Major: Biochemistry & Molecular Biology
“Identification of the Stress-dependent Interactome of O-GlcNAcase”
Cardiovascular disease, including ischemia-reperfusion injury (from heart attacks), is a leading cause of death worldwide. Recent studies of the endogenous protective mechanisms of the heart provide insight into the pathophysiology of heart attacks and highlight possible new directions for the development of innovative cardioprotective therapeutics.
O-linked-β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification of nuclear, cytoplasmic, and mitochondrial proteins that is considered protective as it regulates the cell’s response to various types of stress including ischemia-reperfusion injury. The O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) enzymes are responsible for adding and removing the O-GlcNAc sugar modification from proteins, respectively. However, the mechanism by which cells/tissues communicate with these enzymes during times of stress remains elusive. The ultimate goal is to understand how cells regulate OGT and OGA in response to oxidative stress to promote cell survival and cardioprotection.
Our research focuses on identifying proteins that interact with and regulate OGA in response to oxidative stress. A mass spectrometry screen was previously performed using hydrogen peroxide stressed cells (to mimic ischemia-reperfusion injury), in which the BioID method was used to biotinylate and isolate interacting proteins of an OGA-biotin ligase fusion protein in vivo. This technique was used in conjunction with western-blotting to validate protein-protein interactions. At two hours of oxidative stress treatment, mTOR, a global regulator of metabolism, and GRP75, a mitochondrial protein of the HSP70 family, were biotinylated. Additionally, OGT was biotinylated under normal and stressed conditions. Validating additional protein-protein interactions, as well as understanding the physiological role of these interactions will provide insight into the mechanisms by which OGA is regulated in response to cellular stress, allowing the possibility to improve treatment for patients experiencing ischemia-reperfusion injury.
This research was supported in part by a training grant from the National Heart, Lung, and Blood Institute (NHLBI) to the Zachara Lab at the Johns Hopkins University School of Medicine (P01 HL107153).
What research experiences have you had?
I am conducting research in the lab of Dr. Natasha Zachara, in the Department of Biological Chemistry at Johns Hopkins University School of Medicine.
How did you find the research opportunity?
I got into this lab though the Johns Hopkins University Summer Internship Program, and I loved the environment so much that I decided to stay as a sustained position.
Who did you work with on this project?
I work closely with a graduate student in the lab. Her name is Jen, and I think we work pretty well together. The entire lab is very collaborative, so I always get feedback and advice from everyone. And Dr. Zachara shares her science wisdom when she gets the chance.
Do you get course credit for this work? Paid? How much time do you put into it?
I get two credits a semester for my research. If you can get paid, by all means, go for it. But do not search for a research position for the money. I think that makes the experience feel like a job. I put about 10-12 hours to lab per week, depending on my pace. Also, find a mentor that is flexible with your school schedule.
What academic background did you have before you started?
I completed my freshman year before the summer internship. This included introductory chemistry and biology. I recommend absorbing as much as you can from these classes as possible. It will give a good foundation, and help you start to digest what you are doing in lab.
How did you learn what you needed to know to be successful in this lab?
Questions, questions, questions. Ask them early and often. Do not walk around wondering where a certain enzyme is, or which antibody is which.
“WRITE EVERYTHING DOWN.”
And after a while, learn to be independent. Not secluded, but independent. Do not be that super needy child in the lab. But all in all, I think the type of support you have in the lab is absolutely vital to your success. It will help you be successful in the moment and also grow as a scientist.
What was the hardest part about your research?
Going through so many pull downs, and developing so many western blots just to not getting significant results most of the time was difficult to get used to. Being persistent is very important however, because someday, you will find an interesting protein to interact with your construct, and you will have solid data, and you will win a poster award. =)
What was the most unexpected thing?
I would say that the lab environment and my comfort with being there were both unexpected. This was my first biomedical research experience and I did not know how it was going to go with the Hopkins geniuses. But always, be confident and trust in yourself that you belong there.
How does this research experience relate to your work in other classes?
Currently, BIOL 302 (Molecular and General Genetics) is overlapping with a great deal of concepts to know with my project, which is very helpful. There are some basic laboratory techniques in CHEM 351L (Organic Chemistry Lab I) that overlap at the lab.
What did you gain from presenting your work at the 2014 Annual Biomedical Research Conference for Minority Students (ABRCMS)?
I had many friends and mentors help with my presentation, and I think towards the end, I was able to tell a very effective story. The ability to communicate science is very critical for everything from writing grants to letting your mother know what you have been up to. Presenting at ABRCMS was a huge step in understanding how to do so effectively. I also made great connections along the way with people in my career field.
What is your advice to other students about getting involved in research?
One, be open to new experiences, because you never know what may spark your interest. But more importantly, put in a great deal of energy to thoroughly understand the research you choose to do. Some of the science will inevitably go over your head, but the harder you try to understand every aspect, the sooner things will begin to click, and the more you will get out the experience.
What are your career goals?
I am pursuing a combined M.D.-Ph.D. degree to become a physician-scientist. I will pursue academic medicine and spend most of my time doing research, but also practicing medicine and teaching as well.
What else are you involved in on campus?
I am a Teaching Assistant for Discovery in CHEM 101. I am also on the executive board for the UMBC American Red Cross Club. And I try to stay active in other clubs such as MAPS (Minority Association for Pre-Medical Students) and MALES (Men Achieving Leadership, Excellence, and Success).