People with adult onset (type 2) diabetes typically exhibit misfolded, insoluble protein aggregates in their pancreas. Such aggregates are also seen in other disorders, such as Alzheimer's disease.
Regarding diabetes, these aggregates damage the membrane (outer barrier) of certain pancreatic cells, hindering the production of insulin. Untreated type 2 diabetes often causes many serious complications, leading to an early death.
How do misfolded proteins damage pancreatic cells? There is evidence that an important step is the action of intermediately misfolded and aggregated proteins.
Such results suggest that the transition from an intermediately aggregated protein to a more extensively aggregated protein plays an important role in the progression of type 2 diabetes. It turns out that many experimental techniques are not up to the task of probing the protein aggregation pathway, on the molecular level.
Further compounding the mystery is that while a diabetes-implicated human protein aggregates and is toxic to (facilitates diabetes in) rats, a similar rat-derived protein does not aggregate, and is not toxic to them. Such observations emphasize the need for an experimental technique that can elucidate how proteins misfold and aggregate, for understanding the progression of type 2 diabetes, as well as other disorders characterized by protein aggregation.
Ayyalusamy Ramamoorthy (University of Michigan) and coworkers have worked on this problem. They have utilized two spectroscopy techniques to probe the aggregation of the islet amyloid polypeptide protein.
Experimental techniques.
The scientists used pulsed field gradient nuclear magnetic resonance spectroscopy to probe the molecular-level protein misfolding and aggregation of the islet amyloid poypeptide protein. This is the protein that accumulates and aggregates in the pancreas of those with type 2 diabetes.
Pulsed field gradient techniques give information on the size of large molecules such as proteins, and nuclear magnetic resonance gives information on chemical bonding within proteins. Thus, combining these two techniques provides valuable insight into protein misfolding and aggregation.
Circular dichroism spectroscopy was applied as well. This is a commonly used technique to quickly elucidate the identity and quantity of specific folded geometries in a protein.
Probing protein misfolding and aggregation.
The scientists used these techniques to probe the folding and aggregation of the islet amyloid polypeptide protein derived from humans, as well as a similar protein derived from rats. Clear differences between the two in their three-dimensional structure were observed.
They found that the human islet amyloid polypeptide protein is more compact than the rat variant of the protein at 4°C, while the rat variant is more compact than the human variant at 37°C. In other words, at physiological temperatures (37°C), the human variant of the protein is less compact and is present as small uncoiled strands and chains, instead of aggregates.
Additionally, when heated from 4°C to 50°C, the human protein undergoes local conformational changes not observed in the rat protein. This structural transition may be important in forming aggregates.
Perhaps most importantly, the scientists also found that the human islet amyloid polypeptide protein aggregates more readily at 37°C (physiological temperatures), relative to 4°C. In contrast, the rat variant of the protein does not aggregate.
These results collectively suggest that single units of the human islet amyloid polypeptide protein can coexist with small chains of the proteins, without initiating protein aggregation. They also suggest that a critical step in the protein aggregation process is the development of partially folded structures.
Implications.
Scientists are currently divided as to the nature of the islet amyloid polypeptide protein when they initially associate with pancreatic cell membranes. Ramamoorthy and coworkers now propose that the proteins are initially in a single unit unstructured state, then transition to a structured multiunit state that can progress to aggregates.
This knowledge will assist other scientists in elucidating further molecular details regarding how the islet amyloid polypeptide protein damages pancreatic cell membranes. In the (very) long term, it may provide insight into how to prevent or combat such damage, facilitating a fundamentally new treatment for type 2 diabetes.
for more information:
Soong, R., Brender, J. R., Macdonald, P. M., & Ramamoorthy, A. (2009). Association of Highly Compact Type II Diabetes Related Islet Amyloid Polypeptide Intermediate Species at Physiological Temperature Revealed by Diffusion NMR Spectroscopy Journal of the American Chemical Society, 131 (20), 7079-7085 DOI: 10.1021/ja900285z