If I have to tell you how did I hit upon my topic of research, I have to go back to my childhood.
I grew up in a small village called Bhawanipur in the district of Howrah, West Bengal. In my childhood, there was very little unhealthy competition amongst peers in the village. My family never imposed that sense of competition on me, nor did they ever compel me to choose a specific career path.
As a result, I enjoyed freedom – freedom to decide what I wanted to be. Freedom of speech was an added advantage. Irrelevant, you may think, but it definitely wasn’t so. My present strong sense of independence and freedom are the gifts of my childhood and liberal upbringing, which brought me closer to the scientific philosophy that I try to adhere to in my lab today and sowed the seeds of appreciating the importance of multi-disciplinary approaches in solving a problem.
A few blind men and an elephant
Among the many things that I owe to my childhood, I remember one story that I heard from my grandparents as well as from our science teacher. How do a few blind men perceive an elephant? There are many different versions of the story. Almost all versions, however, begin with a group of blind men who had never encountered an elephant before. Since they were blind, they had to figure out the shape of the elephant by using ‘touch’ alone. So, each of them touched a different part of the animal and declared their interpretation to the rest. The man who touched the trunk thought that the elephant was like a thick snake. Likewise, those who touched the ear, legs, body and the tail thought that the elephant resembled a fan, a pillar, a wall and a rope, respectively.
In some versions of the story, the men then started fighting each other and accusing each other of dishonest practices. In others, they listened to each other, respected different views, and after a collaborative effort arrived at a more accurate picture of the elephant.
I liked the story at that time without much thought; however, it kept coming back to me as I grew older, especially when I decided to be a researcher. I realized at almost every turn in the journey afterwards how significant the story was. As a researcher one has to deal with and tame the entire elephant, and examining just the tail or the leg would not bring any more insight than did the observations of the blind men at the beginning of the story.
I consider myself extremely lucky to have had extraordinary teachers at all stages of my development. Excellent teachers had taught us both at the BS and MS levels. It was while doing my MS in the Department of Biochemistry, University of Calcutta, that I first got attracted towards post-translational modifications (PTMs). Conformational changes caused by PTMs that lead to novel functionalities of a protein fascinated me. This term ‘conformational changes’ would keep reverberating in the classroom lectures, during conversations among friends, and even in my mind. At one point I felt I was entangled with the phrase.
I would receive a battery of responses when I asked around about what exactly did ‘conformational changes’ mean in chemical or physical terms, and how did such physicochemical changes translate to the biological effects downstream. Sadly, most of the responses I got made me feel that something was lacking in these descriptions. Since I couldn’t be satisfied with these explanations, I knew that I had to find about the subject in further detail, from some other source. But how?
The Calcutta University Biochemistry department had a tradition of inviting its ex-students (who were established and renowned researchers by then) to share their experience and insights with the current students. Such encounters were priceless. This was a time before YouTube, before the internet had become a part of our daily lives. It was not possible to witness the excitement of modern, cutting edge research just by the click of a mouse. Personal encounters were thus invaluable.
On one such occasion, we witnessed the beaming passion of Gourisankar Ghosh (University of California, San Diego (UCSD)) who had just published a seminal paper describing the structural basis of NF-kB inhibition by I-kBalpha in the journal Cell. This lecture was a gamechanger in my life. As I left the lecture hall, I knew that I wanted to be a structural biologist.
Understanding the interactions of p300
The course to reach there, however, was not as linear as I would have liked it to be. I joined Siddhartha Roy’s lab at Bose Institute, Kolkata, as a PhD student. I was given a couple of problems to tackle in order to come up with a thesis. One of the problems that I was dealing with aimed to understand the structural basis of stress-induced phospho-p53 recognition by its co-activator p300/CBP. NMR spectroscopy was the preferred method of choice, but we soon realized if we remained adamant about using NMR to understand this system we would either end up with the ‘rope’ or the ‘pillar’ but not the elephant.
p300 is a multidomain protein and many of these domains interact with the N-terminal Trans Activating Domain (TAD) of p53. These interactions were intricately connected to site-specific phosphorylation of p53-TAD. It would be an unfathomable task if we had to study each and every such complex by NMR. We would know a great deal about one or two such complexes, not all, and a comprehensive picture wouldn’t emerge.
