Effect of disulfide bond scrambling on protein stability, aggregation, and cytotoxicity

Proteins are nano-machines that carry out majority of the cellular functions. Thermodynamically they are functional and stable within a very narrow range (1 kcal/mol). External perturbations in the form of pH change, thermal, or oxidative/reducing stress can destabilize the protein resulting in misfolding and aggregation. Prolonged environmental stress can affect the cells adaptive response resulting in loss of ability to refold or recycle proteins. This can lead to accumulation of misfolded or aggregated proteins within the cell. Such accumulation of aggregated proteins have been associated with neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS), Parkinson’s, Huntington’s, and Alzheimer’s disease. There is a general consensus among scientists that aggregated proteins cause disease by a ‘toxic gain of function’. However, there is a huge debate among scientists on what are the toxic forms of protein aggregates. This is largely due to lack of research that have looked at the relationship between morphology of aggregates and their toxicity. Therefore, such studies that can help clarify the relationship between morphologically different forms of aggregates and their associated toxicity are needed. In this dissertation, we study how disulfide reducing environment can impact protein stability, aggregation, and cytotoxicity. We also study effect of molecular crowding agent polyethylene glycol (PEG) (600 and 2000 g/mol) on providing protection against reducing and thermal stress. Disulfide bonds are covalent interactions that provide major stability to the protein in conjunction with several non-covalent interactions such as hydrophobic interactions, hydrogen bonding, Van der Waal and electrostatic interactions. These interactions help the proteins to fold into its native three-dimensional fold. However, the cytoplasmic environment in cells is highly reducing and can compromise the disulfide bonds leading to protein aggregation. To better understand how disulfide bond scrambling can affect protein aggregation, we used insulin as a model protein. We made seeds of insulin by incubating the protein in presence of disulfide reducing agent for varying lengths of time at 37 °C. These seeds were labeled as ‘nascent’, ‘intermediate’, or ‘mature’ based on their ability to induce and promote aggregation of native protein with different kinetics. Nascent seeds promoted fastest insulin aggregation and formed amorphous aggregates. In another related study we used a combination of pH (acidic to basic) and temperature (37 and 65 °C) to generate morphologically different types of insulin aggregates under disulfide reducing/non-reducing conditions. These aggregates were characterized by different techniques and tested for their toxicity on SH-SY5Y cells. Cytotoxicity studies of insulin aggregates on neuroblastoma cells showed that aggregates formed from disulfide reduced proteins at acidic pH were more toxic compared to the aggregates formed at neutral or basic pH. Lastly, we wanted to study how these properties could be impacted by molecular crowding. We mimicked the intracellular crowded milieu in vitro by using PEG to investigate the effect of crowding on lysozyme stability and aggregation under thermal and reducing stress. We observed that PEG-2000 stabilized the molten globule intermediate of lysozyme in the presence of a non-thiol based reducing agent. Overall, the results indicates an intricate relationship of pH, temperature, and reducing environment impacting proteins aggregation and its associated toxicity