Metabolic interplay

fimmu-08-00248-g001
Renner K et al. Front Immunol. (2017)

I recently came across this figure that shows the key metabolic processes that dictates an immune cell behavior and function. Biochemists and pharmacologists sometimes focus on one or two key pathways in a disease model and forget that proteins don’t function in isolation. Protein networks are complex pathways with many overlays. A drug designed to inhibit or activate a specific protein can also affect other proteins in the connected pathways. This figure is focussed on an immune cell (natural killer cell) and its interaction with a tumor cell. The interplay between the different metabolic pathways applies to all kinds of cells in the body.

This figure is also quite interesting to me because I have been studying the arginase-1 (Arg1) pathway in microglial cells and this gives me a brief overview of where my study lies in the spectrum of key cellular metabolic pathways. Arg1 is an enzyme that metabolizes L-arginine to L-ornithine and urea in the urea cycle. With the help of ornithine decarboxylase (ODC), L-ornithine further makes polyamines that are important (? – it depends) for cell growth and survival (? – it depends). I think it is quite interesting to see how Arg1 and ODC would dictate the phenotypes of the microglial cells in the brain. Microglia are the brain’s resident immune cells – they chew up all the toxic stuff and get rid of them (this is known as phagocytosis). We have always studied these cells based on their two active states (M1 or M2). There has been evidence in the recent years to show that these cells in fact may exhibit multiple activated states (not just M1 and M2). Just like many immune cells in the body that exhibit a heterogenous phenotype, microglia in the brain may be no different. I’m curious if Arg1 and ODC may be involved in regulating a similar mechanism in microglial cells during neurodegeneration..

Source: Renner K., Singer K., et al. Metabolic Hallmarks of Tumor and Immune Cells in the Tumor Microenvironment. Front Immunol. 2017; 8: 248.

 

Thoughts on lab rotations

The thing with first-year rotations in a Ph.D. program is that anxiety starts kicking in somewhere along the way when you consciously identify the lab that you want to join and want to get started right away. Having realized that this is going to be a long journey and rushing into things may not help, I am now gaining patience and perspective, and hope to make the most of the remaining time of my first year.

Rotations are a great way to learn about a lab and get involved in the nitty-gritty of research. I was warned at the beginning by a few seniors that I would either love a lab or reject it within the first few weeks of the rotation. Mind you – this has nothing to do with the science pursued in the lab (one wouldn’t decide to rotate in a lab if they didn’t find the research interesting in the first place). This is more about getting comfortable with the way a lab functions and deciding if the environment is a good fit for you. An eight-week lab rotation is really like an eight-week long interview with a potential PI and the lab! It is essential to identify the kind of relationship you foresee having with your advisor for the next couple of years (and beyond). This is perhaps one of the most important aspects of a rotation for me, next to the research work. A good mentor-mentee relationship can go a long way and can be extremely beneficial to one’s academic/professional career. I prefer having an open channel of communication with my mentor and learn as much as possible from him/her.

Not all graduate programs require laboratory rotations. Many departments or programs accept or reject students simply based on their application and/or an interview. In the UK for example, students are recruited to work on specific projects and grants as a part of their Ph.D. for the time period of around 3 years. This may not benefit the candidates who wish to propose their own ideas and develop their own thesis based on their individual research interests. In the US, for most graduate programs in the life sciences (mainly biology and chemistry), the average time for graduation is around 5-6 years. I believe that the freedom and independence of this system trump the short graduation time of the other systems. Although I am certain that both sides have their set of merits and demerits, at the end of the day, the journey is unique to each one of us and what we make of the experience matters the most.

Orientation week: What do I want out of grad school?

The first week of grad school was intense and exhaustive with all kinds of information being tossed at us from all directions. We started off with a formal introduction to the school, the department, and all the resources available at our disposal like the libraries, mentors, health benefits, and so on. Besides all this, a main objective of the orientation week was to decide the first two labs that we are interested to rotate in. The process involved meeting with several professors, going on lab tours, meeting other grad students and evaluating if a lab was a good fit for us or not. Although I knew the direction of research I wanted to pursue, discovering so many options and learning about cool new research areas left me wondering if I really knew what I wanted to be doing for the next five years! Right now, I feel like a first grader starting school for the first time and constantly being exposed to many things I never knew existed.

Grad school 101 - What I don't know
Grad school 101 – What I don’t know

Being in a big umbrella program, there are ten different training groups to choose from. First year graduate students pick four labs within any of the groups to rotate in during their first year. This is very different from a departmental graduate program where a student can only rotate in labs within that respective department. After all the decisions and evaluations, I have chosen my first two labs for the semester and I am looking forward to be officially starting next week.

This process has made me question some decisions that I’ve taken in the last couple of years. “What do I want out of grad school?” seems to be the most significant one. Before beginning my journey, I knew that I wanted to train to be a good scientist, learn how to think, develop skills unique to my field, master techniques that will make me employable, learn how to learn, and be an overall well rounded researcher. Now I’m not sure if there is a definite answer to the question. It is something that I’d have to figure out on-the-go.

G protein-coupled inwardly-rectifying potassium (GIRK) channels

My current Master’s thesis research is focussed on understanding the structural and functional properties of G protein-coupled inwardly-rectifying potassium (GIRK or Kir 3.x) channels. This class of potassium ion channels are responsible for regulating the heart rate and modulating the neuronal excitability of certain neurons.

