proteins to become unstable
Damage (red devils) like drying out, harsh chemicals or heat normally causes proteins to become unstable and lose their proper shape and function (left side, orange). Researchers at the University of Tokyo have characterized Hero proteins (pink, purple, green), long, flexible proteins that protect other proteins (right side, orange). [Illustration by Kotaro Tsuboyama, CC BY 4.0]

Researchers at the University of Tokyo have discovered a group of unusually shaped, heat-resistant proteins, dubbed “Hero” (heat-resistant obscure) proteins, which have the ability to block the formation of pathological protein clumps in lab-grown cells and in live fruit fly models that model neurodegenerative diseases. The Hero proteins, which appear to be widespread in different animals including insects and humans, were also found to extend lifespan in fruit flies.

Reporting in PLOS Biology on experiments with several of the new-found Hero proteins, the scientists suggest that as well as offering new insights into protein stability and function, their discoveries may also “highlight potential biotechnological and therapeutic applications of Hero proteins.” They reported their research in a paper titled, “A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation.”

Proteins are essentially polymers composed of 20 different amino acids, with side chains that have different properties. The diversity of protein molecules allows them to fold into three-dimensional structures that determine their activity and function, the authors explained. Proteins are generally stable at physiological temperatures or even up to about 50–60° C, but most are denatured when heated to near-boiling point. There are some exceptions, typically among organisms that might have to survive in harsh environments. “Currently, these heat-resistant proteins are viewed as special cases required by organisms living in extreme conditions to protect their functional proteins,” the authors wrote. “Although mammals have been reported to produce some highly heat-soluble proteins, their functions remain largely unexplored.”

The first Hero protein was identified by accident in about 2011 when then-graduate student Shintaro Iwasaki encountered an unusually heat-resistant protein in Drosophila that increased stability of another protein, Argonaute, which was the focus of the lab’s studies. Iwasaki now leads his own lab at RIKEN. “It was kind of cool to know that a strange, extremely disordered, heat-resistant protein improved the behavior of Argonaute, but its biological relevance was unclear and, moreover, the protein’s sequence seemed unrelated to anything else,” said Yukihide Tomari, PhD, who heads the University of Tokyo lab and is co-corresponding of the paper published in PLOS Biology. “So, we didn’t know what to do next and just decided to put it on the shelf until years later.” The researchers named the protein using a combination of an informal Japanese word meaning weak or not rigid, and the diminutive suffix normally attached to young boy’s names, “hero-hero kun.”

Years later, the name coincidentally also fits the English meaning of hero, as a brave defender. The University of Tokyo team’s continued work has shown how Hero proteins can protect other proteins from degradation, extend the life span of fruit flies by 30%, and protect both fruit flies and lab-grown human motor neurons from pathological protein aggregates similar to those that are found in patients with neurodegenerative diseases.

To try and identify additional Hero proteins, the researchers derived extracts from lab-grown human and fruit fly cells, then simply boiled them. High temperatures normally weaken the chemical interactions that support a protein’s structure, causing it to unfold and clump together with other unfolded proteins. But this didn’t happen with the Hero proteins. “Proteins are generally damaged by heat, but we found that Hero proteins remain intact even at 95° C [203° F] without losing function,” commented co-author Kotaro Tsuboyama, PhD, postdoctoral fellow at the Univerity of Tokyo. “It is a bit strange, which is why I think no one has carefully characterized these proteins before.” When the team then used mass spectrometry to identify any proteins that remained in the boiled test tubes, they found hundreds of Hero proteins in fruit flies and in humans.

Proteins with similar functions usually have similar amino acid sequences even between different species. This is known as evolutionary conservation. However, the lack of evolutionary conservation that Tomari’s team encountered when they first identified hero-hero kun, seems to be a defining characteristic for Hero proteins, making it difficult to predict their function or even identity. And interestingly, while most proteins have well-defined folds and twists that form a rigid 3D structure, the newly identified Hero proteins have a long, flexible string-like structure.

Tsuboyama selected six Hero proteins to study in detail. The team’s experiments showed that when some of the Hero proteins were mixed with other “client” proteins, the client kept its shape and function when exposed to high heat, drying, or harsh chemicals that would normally destroy them. “Hero proteins are hydrophilic and highly charged, and function to stabilize various ‘client’ proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents,” the authors wrote.

The liquid portion (lysate) of cells is clear at normal temperature (left) but becomes cloudy after boiling (center). After researchers spin the tubes at high speeds (centrifugation), the cloudy liquid separates into a white clump at the bottom of the tube, made of normal proteins that became unstable and misfolded due to the heat, and the remaining clear liquid containing anything that was unaffected by the heat (right). The remarkable Hero proteins are part of the remaining clear liquid and were studied by University of Tokyo researchers. [Photo by Kotaro Tsuboyama, CC BY 4.0]
In experiments using lab-grown cells, including human motor nerve cells, high levels of Hero proteins also stopped the development of protein clumps, including aggregates that are characteristic of the neurodegenerative disease amyotrophic lateral sclerosis (ALS), and restored normal cell growth patterns. The studies indicated that different Hero proteins acted with different client proteins. “Interestingly, among the six Hero proteins tested, there was no “super-Hero” protein able to function for all proteins,” the team wrote. “Instead, distinct subsets of human Hero proteins were more effective in preventing different types of aggregations … Apparently, there was no common rule for the effective combination between different Hero proteins and different client proteins.”

As Tsuboyama further commented, “We saw many positive effects, but so far, we did not find any ‘superhero’ among those six Hero proteins that can stabilize all client proteins. Some Hero proteins are good for some clients, and others are good for other clients.”

The team also tested the Hero proteins in live Drosophila fly models. The large, sensitive eyes of fruit flies are often used as disease models, because they are deformed by mutations that cause neurodegeneration in humans. The researchers’ experiments in these models also observed that enhancing Hero activity protected the flies’ eyes from deformation caused by protein clumps associated with ALS. Conversely, eliminating normal Hero activity caused defects in the development of the fly eye. “The finding that some Hero proteins can block pathogenic protein aggregates in cells and in fly models for neurodegenerative diseases may also serve as a starting point for future development of therapeutics or diagnostic methods,” the scientists suggested.

In further experiments, the investigators found evidence that genetically modified healthy fruit flies to have high levels of individual Hero proteins throughout their whole bodies prolonged longevity. Remarkably, some of the Hero proteins caused flies to live about 30% longer than control flies.

The scientists are planning future experiments to identify any patterns or rules about which Hero proteins assist which client molecules in living organisms. “It appears that Hero proteins naturally exist to keep other proteins happy,” said Tomari. “We hope that, in the long run, Hero proteins can be useful for biotechnological and therapeutic applications.” As the authors concluded, “Each organism appears to encode hundreds of Hero proteins with no apparent common motifs, and each Hero protein is likely to deal with many different client proteins, thus providing a rich but complex source to mine.” They suggest that a combination of different approaches, including genetics, biochemistry, and biophysics, will be needed to help unpick the relationships among and between Hero proteins and their clients.

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