This offered me the opportunity to embrace the possibility of employing a number of other experimental techniques and realize the importance of an open mind to appreciate the value of something that wasn’t my forte. We took help of chemical biology tools and fluorescence spectroscopy and studied almost all of those combinatorial complexes. We found that different domains of p300 differentially recognize differentially phosphorylated p53-TADs. It helped embolden the thought that different post-translationally modified p53 fragments may form transcription-initiating complexes of different configurations, leading to the activation of different promoters and hence different gene-expression programs.
Chasing the structure of IKK
After completing my PhD, I joined the field of NF-kB research in the Gouri Ghosh lab in UCSD. I was assigned a project aimed at unravelling the structural basis of IKK-activation, primarily by using X-ray crystallography. IKK-structure had remained an enigma since its discovery in 1998, despite its importance in metazoan biology. Even though it was recognized as one of the most attractive drug targets of that time, several big pharma companies and academic labs alike had failed to determine its structure. Ghosh lab was no exception.
Everything was new to me - the molecule, related biology, experimental techniques, the expression system, new society, new culture - everything! Gradually I realized what a beast the molecule was, and I readily sympathized with everyone else who had failed in this project in the past since its discovery in 1998. And, slowly but steadily I made a connection with the molecule and the project grew on me. I had taken on the challenge to conquer it.
It was a strange relationship with a strange molecule. Finally, in 2013, we managed to determine the first X-ray crystal structure of the human IKK2/beta. In the process, I discovered a number of seemingly disparate yet intriguing characteristics that made me look at the molecule differently. I was awestruck by kinase signalling and regulation, especially the pathways that behave differently depending upon the upstream signal, and often lead to devastating outcomes as they go awry.
During this period, I had the opportunity to work/collaborate with a number of renowned scientists: Inder Verma (Salk Institute, Cancer biologist), Alexander Hoffmann (UCSD and now at University of California, Los Angeles, Systems Biologist), and Dmitry Lyumkis (Salk Institute, Cryo-EM). Close encounters with a group of people with different expertise yet overlapping interests helped broaden my thought process tremendously. And it served my cause well - to describe the whole elephant!
And the story continues…
I remained focused on understanding the IKK-system, and that’s what we primarily do right now in the lab. I must tell you that IKK is no longer considered a lucrative drug target given that it is too important physiologically to be inhibited indiscriminately. In fact, the related biology is overwhelmingly complex. Context-independent, indiscriminate tweaking of IKK-activity often causes havoc. Still, IKK remains an interesting target as it is involved in so many pathological scenarios along with its protective and homeostatic roles. Any clue to the possibility of tweaking IKK-activity in a context-dependent manner (i.e., only in a pathological scenario when it has gone awry) will be invaluable, which makes it a highly intriguing problem.
So, a complex mixture of passion, personal entanglement, and the depth and complexity of IKK made me stay with the system. We use a number of techniques that come handy in finding a clue to solving the mystery. In addition, we are also dealing with a MAPK module that plays vital yet contradictory roles in neuronal degeneration as well as regeneration in worms.
We also deal with two other programs in the lab that are not related to kinase-biology. These non-kinase projects bring a different aura to the lab, give us opportunities to practice a different kind of science. It helps assess our abilities to move away from our comfort zone. I had not practised this kind of science ever before in my life, yet I believed that my present skill-set and thought process can be useful in dealing with those problems. More than anything else, these projects will allow us to learn something different.
So, how should a young investigator choose their research problem?
To be very honest, I don’t want to preach. Only thing I can tell you is that, don’t let others decide what’s going to be the focus of your lab. You are the master of your own research. Be resilient. Ask yourself: what is it that you are looking forward to the most when you start your own lab? What is ‘success’ according to you? Don’t look around for an answer; the answer should come from within. Be true to yourself. This answer will tell you what problem should you deal with in your lab. I wish I could be more elaborate, but this is a very individual decision. Choose to do something that would excite you every morning and help you carry on no matter what happens. Personally, I enjoy the journey more than the destination. Do you? Cheers!
This is a companion discussion topic for the original entry at https://indiabioscience.org/columns/journey-of-a-yi/on-choosing-a-research-problem