GIRK channels are activated by G-protein coupled receptors (GPCRs) including the muscarinic, dopamine, serotonin, GABA, opiod, and acetylcholine receptors, which are involved in many signal transduction pathways in the cell. The activation of a GPCR by its ligand (neurotransmitter or hormone) results in the release of Gα and Gβγ, two intracellular effector molecules. The activated Gβγ binds to the GIRK channel and opens it up to potassium ions resulting in the hyperpolarization of the cell (increased negative charge due to efflux of K+ ions).

Activation of GIRK channel
GIRK channels are activated upon GPCR stimulation by direct interaction with Gβγ.

Molecular cloning techniques have led to the discovery of four channel subunits – GIRK1 (Kir 3.1), GIRK2 (Kir 3.2), GIRK3 (Kir 3.3) and GIRK 4 (Kir 3.4). GIRK1 through 3 can be found in the central nervous system and GIRK4 is primarily found in the heart. Four of these subunits assemble either as homomers or heteromers (in 1:1 subunit ratio) to form a tetrameric functional channel.

Structurally, the channel is divided into cytoplasmic and transmembrane domains. The amino- (NH2) terminus and the carboxyl- (COOH) terminus are present in the cytoplasm and contribute to the formation of the intracellular/cytoplasmic domain. Each subunit is composed of two transmembrane domains separated by a P-loop containing the “ion-selectivity filter”. This type of channel assembly results in significant interactions between the cytoplasmic domains of the four subunits.

Crystal Structure of GIRK channel. Left - Front view of four GIRK2 subunits (color coded) channel assembly. Right - Top view of the cytoplasmic domain forming the selectivity filter.
Crystal Structure of GIRK channel. Left – Front view of the channel comprised of 4 subunits (color coded). Right – Top view of the cytoplasmic domain forming the selectivity filter. Protein Data Base ID – 3SYQ and 2QKS (Whorton, M. R., Mckinnon, R., 2011)

I have been specifically involved in understanding how certain amino acid residues residing in the hydrophobic pockets of the subunits influence channel activation and function. I use multiple experimental methods to investigate the interaction between the N- and C- termini of the GIRK1 and GIRK4 channel subunits to analyse protein expression and domain association. Previous research (Sarac et al, 2005) has revealed that certain mutations in the amino acid residues of these two subunits alters channel function.

Understanding how the interaction between the different GIRK channel subunits influences the channel formation and activity is critical for the elucidation of certain cellular mechanisms involved in cell physiology as well as in various channelopathies. New studies also suggest that ethanol binds to a hydrophobic pocket in the channel and activates it. (Bodhinathan, K., Slesinger, P. A., 2013 and Aryal, P., et al, 2009) Ethanol activation of the channel can be utilised for developing selective therapeutics to treat alcohol-related disorders like alcohol addiction and abuse.

Selected Resources:

Stress – it’s all in your head!

Stress is an interesting body response that is stimulated by our brain due to incoming auditory, visual and/or somatosensory signals. It is how we feel and how our body reacts when we encounter an imbalance in the normal rhythm of life. Watching a horror movie, coming face to face with a deadly creature or simply feeling overwhelmed due to daily tasks may all evoke stress. How does our brain respond to a stimulus that elicits fear and anxiety?

The key areas of the brain that are involved in stress are the thalamus, hippocampus, amygdala, and the prefrontal cortex. The thalamus located in the forebrain processes the incoming visual and auditory signals and relays them to the prefrontal cortex and the amygdala. The prefrontal cortex is the hub for executive function. With respect to stress, it gives meaning to the relayed signals and makes us conscious of what we see and hear. This part of the brain is also critical for ‘turning off’ the stress response once the condition is passed.

The Neurobiology of Stress - Brain regions involved in stress response
The Neurobiology of Stress – Brain regions involved in stress response

The amygdala is the emotional center of the brain and is responsible for triggering the stressful response. It is a part of the limbic system and is located deep within the temporal lobes of the brain. The amygdala also drives the body’s sympathetic nervous system to initiate anxiety that is associated with stress. This includes increasing the heart rate, blood pressure, hyperventilation of the lungs and increasing perspiration.

Finally, the hippocampus located in the medial temporal lobe stores the memory linked to a particular stress response and allows the brain to access these memories when the same visual and auditory triggers of stress are encountered later on.

It is also essential to mention the role of the hypothalamus and the linked pituitary gland that pumps out high levels of cortisol – “the stress hormone”. Recent studies suggest that cortisol can damage and kill brain cells, especially that in the hippocampus. (The hormonal response of stress is in fact a huge area of study with lots of factors involved.)

A critical question in this area of study that interests me is, “How much stress is bad for us? Can a little stress actually be helpful?” It turns out that acute stress (short-lived, unlike chronic stress) may actually be good for us. New research suggests that it conditions the brain for improved performance by inducing an increased level of alertness, behavioural and cognitive performance. This may explain why we get most of work done when we’re under pressure!

Completed my first MOOC – Understanding the Brain: The Neurobiology of Everyday Life

I am excited to have successfully completed my first ever massive online open course, “Understanding the Brain: The Neurobiology of Everyday Life” with distinction. Taught by Professor Peggy Mason at The University of Chicago, this 10 week course was one of the most interesting and engaging classes that I’ve been a part of. The best aspect of the course was how involved everyone was, including Dr. Mason and her student assistants. The lectures were well organized with weekly quizzes and corresponding lab videos (which included sheep brain dissection among many other things) and discussions related to current news revolving around neurobiology.

Coursera neurobio 2014
Statement of Accomplishment

It is motivating to be a part of a community that takes time out to learn something new. E-learning is an awesome platform to explore diverse subjects along with people from all over the world. It is also a great opportunity to learn from some of the best minds in the respective fields. I am already looking forward to my next course!