structural information for the other family members is yet, unknown. The current study is aimed to determine the structure of the 11‐mer region in Perilipin 5. The 11‐mer region is implicated in the interactions between Perilipin 5 and lipids. Bioinformatics data were obtained on the 11‐mer sequence. The secondary structure of the 11‐mer region was characterized using Phyre2, and RaptorX and viewed using molecular visualization software including PyMol and NGL. These models predicted the 11‐mer region to have an amphipathic alpha helix which allowed Perilipin 5 to bind to lipid droplets. Additional studies are being performed using protease protection assays and characterization of a synthetic peptide that mimics the 11‐mer region. Characterization on the 11‐mer region of Perilipin 5 will further help elucidate how this region of perilipin 5 associates with lipid droplets.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.