Sally Zhang is a junior who is majoring in Biology. She was awarded a Fall 2018 Independent Grant which she used to conduct research on ubiquination and Alzheimer's disease under Dr. Hyojung Choo.
I started doing my project at the Seyfried Lab of the Biochemistry department and Center for Neurodegenerative diseases of Emory School of Medicine in April 2018. My project aims to examine the mechanisms underlying the turnover of insoluble neurofibrillary tangles of Tau, a key type of pathological protein aggregate in Alzheimer’s disease. Alzheimer’s disease is characterized by the presence of protein aggregates and a loss of protein homeostasis. Ubiquitin (Ub) is a 76 amino acid peptide that marks proteins for degradation mainly through the ubiquitin-proteasome system (UPS) and autophagy. Modification by Lys-48-linked poly-Ub chains has been reported as means of selecting proteins for degradation by the UPS. Whereas, Lys-63-linked poly-ubiquitination is associated with the clearance of proteins by autophagy.
The project involves affinity capturing poly-ubiquitinated proteins by tandem ubiquitin binding entities, which has a nano-molar affinity for poly-ubiquitinated substrates. So far, we have used TUBE 1, which is captures all poly-Ub chains regardless of linkage type, and analyzed the affinity captured proteins by label-free mass spectrometry. After controlling for non-specific binding, we identified about 200 proteins that were at least two-fold enriched between control and AD cases. Several targets, such as Tau, p62/SQSTM1 and 14-3-3 proteins, which are known to be associated with AD have been identified as ubiquitinated more than two-fold compared to control. Compared to a previous study which I was a part of that was published in the journal, proteomics, over 50% of the above mentioned 200 or so proteins have increased site-specific levels of ubiquitination in AD brain. Compared to that same study, interestingly, we identified less ubiquitinated proteins. We hypothesize that the reason that there may be less identified may be due to the TUBEs’ preference for poly-ubiquitin chains whereas the di-Gly antibody used in that study can be used to identify all Ub molecules.
It has been a challenging but rewarding experience for me. As someone who worked for two years in a microbiology lab and published a paper in PLoS One as the primary author in the same field, I found there to be quite a few key differences that were difficult to adjust to right away. For instance, differences in what to watch out for during sample preparation. There are different things to watch out for when working with different types of samples. When working with bacterial samples it is very important to make sure everything is sterile and kept under a fume food for experiments so that the samples will not be contaminated while these samples are not very sensitive to temperature. On the other hand, when dealing with brain tissue samples it is extremely important that samples are kept on ice. It felt somewhat strange to not work under a fume hood for almost all experiments initially. As well, at first I underestimated tissue samples’ sensitivity to temperature and ended up with not very ideal results for a while. I very much enjoyed trying research in a very different field. I think that it has been quite challenging but I was able to learn many interesting things.
Visit the Undergraduate Research Programs website to learn more about applying for Independent Research Grants.
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