Discovery Reveals Key Mechanism All Living Things Use to Transport Trace Mineral Essential for Survival
Most people don’t think much about zinc. But all living things need zinc to survive. This trace mineral helps many proteins fold into the right shapes to do their job. And in proteins known as enzymes, zinc helps catalyze chemical reactions, many of which are important for providing energy to cells. If zinc is missing, people, pets and plants do not thrive.
This is one of the reasons why biologists at the US Department of Energy’s Brookhaven National Laboratory are so interested in this element.
“We’re looking at ways to grow bioenergy plants – either plants that produce biofuels or whose biomass can be converted into fuel – and do it on land that isn’t suitable for growing food crops,” he said. said Brookhaven Lab biologist Crysten Blaby, who also holds an adjunct position at Stony Brook University. “So we’re interested in the strategies nature uses to survive when zinc and other micronutrients are lacking.”
In an article just published in the journal Cell Reports, Blaby and his colleagues describe one such strategy: a so-called chaperone protein that delivers zinc where it’s needed, which could be particularly important when access to zinc is limited. Although scientists, including Blaby, have long suspected the existence of a zinc chaperone, the new research provides the first definitive proof in identifying a “destination” for its deliveries.
Through a series of biochemical tests and genetic experiments, the team identified a zinc-dependent protein that cannot function properly without the chaperone. This protein, called MAP1, exists in all species, from yeasts and mice to plants and humans. This means the findings are relevant not only to plants, but also to human health, where zinc deficiency leads to impaired growth and development.
“Our goals are the sustainability of bioenergy crops, but because the proteins we study are found almost everywhere, our research has very broad applications,” Blaby said.
Track down a discovery
Other trace metals, such as nickel and copper, are transported into cells by chaperones because they can be toxic. Chaperones prevent reactive metals from engaging in “undesirable associations”. Reactions between certain trace metals and oxygen generate free radicals that damage cells. But zinc does not seem to have the propensity for such dangerous bonds.
“Zinc is a relatively harmless metal ion. Since it does not react with oxygen to create reactive oxygen species, we thought it might just be diffusing out to where it needed to go without need a chaperone,” Blaby said. But that hasn’t stopped scientists from looking for one.
When Blaby was a graduate student at the University of Florida in the early 2000s, she worked with Professor Valérie de Crécy-Lagard, who first predicted that members of a protein family called CobW were the chaperones of zinc missing. “My research as part of this group provided evidence that if one existed, it was probably a protein from this family. But to prove that it functions as a zinc chaperone, we needed to identify the destination – the protein it was delivering zinc to,” Blaby said.
Many groups have been working on this challenge for years but still couldn’t find and prove the alleged chaperone’s target.
Data mining reveals clues
Fast forward to when Blaby began building her research group at Brookhaven in 2016. As she explored data on protein interactions that had been deposited in searchable databases over the past decade , she found evidence of an interaction between a protein from the so-called zinc chaperone family and a protein called methionine aminopeptidase, or MAP1. And she found the interaction in both yeast and humans.
“Any time you see a conserved protein interaction like this, in very different organisms, it usually means it’s important,” Blaby said.
It turns out that MAP1 modifies many proteins in the cell – in almost all species. If MAP1 doesn’t work, unmodified proteins have problems. And MAP1 depends on zinc to function.
“The pieces were starting to fall into place,” Blaby said. “Then the real fun began – which was to test our very specific hypothesis: that this protein that we have come to call ZNG1 (pronounced zing 1) is the chaperone that supplies zinc to MAP1.”
Blaby worked with Brookhaven postdocs Miriam Pasquini and Nicolas Grosjean, who designed and conducted a series of experiments to nail the case. The two share first paternity on paper.
“It was a very good team to bring together to do both the live and mein vitro work needed to finally provide experimental evidence for the function of these proteins,” Blaby said.
The proof is in the bottle
First, using fast-growing yeast cells, Grosjean knocked out the gene that tells cells how to make ZNG1. If this protein is the chaperone that delivers zinc to MAP1, then MAP1 should not function properly in knockout cells.
And when zinc is lacking in the environment, the malfunction of MAP1 should worsen.
“When many proteins are competing for limited zinc, that’s a situation where, if there’s a chaperone, it could help decide which of many zinc-dependent proteins should get this valuable resource,” Grosjean explained. . In other words, when zinc is limited, the absence of the chaperone should be felt more.
The results came out as expected: cells without the ZNG1 gene showed defects in MAP1 activity, and the level of defect increased in the low-zinc environment.
Next, Pasquini led a project to purify the two proteins – ZNG1 and MAP1 – in isolation. First, it showed that when no zinc is present, as expected, MAP1 by itself does not work.
Then she mixed MAP1 with ZNG1 which had been loaded with zinc. But again, there was no MAP1 activity. The scientists felt that something else must be missing.
Through a series of experiments, they demonstrated that ZNG1 must be activated to deliver its cargo of zinc. This activation comes from an energy molecule known as GTP.
“What we think is happening is that the chaperone binds to the GTP and has a certain conformation or shape,” Pasquini said. “When it releases energy from GTP, it changes shape. We think the change in conformation might be important for binding and releasing zinc.”
When Pasquini added GTP to the mixture of ZNG1 and zinc-loaded MAP1, she finally observed MAP1 activity.
“It’s only after you add the energy molecule that you see evidence of zinc transfer to MAP1,” she said.
Together, these experiments provided evidence that the long-suspected protein now known as ZNG1 functions as a chaperone to supply zinc to MAP1.
Larger scale implications
The team also collaborated with scientists from the Molecular Environmental Sciences Laboratory, a DOE Office of Science user facility at the Pacific Northwest National Laboratory, on larger-scale “proteomics” experiments. And they worked with Estella Yee at 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 chaperone of zinc and MAP1.
“Our live and in vitro the experiments involved only a few players. What proteomics has allowed us to do is see how deleting the zinc transferase gene affects everything proteins – and study the impact of these actors on the rest of the cell and the organism,” Blaby said.
One of the main impacts is that cells can no longer adapt to low levels of zinc.
“Cells have evolved so that when zinc levels get too low, a cluster of genes kick in to respond to that change in circumstances. But when you get rid of ZNG1, a lot of those genes stay turned off,” said said Blaby.
“We are now building on this fundamental work done in the fast-growing yeast model organism to understand how these proteins and their functions are conserved in bioenergetic cultures,” Blaby said. “This work sheds light on a previously unknown strategy that plants use to thrive when zinc is limited in the soil. Understanding these strategies can help us design ways to optimize crop productivity and achieve environmentally sustainable bioenergy.”
Pasquini added: “The ability for plants to gain resilience in low zinc soils also means that we would be able to exploit non-arable land for growing bioenergy crops, leaving dedicated fertile soils for other agricultural purposes. Triggering plant cells to produce more ZNG1 would likely allow superior growth on marginal zinc-depleted land.”