Unstable proteins are crucial factors in a variety of inherited diseases, as highlighted by a recent study. These include genetic conditions that result in cataracts and various rare neurological, developmental, and muscle-wasting disorders. Unstable proteins tend to misfold and break down, leading to loss of function or harmful accumulation in cells.
A comprehensive study published today in the journal Nature reveals that most mutations causing diseases do so by substituting one amino acid for another, which compromises protein stability. When proteins become unstable, they are more prone to misfolding and degradation, resulting in a failure to function properly or harmful buildup in cells.
This research clarifies why even minor changes in the human genome, known as missense mutations, can lead to diseases at the molecular level. The findings show that instability in proteins is a major factor in heritable cataract formation and also plays a role in various neurological, developmental, and muscle-wasting conditions.
Scientists from the Centre for Genomic Regulation (CRG) in Barcelona and BGI in Shenzhen investigated 621 established disease-related missense mutations. They found that 61% of these mutations led to a measurable reduction in protein stability.
The study delved deeper into some specific mutations linked to diseases. For instance, beta-gamma crystallins are proteins vital for maintaining the clarity of the eye lens. The researchers found that 72% (13 out of 18) of the mutations associated with cataracts destabilized crystallin proteins, increasing their likelihood of clumping together and creating cloudy regions in the lens.
Furthermore, the study directly linked protein instability to the emergence of reducing body myopathy, a rare disorder that results in muscle weakness and wasting. It also associated instability with Ankyloblepharon-ectodermal defects-clefting (AEC) Syndrome, which is marked by the presence of a cleft palate alongside other developmental issues.
However, not all mutations causing diseases destabilized proteins, revealing alternative molecular mechanisms at work.
Rett Syndrome, a neurological disorder that leads to severe cognitive and physical challenges, is caused by mutations in the MECP2 gene. This gene produces a protein that regulates gene expression in the brain. The study found that many mutations in MECP2 do not destabilize the protein but instead occur in regions that influence how MECP2 interacts with DNA to regulate other genes. This disrupted function may impair brain development and performance.
“We demonstrate on a previously unseen scale how mutations lead to disease on a molecular level,” states Dr. Antoni Beltran, the study’s first author and a researcher at CRG in Barcelona. “By identifying whether a mutation destabilizes a protein or alters its function without affecting stability, we can create more targeted treatment strategies. This could differentiate between developing drugs that stabilize proteins and those that block harmful activities. This is a major advance toward personalized medicine.”
The study also identified that the impact of mutations on diseases frequently corresponds to whether the condition is recessive or dominant. Dominant genetic disorders arise when a single mutated gene copy is sufficient to cause the disease, regardless of the normal copy, whereas recessive disorders occur when an individual inherits two mutated copies—one from each parent.
Mutations linked to recessive disorders were found to be more likely to destabilize proteins, while mutations associated with dominant disorders typically affected other protein functions, such as their interactions with DNA or other proteins, rather than just stability.
For example, the research revealed that a recessive mutation in the CRX protein, crucial for eye function, significantly destabilizes the protein. This instability could lead to hereditary retinal dystrophies as the absence of a stable, functional protein hinders normal vision. Conversely, two different dominant mutations preserved the protein’s stability but caused it to malfunction, leading to retinal disease despite intact protein structure.
These findings were made possible by the development of Human Domainome 1, a vast library of protein variants. This resource features over half a million mutations across 522 human protein domains, which are the segments of proteins that determine their function. It is the most comprehensive catalog of human protein domain variants created to date.
Protein domains consist of specific sections that can take on a stable form and function independently from the rest of the protein. The Human Domainome 1 was established by systematically substituting each amino acid in these domains with every other possible amino acid, assembling a complete catalog of potential mutations.
To assess how these mutations affected protein stability, the researchers introduced mutated protein domains into yeast cells. Only one specific type of mutated protein domain was produced in these yeast, and the cultures were cultivated in scenarios that linked protein stability to yeast growth. If the protein was stable, the yeast grew well; if unstable, growth was poor.
By employing a unique technique, the researchers ensured that only yeast cells that produced stable proteins could thrive and multiply. By comparing mutation frequencies before and after yeast growth, they were able to ascertain which mutations resulted in stable proteins and which led to instability.
Although Human Domainome 1 is approximately 4.5 times larger than previous protein variant libraries, it still only encompasses 2.5% of known human proteins. As researchers continue to expand this catalog, the precise role of disease-associated mutations in protein instability will become increasingly clearer.
Meanwhile, scientists can use data from the 522 protein domains to make inferences about similar proteins. This is because mutations often have analogous impacts on proteins that share structural or functional similarities. By examining a varied collection of protein domains, the researchers identified patterns in how mutations influence protein stability that are applicable across related proteins.
“This implies that information from a single protein domain can predict how mutations will affect other proteins within the same family or with comparable structures. The insights from these 522 domains are sufficient to help us make informed predictions about many more proteins than those represented in the catalog,” explains ICREA Research Professor Ben Lehner, the corresponding author of the study, who is affiliated with both the Centre for Genomic Regulation and the Wellcome Sanger Institute.
The study has its limitations. The researchers analyzed protein domains in isolation rather than within complete proteins. In living organisms, proteins interact with other components and molecules in the cell. Thus, the study might not fully reflect how mutations influence proteins in their natural environment inside human cells. The research team plans to address this by examining mutations in larger protein domains and, eventually, full-length proteins.
“Ultimately, our aim is to chart the effects of every possible mutation on all human proteins. This is an ambitious goal that could revolutionize precision medicine,” concludes Dr. Lehner.
