Scientists identify 'destination' for protein that provides zinc

Scientists identify ‘destination’ for protein that provides zinc

Close-up of a plate sample filled with yeast cell cultures used in these studies. These panels allow scientists to run experiments on many duplicate samples simultaneously to increase confidence in the results. Credit: Brookhaven National Laboratory

Most people don’t think much about zinc. But all living things need zinc to survive. This trace element helps to fold many proteins into the correct shapes to carry out their functions. And in proteins known as enzymes, zinc helps catalyze chemical reactions — including many that are important for providing energy for cells. If zinc is absent, people, pets, and plants will not thrive.

That’s one reason biologists at the US Department of Energy’s Brookhaven National Laboratory are so interested in this element.

“We are looking at ways to grow bioenergy plants — either plants that produce biofuels or whose biomass can be converted into fuel — and to do so on land that is not suitable for growing food crops,” said Brookhaven Lab biologist Christine Blaby, who also works as a holder An assistant appointment at Stony Brook University. “Therefore, we are interested in the strategies that nature uses to survive when zinc and other micronutrients are deficient.”

In a paper just published in the magazine cell reports, Blaby and colleagues describe one such strategy: a so-called “helper” protein that gets zinc where it’s needed, which may be especially important when access to zinc is limited. Although scientists, including Blaby, have long suspected zinc facilities exist, the new research provides the first conclusive evidence by defining a “destination” for their delivery.

Through a series of biochemical tests and genetic experiments, the team identified a zinc-based protein that cannot function properly without the chaperone. This protein, called MAP1, is found across species — from yeast and mice to plants and humans. This means that the findings are relevant not only to plants but also to human health, as zinc deficiency leads to growth and poor growth.

“Our goals are to sustain bioenergy crops, but because the proteins we study are almost ubiquitous, our research has very broad applications,” said Blaby.

discovery tracker

Other trace metals, such as nickel and copper, are carried around cells by chaperones because they can be toxic. Companions prevent reactive metals from engaging in “unwanted associations”. The interactions between certain trace minerals and oxygen generate free radicals that damage cells. But zinc does not seem to tend to such dangerous relationships.

“Zinc is a relatively harmless metal ion. Since it doesn’t react with oxygen to form reactive oxygen species, we thought maybe it just diffuses to get where it needs to go without a companion,” said Blaby. But that hasn’t stopped scientists from looking for one.

When Bellaby was a graduate student at the University of Florida in the early 2000s, she worked with Professor Valerie de Cressi-Lagarde, who first speculated that members of a protein family called CobW were the missing zinc companions. “My research as part of that group provided evidence that if there was one, it likely was a protein in this family. But to prove that it acts as a zinc chaperone, we needed to identify the destination — the protein that was transporting zinc to it,” said Blaby.

Several groups have worked on this challenge for years but still have not been able to find and prove the companion’s purported target.

Data mining provides clues

Fast forward to the time when Blaby began building her research group at Brookhaven in 2016. While mining data on interactions between proteins that have been deposited in searchable databases over the past decade, I found evidence of an interaction between a protein in the zinc chaperone family. The purported protein is methionine aminopeptidase, or MAP1. The interaction was found in both yeast and humans.

“When you see a conserved protein interaction like this, in very different organisms, it usually means it’s important,” said Blaby.

It turns out that MAP1 modulates many proteins in the cell in almost all species. If MAP1 is not working, the unmodified proteins have problems. And MAP1 relies on zinc to function.

“The pieces are starting to come together,” Balabi said. “Then the real fun began — which was testing our very specific hypothesis: that this protein that we came to call ZNG1 (pronounced Zing 1) is the helper that delivers zinc to MAP1.”

Scientists define

Brookhaven Lab biologist Christine Blaby and postdoctoral fellow Nicholas Grosjean and colleagues conducted genetics experiments, biochemical assays and computational modeling studies that identified ZNG1 as a zinc helper protein. Credit: Brookhaven National Laboratory

Blaby worked with Brookhaven postdocs Miriam Pasquini and Nicholas Grosjean, who designed and conducted a series of experiments to highlight the case. The two share the first authorship on paper.

