Throughout the year, Department of Ophthalmology faculty members welcome medical student involvement in research projects. Medical students interested in pursuing research in the Department of Ophthalmology should review the topics below and contact the faculty member directly.
Identification of the subcellular localization of wild-type and mutant fibulin-3
Mentor: John D. Hulleman, Ph.D.
The rare retinal dystrophy, Malattia Leventinese, is caused by a mutation of an arginine residue at the 345th position in a protein called fibulin-3. Instead of incorporating an arginine residue, a change in the DNA code results in the insertion of a large, bulky tryptophan residue, thus the mutation is called Arg345Trp (R345W). This mutation site occurs in a specific region of the protein called an epidermal growth factor (EGF) domain.
The presence of this large tryptophan residue causes problems with fibulin-3 protein folding and its eventual secretion from cells. As a result, the disease-causing mutant protein is poorly secreted and has a tendency to activate the cell’s stress responsive signaling pathways.
In healthy individuals, misfolded proteins are normally recognized by the cellular quality control machinery and targeted for degradation, effectively removing them from the cell. However, an interesting aspect of this R345W mutation is that it is not effectively degraded within the cell. This observation begs the question: Where is mutant fibulin-3 located within the cell that renders it resistant to degradation? Is it localized in vesicles? Is it trapped in the Golgi apparatus? Can we identify ways to target mutant, disease-causing fibulin-3 for degradation?
The overall goal of this project is to determine the intracellular localization of wild-type (normal) and R345W (mutant) fibulin-3 in human retinal pigmented epithelial cells.
The student who undertakes this study will develop a skill set enriched in molecular biology and cell biology techniques including: aseptic cell culturing, mammalian cell transduction, immunohistochemistry, epifluorescence microscopy, and confocal microscopy.
Generation of high-throughput-capable fusion proteins for identifying new drugs to treat retinal diseases
Mentor: John D. Hulleman, Ph.D.
A number of retinal disorders are caused by genetic mutations in genes encoding for secreted proteins. Many of these mutations compromise protein folding. This results in a defect in the protein’s secretion efficiency. Identifying compounds which rescue the mutant protein’s secretion defect is of substantial interest to my lab. However, since a cell at any given time secretes tens of thousands of proteins, it is difficult to specifically monitor the secretion of the one protein.
Researchers have therefore developed reporter assays to follow a single protein by modifying it and making it unique compared to the rest of the cell’s proteome. One such reporter assay strategy is to fuse is a light-generating enzyme (luciferase) to the protein of interest. Then, using an assay to detect the amount light given off in a sample, researchers can infer how much protein of interest is present. Such an approach can be useful for identifying new potential drugs which rescue the secretion defects using unbiased screening techniques in a high-throughput manner.
The goal of this project will be to use the Gaussia luciferase or Nano luciferase as a way to follow the secretion of retinal disease-associated proteins. Following the successful establishment and validation of this assay, the student will perform small-scale high-throughput screening experiments to identify drugs which may rescue the secretion of the mutant protein.
The student who is assigned to this project will develop skills associated with molecular biology, cell biology, and high-throughput screening. Specifically, s/he will be taught molecular cloning, luciferase assay development, cell culture, high-throughput screening, secondary assay verification, and quantitative PCR.
These projects will be posted midway through the 2017-18 academic year.