Grant Daskivich, Brodsky Lab
Investigating potential sources of protein disaggregation in mammalian cells
About one third of all proteins are translocated into the endoplasmic reticulum (ER) for folding and processing, including te majority of secretory proteins. Regardless of their location, proteins must fold correctly in order to carry out their function. Misfolded proteins are not only compromised in function but can aggregate, leading to harmful diseases like Alzheimer’s, Parkinson’s, and Huntington’s. One way that the cell handles misfolded, aggregation-prone proteins in the ER is through endoplasmic reticulum-associated degradation, or ERAD. A molecular chaperone in yeast, Hsp104, has been shown to completely resolve otherwise insoluble aggregated ERAD substrates. Specifically, previous work in the Brodsky Lab has shown that Hsp104 is required for the degradation of an artificial ERAD substrate that aggregates upon a temperature shift in yeast. Mammalian cells do not express Hsp104, and the mechanism by which they triage similar aggregates is poorly understood. By expressing the model substrate, known as GD*, in mammalian cells, I plan to identify compensatory sources (if any) of disaggregation activity in the absence of Hsp104. Thus far I have successfully expressed GD* in HEK293H cells and shown with a detergent solubility assay that a population of protein shifts to the insoluble fraction at higher temperatures. Furthermore, I am continuing previous work in the lab that has begun to visualize GD* puncta formation at higher temperatures by indirect immunofluoresence microscopy. Potential disaggregase chaperones will also be investigated by their impact on GD* stability and solubility using siRNA knockdown. Identifying how mammalian cells handle protein aggregates is vital to understanding how to better treat protein aggregation-related diseases in the future.
Matthew Googins, VanDemark Lab
Biochemically characterizing the activity and structure of GDAP1
Charcot-Marie-Tooth (CMT) disease is a neuropathy linked to neuron damaged cause by axonal loss and axon demyelination. Mutations to mitochondrial proteins have been found in patients suffering from several subtypes of CMT. Mitochondria are involved in key metabolic pathways of the cell that generate oxidative byproducts, which can damage the cell. Many families of proteins are utilized by cells to alleviate the stress caused by these byproducts, such as Glutathione-S-transferases (GSTs). The focus on my project is on a unique putative GST called the Ganglioside-induced Differentiation-Associated Protein 1 (GDAP1). Primary sequence analysis of GDAP1 indicated that it contained functional domains also found in canonical GSTs. The analysis also indicated that it contained three regions not found in canonical GSTs: an n-terminal extension, a linker region, and a hydrophobic domain. The function of these domains is unknown. Interestingly, recent research has indicated that GDAP1 expression is upregulated by oxidative stress and that it plays a role in regulating the process of mitochondrial fission. It is from this work that I have developed my hypothesis that GDAP1 functions as a sensor of oxidative stress that works to alleviate said stress through the recruitment of mitochondrial fission proteins. To test this hypothesis, I will biochemically characterize the activity of GDAP1 and determine its unique structural characteristics. Currently, assays examining the enzymatic activity of GDAP1 with known GST substrates are ongoing, and I have crystallized GDAP1 and am working on resolving its structure with molecular replacement.
Friday, September 7th 12 PM, Langley Hall A219B