“This was a really great team to put together to do both in vivo and men lab The work needs to finally provide experimental evidence of the function of these proteins.”

The proof is in the bottle

First, using fast-growing yeast cells, Grogan knocked out the gene that tells the cells how to make ZNG1. If this protein is the moderator that delivers zinc to MAP1, then the MAP1 protein should not function properly in excisable cells.

And when zinc is deficient in the environment, the defect in MAP1 function must worsen.

“When a lot of proteins are competing for a limited amount of zinc, this is a case where, if there is an accompaniment, it may help to choose which of the many zinc-based proteins should get this valuable resource,” Grogan explained. In other words, when zinc is limited, the absence of utilities should be felt even more.

The results were as expected: cells without the ZNG1 gene had defects in MAP1 activity, and the level of defect was increased in the low zinc environment.

Next, Pasquini led a project to purify the two proteins – ZNG1 and MAP1 – separately. First, it showed that in the absence of zinc, as expected, MAP1 does not act on its own.

Then she mixed MAP1 with zinc-loaded ZNG1. But again, there was no MAP1 activity. The scientists concluded that something else must be missing.

Through a series of experiments, they demonstrated that ZNG1 needs activation to deliver its zinc charge. This activation comes from an energy molecule known as GTP.

“What we think is happening is that the flanking binds to the GTP and has a certain shape or form,” Pasquini said. “When it releases energy from GTP, it changes shape. We think the harmonic change could be important for zinc binding and release.”

When Pasquini added GTP to the zinc-loaded ZNG1 and MAP1 mixture, she finally noticed MAP1 activity.

“Only after you add the energy molecule do you see evidence of zinc transfer to MAP1,” she said.

Together, these experiments provided evidence that the long-suspected protein now known as ZNG1 acts as a chaperone for zinc delivery to MAP1.

wider implications

The team also collaborated with scientists at the Environmental Molecular Sciences Laboratory, a user facility of the Department of Energy’s Office of Science at Pacific Northwest National Laboratory, on a larger scale of “proteomics” experiments. And they worked with Estella Yee at the Brookhaven Lab’s National Synchrotron Light Source II (NSLS-II), another DOE Office of Science user facility, on computer modeling studies to understand the protein complex that forms between the zinc chaperone and MAP1.

“for us in vivo And in the laboratory The experiments were looking at players only. What the proteins allowed us to do was see how deleting the zinc transferase gene affected everyone The proteins — and the study of the effect these players have on the rest of the cell and the organism, said Blaby.

One of the main effects is that cells can no longer adapt to low levels of zinc.

“Cells have evolved so that when zinc concentrations drop drastically, a set of genes are turned on in response to this change in conditions. But when you take out ZNG1, many of those genes remain disabled,” said Blaby.

“We are now building on this foundational work done in a rapidly growing yeast model organism to understand how these proteins are conserved and functioned in bioenergy crops,” said Blaby. “This work highlights a previously unknown strategy that plants use to grow when zinc is limited in the soil. Understanding such strategies may help us devise ways to improve crop yields and achieve environmentally sustainable bioenergy.”

Pasquini added: “The possibility of plants gaining resilience in low zinc soils also means that we will be able to exploit non-arable land to grow bioenergy crops, leaving fertile soils for other agricultural purposes. Pushing plant cells to produce more ZNG1 can conceivably achieve superior growth in Zn-depleted marginal lands.

Autophagy: balancing zinc and iron in plants

more information:
Crysten E. Blaby-Haas, Maintenance of protein activity during zinc starvation using GTP-dependent metal transfer, cell reports (2022). DOI: 10.1016/j.celrep.2022.110834.… 2211-1247 (22) 00607-6

Provided by Brookhaven National Laboratory

the quote: Scientists identify ‘destination’ for protein that delivers zinc (2022, May 17) Retrieved May 17, 2022 from

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2022-05-17 15:00:02

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