n 11.04.23 – Modifying a new technique, scientists at EPFL have uncovered how evolution impacts the way our cells deal with oxidative stress from the things we eat.
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Eating our favorite foods, inhaling tobacco smoke, and even exercise can produce reactive small molecules in our bodies. These reactive species are usually mopped up by detoxifying enzymes or break down because of their intrinsic instability.

Nonetheless, some of these small molecules, under the right conditions, can trigger a domino effect of molecular and biochemical reactions known as “signaling cascades”, which can have a wide variety of physiological effects. Unfortunately, because of their inherent instability and chemical “promiscuity”, these reactive molecules, have proven particularly difficult to study in living systems.

Now, scientists led by Yimon Aye at EPFL have developed a method to trigger specific signaling pathways in live zebrafish, which are often used in genetics and molecular biology research. The work is published in two journals, eLife and Nature Protocols.

The method is a modification of a chemical biology technique developed by Aye in 2016 called called “targetable reactive electrophiles and oxidants” or T-REX for short. The idea behind T-REX – and its zebrafish-specific version used in these studies, Z-REX – is to release a specific reactive compound called an electrophile onto a target protein and see its effects.

In chemistry, electrophiles are highly reactive molecules that are “eager” to form bonds with atoms or other molecules that have an available electron pair. “This protocol opens up the field to study so-called “electrophiles” and drug-like ‘electrophilic’ fragments, not only in live fish, but also in specific tissues, such as the heart,” says Aye.

The second paper uses Z-REX to study the function of the two paralog genes of the protein keap1. Paralog genes have similar sequences but have diverged from each other because of genetic duplication. Because the paralogs share a common evolutionary ancestry, the two gene products have broadly similar structures and functions in related signaling cascades and protein complexes.

The researchers used Z-REX to study the two paralogs of keap1, a protein involved in both the human and fish responses to oxidative stress. Specifically, keap1 interacts with another protein, Nrf2, which is a master regulator of the cell’s antioxidant response. In that interaction, keap1 anchors Nrf2 to the cytoplasm and ushers Nrf2 breakdown.

“Due to an ancient genome duplication that happened in the lineage of zebrafish, they possess two copies of many of their genes,” says Aye. “These copies often show tissue- or function-specific capacities, relative to their human counterparts, of which there is usually a single gene. In this case, the first Keap1 paralog has lost the ability to respond to modification by reactive species, whereas the second paralog of Keap1 functions identically to the human version, upregulating Nrf2 activity upon electrophile modification.”

The scientists used Z-REX to show that both paralogs of Keap1 in zebrafish indeed suppress the activity of Nrf2 – essentially performing their “canonical” function. In addition, they also found that the first, unreactive paralog of Keap1 appears to behave as a “dominant negative”, which means that its mere presence prevents the function of the second, active paralog. “These discoveries explain tissue-specific responsivities of different tissues to small-molecule electrophilic metabolite stress in developing fish,” says Aye.

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