Unravelling the foundations of type 2 diabetes
For the first time, researchers have used a single-molecule technique to study the potential origins of type 2 diabetes.
Type 2 diabetes is caused either by the body not making enough insulin, or that the insulin it does make not working properly to reduce blood sugar levels. There is no cure, only medicines that control blood sugar levels alongside lifestyle changes.
Unravelling the molecular mechanisms behind the condition, however, could lead to new drug targets and potential therapies. One promising target is a protein called amylin, which is known to aggregate in the pancreas of people with type 2 diabetes, forming fibrils that can break cells apart.
Now, a team led by Dr Jinqing Huang from the Department of Chemistry at Hong Kong University of Science and Technology has examined these amylin proteins in new detail. They were able to analyse individual proteins, molecule-by-molecule, and discover rare protein structures that appear to be involved in aggregation. The results were published in Nature Communications.
One by one
Amylin proteins are produced in the pancreas, so are found in high concentrations in pancreatic cells. However, they don’t aggregate in this environment. When they move to the extracellular environment, despite being lower in concentration, this is where they aggregate. One difference between the two environments is the pH, with the cells being more acidic inside than outside.
Finding out whether pH may be a determining factor in causing proteins to misfold and aggregate, however, is not straightforward. In a complex and heterogeneous system of molecules, such as is found in the pancreas, techniques that analyse the whole mixture are likely to get an average, rather than finding potentially rare and transient molecular structures that could be crucial.
So Huang and her team turned to a single-molecule technique for surface-enhanced Raman spectroscopy (SERS) – a method of boosting the signal from target molecules in order to detect them more easily. With a stronger signal, less target volume is needed, meaning single molecules can be analysed on their own.
In order to analyse each molecule, they have to be trapped one-by-one. To achieve this, the researchers created a tiny trap controlled by light. Two microbeads made of silica were covered in a layer of silver nanoparticles and connected in stable conjunction. When irradiated with a laser, the local electromagnetic field of the microbeads was enhanced, trapping a free silver nanoparticle and forming a nanocavity that can trap and detect single molecules.
Since this trap is created by laser irradiation, turning the laser on and off forms and dismantles the cavity, trapping one molecule at a time. With the light switching on and off automatically and the spectrum continuously being analysed, the method is fast and high throughput.
“It works like a needle catching things in water,” says Huang. “The tiny space that traps molecules both enhances their signal and means only one is analysed at a time. This is crucial for analysing a meaningful number of even rare amylin protein structures.”
And rare structures they found. Between the acidic conditions that mimic the pancreatic cellular environment, and the neutral conditions found in the extracellular environment, they found rare proteins that carried one small difference: one amino acid changed its charge from positive to neutral. This is enough to change the protein structure in these rare cases.
“The majority of proteins we measured did not change in this way, but the rare cases appear to be enough to shift the equilibrium of the whole mixture, tipping the proteins towards aggregation,” says Huang. Even if the altered proteins were put back in an acidic environment, they stayed misfolded and still induced aggregation.
This is an exciting result, but it is only the beginning. The samples used were a model system, with pH as the only environmental factor. In future, Huang wants to use more complex environments, testing factors like metal ion concentrations and membrane compositions, and the presence of other proteins like insulin that co-synthesise with amylin.
She also wants to map how amylin interacts with RNA and DNA, and what happens when these proteins are overproduced. This is akin to current studies in another, similar system: the amyloid plaques that form in the brains of people with neurodegenerative diseases like Alzheimer’s and Parkinson’s.
Plaques have been the main targets for potential neurodegenerative disease treatments for the past 10-15 years. But even when they are destroyed, the symptoms of sufferers are not reversed. This has led to speculation that the plaques are a result, rather than a cause, of the neurodegeneration. The same is likely true of amylin aggregates in type 2 diabetes: they may be a symptom rather than a cause.
A single-molecule future
To find the root of both the recognised symptoms of these diseases and the protein aggregates associated with them, researchers are looking further and further back in the process: looking at how proteins aggregate, how they misfold, and what molecular changes lead there.
This all requires single-molecule analysis, which has gained prominence as techniques have emerged that make it viable.
Techniques like cryo-electron microscopy or X-ray diffraction are the gold standard for discovering molecular structure at the atomic level, but single-molecule SERS techniques allow dynamic systems to be analysed with efficiency. To make up for the shortfall in structural information, Huang combined the spectroscopy data from her SERS experiments with molecular dynamic simulations.
“The ‘blurred’ picture we get from the experiments can be combined with computations to find matching explanations, helping elucidate more details about the molecule’s structure,” says Huang.
These studies are only at the beginning, but they have enormous power that could one day enable researchers to uncover the molecular roots of diseases like type 2 diabetes and Parkinson’s, and ultimately, new ways to treat them.