Research

Protein Misfolding and Stabilization

        Work in our laboratory focuses on aggregation of proteins under all kinds of scenario. The delicate balance of forces which maintains the three-dimensional architecture of proteins leaves them prone to misfolding and/or aggregation even with any mild change in environmental conditions. An increase in protein aggregation is usually associated with a higher rate of intermolecular interactions due to increase in hydrophobic interactions. Proteins can aggregate in the initial expression stage of product development. This could be a change in pH, temperature (high/low), shaking/stirring, agitation, etc. in in vitro and/or in cell situations. It could also result from overexpression of proteins in foreign hosts.

Various protein deposition diseases are known. These are commonly referred to as amyloid diseases (amyloidoses), such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, prion diseases such as bovine spongiform encephalopathy (BSE) or mad cow disease, sporadic inclusion body myositis (sIBM) and Creutzfeldt-Jacob disease (CJD) in humans. There is no sequence similarity among the implicated proteins. However, the mature aggregated protein forms show characteristic ordered structures, specifically amyloid fibrils. The accumulated proteins typically form intracellular inclusions or extracellular aggregates in specific areas of the brain and exhibit characteristic pathological hallmarks of the diseases. These accumulated proteins are misfolded and yield a ß- sheet structure that promotes fibrillation and aggregation.

Osmolytes as protein stabilizers

The advantages of using osmolytes have been visible in overexpressing heterologous proteins in the soluble form. The attempt was to inhibit formation of inclusion bodies, thus, minimizing number of unit processes and increasing yield. Under normal growth conditions, green fluorescent protein (GFP) and the N-terminal domain of HypF (HypF-N) were expressed as inclusion bodies. These could be expressed in the soluble form when grown in the presence of osmolytes such as sorbitol, arginine, etc.

The advantages of using osmolytes have been visible in overexpressing heterologous proteins in the soluble form. The attempt was to inhibit formation of inclusion bodies, thus, minimizing number of unit processes and increasing yield. Under normal growth conditions, green fluorescent protein (GFP) and the N-terminal domain of HypF (HypF-N) were expressed as inclusion bodies. These could be expressed in the soluble form when grown in the presence of osmolytes such as sorbitol, arginine, etc.

Aptamers as protein stabilizers

Our studies with adjuvanted tetanus toxoid showed that when subjected to freeze-thawing, the non-reducing disaccharide trehalose was able to retain the antigenicity of the protein although the integrity of the alumina matrix was destroyed. On the other hand, glucose was unable to stabilize the toxoid against freeze-thawing. When subjected to agitation, however, trehalose was unable to stabilize the toxoid while glucose and sorbitol showed success. Thus, depending on the stress the protein is exposed to, the requirement of the osmolyte changes.

Paradigm shift 

            Solvent-centric to protein centric approach, i.e. a common stabilizer for a protein which is active against a wider variety of stress conditions. This is where aptamers come in. Aptamers are relatively short single-stranded oligonucleotides (ssDNA/RNA) or peptides, which assume specific, stable three dimensional conformations and bind tightly to specific targets such as ions, proteins, low molecular weight metabolites, sugar moieties, lipids and even whole cells by shape complementarity. Our results show that specific and high affinity aptamers against tetanus toxoid are able to protect proteins like tetanus toxoid and insulin against a variety of stress conditions. This then makes them likely candidates to be called as “universal stabilizers” of proteins.

Aptamers for intracellular protein stabilization

Aptamers can be used intracellularly (‘intramers’) which binds specifically to proteins. They thus inhibit protein-protein interaction and hence protein aggregation inside the cell. Non-toxic and non-immunogenic nature of aptamers makes them ideal candidates for in vivo administration.

            Selected aptamers were able to inhibit aggregation of mutant huntingtin in vitro and in a yeast model of Huntington’s disease. Aptamers were also able to rescue yeast cells from endocytotic defect due to protein aggregation, with improved cell survival.  A combination of aptamers, which bind to different ‘aptatopes’ on mutant huntingtin, showed enhanced inhibitory effect against aggregation.

Understanding epidemiological observations with Parkinson’s disease

            Coffee drinking and cigarette smoking are associated with beneficial effect on development of Parkinson’s disease. Our results show that caffeine, the major component of coffee, is associated with reducing the rate of fibrillation of alpha-synuclein, thus decreasing the amount of aggregates formed. Caffeine is also successful in altering the fibrillation landscape by changing the nature of aggregates from fibrillar to amorphous. Using a yeast cell model of Parkinson’ disease, we showed that caffeine decreases intracellular toxicity and increases cell viability.

Extramural Funding