Meghana Chithirala
Author: Meghana Chithirala | Majors: Chemistry/Biochemistry and Economics
My name is Meghana Chithirala. I am an honors college fellow in the Fulbright College majoring in chemistry/biochemistry and economics. During the 2021 fall grant term, I worked with my honors mentor, Dr. Yuchun Du, and graduate research assistant, Brihget Sicairos-Meza, to identify cellular proteins that determine the activation state of the proapoptotic protein Bax.
I chose my topic by exploring the University of Arkansas website to gain insight of the faculty’s research interests. Dr. Du’s research activity especially interested me, so I reached out to him via email to learn more about his research and gain a position in his lab. The work for my specific research topic had already begun, so I was caught up to speed and taught about the different procedures involved in the project by Brihget when I first joined. Mainly, I work alongside Brihget and consult her with any issues I have. She is an excellent instructor and explains everything very clearly and precisely. I have also made plenty of mistakes since joining, but she has never looked down upon me and, instead, always assured me that making mistakes is normal in a lab. Last semester was mostly spent with Brihget right next to me for all of the procedures to make sure I did them correctly, but I have been given a lot more independence this semester. I mostly do everything on my own unless I am doing something new or need help.
Dysregulation of apoptosis, a form of programmed cell death, is involved in many human diseases. Bax is an essential gateway to mitochondrion-dependent apoptosis and plays a role in several major types of diseases, but it is unknown how Bax is activated during apoptosis. The traditional methods to identify protein-protein interactions use biochemical and genetic approaches and have led to the accumulation of a significant amount of information about protein interactions. However, they are not without limitations. A proximity-dependent biotin identification (BioID) method has been developed to overcome these limitations based on fusion of a protein of interest to a promiscuous biotin protein ligase.
The objective of this project is to understand the molecular mechanism of Bax activation. My hypothesis is that one or more cellular proteins interact with the Bax protein and keep Bax in the inactive state in healthy cells, leading Bax to change its conformation and migrate to the mitochondria to induce apoptosis once the interacting protein(s) is stripped off. The two aims I have for this project are to identify cellular proteins that control the activation state of Bax in human cells using a proteomic method and verify the interaction of identified proteins with Bax using immunoprecipitation and Western blotting. The research design and methods for the first aim includes the coupling of the TurboID-based biotin affinity capture with a SILAC-based quantitative proteomic method to identify the proteins that interact with or are proximal to Bax protein. For the second aim, the verification of interactions between Bax and ~10 identified proteins, after proteomic identification, will be analyzed using immunoprecipitation and Western blotting.
The expected outcome of the proposed project is to reveal the cellular proteins that interact with or are proximal to Bax protein. This information will provide novel insights into the mechanisms by which Bax is kept in-check in the inactive state in healthy cells and activated during apoptosis under stress conditions. The knowledge will help understand how certain human diseases develop and may provide novel therapeutic strategies to treat those diseases.
The main challenge I have faced with this project has been due to COVID-19 safety regulations. I have not been able to work in the lab as often as I would like to, and that has postponed my research a little bit since I have not been able to achieve the specific aims of the project as quickly as I would have liked. To combat this challenge, I worked in the lab more often this semester and will continue to devote my time to this project next semester in hopes of fulfilling my research goals. Next semester, I also hope to attend a research conference and present my research, and I will write and defend my thesis to an honors committee.
Participating in undergraduate research has been an eye opener in multiple ways. I have had to learn how to prioritize and allocate my time as research procedures can take many hours or, even, days to complete. I have also learned the value of teamwork and the importance of hands-on experience to further one’s knowledge. The difficulty of some research procedures can’t be understood through a textbook, and being able to work with research equipment and materials to further specific research goals has been a very appreciated learning experience. For example, I have also been able to apply concepts I have learned in my undergraduate classes to a practical setting, which should enable me with skills to take to medical school. Furthermore, presenting my thesis in front of a committee or a conference provides me with the opportunity to advance my presentation and critical thinking skills. Thanks to the Honors College Research Grant, I have been able to become more involved in research than I had ever thought to further my professional and academic goals.