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Project 1. Study the mechanism of intercellular communication

 

 

Chemical messengers (like - hormones, neurotransmitters, neuropeptides, cytokines etc.) are entrapped inside the membrane enclosed vesicles within the cell. These vesicles fuse with the plasma membrane in spatiotemporally coordinated manner, to release their content outside the cell, thus establishing the intercellular communication. This process is called exocytosis. Much of the functional repertoire of exocytosis depends on the fusion pore, the first aqueous connection that forms between the lumen of secretory vesicles and the cell exterior (for further reading: Lindau M. & Almers W., Curr Opin Cell Biol. 1995,7(4):509-17; Jackson M.B. & Colleagues, J. Gen. Physiol. 2017,149(3):301-322). These are nm-scale transient structures, lasting only milliseconds before they either close, or dilate such that the vesicle membrane collapses into the plasmalemma. The soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) are proteins that catalyze the formation of fusion pore and serve as the minimal machinery required for vesicle fusion (for further reading: Sudhof T. & Rothman J., Science 2009, 323(5913): 474-477). A number of groundbreaking studies have improved our present understanding of the SNARE mediated vesicle fusion. Still we have very limited knowledge about the structure-function relationship of the fusion pores and how accessory proteins directly or indirectly modulate the pore properties. This information is crucial to understand, how, chemical messengers are secreted constitutively and regulated manner. In depth understanding of which will, in turn, help to therapeutically modulate the cellular secretion under various pathological conditions.

 

In our lab we investigate:

(1) Whether fusion pore acts as a pathway for passive diffusion of chemical messengers or it is an active valve that regulates the release of vesicular content through it.

(2) How the structure of SNAREs are related to their function? What role the accessory factors play in the SNARE complex assembly /disassembly?

(3) What is the molecular basis of abnormal vesicular secretion?

 

           Our lab utilizes a range of steady-state and time resolved ensemble measurements, to address the questions, which are otherwise impossible to understand within the cell. Additionally, we use lipid scaffold protein (e.g. apolipoprotein variants, saposins etc.)  based planar bilayer electrophysiology technique, to study the open and close dynamics of a single recombinant fusion pore, in micro-second time resolution. Finally, we use in vivo approaches, to further justify our in vitro observation.  

Project 2. Protein quality control mechanism inside the cell

 

 

Improper folding of proteins has severe pathological consequences, e.g. amyloidosis, such as Alzheimer and Creutzfeldt-Jakob disease; lung diseases, such as cystic fibrosis or hereditary emphysema; diabetes, in which misfolded proteins disrupt carbohydrate metabolism, or even accumulate in the ER; liver diseases, in which proteins needed in signaling or enzyme regulation are retained in the ER etc.

     The most direct way for a cell to avoid the accumulation of damaged proteins is to avoid creating flawed proteins (for further reading: Weissman JS and colleagues, Cell. 2014,157(1):52-64). It does so by recognizing and controlling the errors as the polypeptides are being synthesized on the ribosome. Despite of the presence of such quality control mechanisms, we still don’t know why they are apparently ineffective in case of diseases.

 

A key step for the therapeutic intervention of these diseases would be to improve our present understanding of the protein quality control mechanism. Inside the cell, this quality control operates mainly at two stages in the life cycle of individual proteins – first, during the biogenesis of proteins on the ribosomes and second, when proteins reside inside the cellular organelles (like, Endoplasmic Reticulum (ER), Golgi etc.) or in cytosol. Either one of these quality control measures, or all of them, could be affected under disease conditions where non-functional protein structures are formed. In this project, our goal is to study the quality control mechanism, during the biogenesis of proteins. It has been proposed, that the most direct way for a cell to avoid accumulation of cytotoxic protein structures, is to merely avoid their formation.

We aim to investigate:

(1) How functional protein structures are protected during their biogenesis?

(2) How protein biosynthetic machinery is functionally linked to the chaperone network of cell?

(3) What role ribosome associated protein quality control machinery plays to produce cytotoxic protein structures?

   
         
We use a range of biochemical and biophysical measurements, to address the questions, which are difficult to study in cell-based experiments. Techniques include, but not limited to, the steady state measurements as well as the fast kinetic measurements using stopped flow, quench flow etc. Finally we validate our in vitro observation, in vivo.