Building on this basic knowledge, the search is underway for cellular mechanisms that could serve as gateways for new therapies. These could lead to precise treatments for disease — targeting a specific cellular function or gene with fewer unintended side effects. Ideally, these effects would also be temporary, returning cells to normal operation once the underlying condition has been treated.

A team of researchers from the 91Ě˝»¨ and the University of Trento in Italy announced findings that could pave the way for these therapies. In a paper July 18 in , they unveiled an engineered protein that they designed to repress a specific cancer-promoting message within cells.

And that approach to protein design could be modified to target other cellular messages and functions, said senior author and 91Ě˝»¨chemistry professor .

“What we show here is a proving ground — a process to determine how to make the correct changes to proteins,” he said.

For their approach, Varani and his team modified a human protein called Rbfox2, which occurs naturally in cells and binds to microRNAs. These aptly named small RNA molecules adjust gene expression levels in cells like a dimmer switch. Varani’s group sought to engineer Rbfox2 to bind itself to a specific microRNA called miR-21, which is present in high levels in many tumors, increases the expression of cancer-promoting genes and decreases cancer suppressors. If a protein like Rbfox2 could bind to miR-21, the researchers hypothesized, it could repress miR-21’s tumor growth effects.

But for this approach to be successful, the protein must bind to miR-21 and no other microRNA. Luckily, all RNA molecules, including microRNAs, have an inherent property that imbues them with specificity. They consist of a chain of chemical “letters,” each with a unique order or sequence. To date, no other research team had ever successfully altered a protein to bind to microRNAs.

“That is because our knowledge of protein structure is much better than our knowledge of RNA structure,” said Varani. “We historically lacked key information about how RNA folds up and how proteins bind RNA at the atomic level.”

91Ě˝»¨researchers relied on high-quality data on Rbfox2’s structure to understand, down to single atoms, how it binds to the unique sequence of “letters” in its natural RNA targets. Then they predicted how Rbfox2’s sequence would have to change to make it bind to miR-21 instead. Elegantly, altering just four carefully selected amino acids made Rbfox2 shift its attachment preference to miR-21, preventing the microRNA from passing along its tumor-promoting message.

The 91Ě˝»¨team spent several years proving this, since they had to test each change individually and in combination. They also had to make sure that the modified Rbfox2 protein would bind strongly to miR-21 but not other microRNAs. Since microRNAs have many functions in cells, it would be counterproductive to repress miR-21 while disrupting other normal microRNA-mediated functions.

The researchers also engineered a second protein that should clear miR-21 from cells entirely. They did this by grafting the regions of Rbfox2 that bound to miR-21 onto a separate protein called Dicer. Dicer normally chops RNAs into small chunks and generates functional microRNAs. But the hybrid Rbfox2-Dicer protein displayed a specific affinity to slice miR-21 into oblivion.

Varani and his team believe that Rbfox2 could be redesigned to bind to microRNA targets other than miR-21. There are thousands of microRNAs to choose from, and many have been implicated in diseases. The key to realizing this potential would be in streamlining and automating the painstaking methods the team used to model Rbfox2’s atomic-level interactions with RNA.

“This method relies on knowledge of high-quality structures,” said Varani. “That allowed us to see which alterations would change binding to the microRNA target.”

Not only would these be useful laboratory tools to study microRNA functions, but they could — in time — form the basis of new therapies to treat disease.

Lead author on the paper is former 91Ě˝»¨researcher Yu Chen, who is now at the Seattle Children’s Research Institute. Other 91Ě˝»¨chemistry co-authors were Fang Yang, Tom Pavelitz, Wen Yang, Katherine Godin, Matthew Walker and Suxin Zheng. Co-authors from the University of Trento include Lorena Zubovic and Paolo Macchi. The research was funded by the National Institutes of Health, the University of Trento and the government of Trento province.

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For more information, contact Varani at 206-543-7113 or varani@chem.washington.edu.

Grant number: R01-GM103834.

91Ě˝»¨ bioengineers have designed a peptide structure that can stop the harmful changes of the body’s normal proteins into a state that’s linked to widespread diseases such as Alzheimer’s, Parkinson’s, heart disease, Type 2 diabetes and Lou Gehrig’s disease. The synthetic molecule blocks these proteins as they shift from their normal state into an abnormally folded form by targeting a toxic intermediate phase.

The discovery of a protein blocker could lead to ways to diagnose and even treat a large swath of diseases that are hard to pin down and rarely have a cure.

“If you can truly catch and neutralize the toxic version of these proteins, then you hopefully never get any further damage in the body,” said senior author , a 91Ě˝»¨professor of bioengineering. “What’s critical with this and what has never been done before is that a single peptide sequence will work against the toxic versions of a number of different amyloid proteins and peptides, regardless of their amino acid sequence or the normal 3-D structures.”

The were published online this month in the journal .

More than 40 illnesses known as diseases – Alzheimer’s, Parkinson’s and rheumatoid arthritis are a few – are linked to the buildup of proteins after they have transformed from their normally folded, biologically active forms to abnormally folded, grouped deposits called fibrils or plaques. This happens naturally as we age, to a certain extent – our bodies don’t break down proteins as quickly as they should, causing higher concentrations in some parts of the body.

Each amyloid disease has a unique, abnormally folded protein or peptide structure, but often such diseases are misdiagnosed because symptoms can be similar and pinpointing which protein is present usually isn’t done until after death, in an autopsy.

As a result, many dementias are broadly diagnosed as Alzheimer’s disease without definitive proof, and other diseases can go undiagnosed and untreated.

The molecular structure of an amyloid protein can be only slightly different from a normal protein and can transform to a toxic state fairly easily, which is why amyloid diseases are so prevalent. The researchers built a protein structure, called “alpha sheet,” that complements the toxic structure of amyloid proteins that they discovered in computer simulations. The alpha sheet effectively attacks the toxic middle state the protein goes through as it transitions from normal to abnormal.

The structures could be tailored even further to bind specifically with the proteins in certain diseases, which could be useful for specific therapies.

The researchers hope their designed compounds could be used as diagnostics for amyloid diseases and as drugs to treat the diseases or at least slow progression.

“For example, patients could have a broad first-pass test done to see if they have an amyloid disease and then drill down further to determine which proteins are present to identify the specific disease,” Daggett said.

The research team includes , Jackson Kellock and of 91Ě˝»¨bioengineering; and Ravi Pratap Barnwal of 91Ě˝»¨chemistry; Peter Law, a former 91Ě˝»¨graduate student; and Byron Caughey of the National Institutes of Health’s .

Working with the UW’s , they have a patent on one compound and have submitted an application to patent the entire class of related compounds.

This research began a decade ago in Daggett’s lab when a former graduate student, Roger Armen, first discovered this new secondary structure through computer simulations. Daggett’s team was able to prove its validity in recent years by designing stable compounds and testing their ability to bind toxic versions of different amyloid proteins in the lab.

The research was funded by the National Institutes of Health (General Medicine Sciences), the National Science Foundation, the Wallace H. Coulter Foundation and Coins for Alzheimer’s Research Trust.

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 For more information, contact Daggett at daggett@uw.edu.