First Chaperone Family to Load Zinc on Client Metalloproteins Identified

Zinc deficiency, linked to immune defects, neurological disorders, and cancers, is the top fifth risk factor for death in developing countries and affects nearly 50% of the global population. This micronutrient’s role as a structural or catalytic cofactor is indispensable for a tenth of all proteins in cells. Yet, cellular response to zinc deficiency remains difficult to parse out because it is unclear how zinc is included in proteins.

A study led by scientists at Vanderbilt University has now identified the first family of evolutionarily conserved proteins to chaperone zinc to a client protein. The researchers show zinc metallochaperones that they named ZNG1 (Zn-regulated GTPase metalloprotein activator), interact with a client protein called METAP1 (Zn metalloprotease methionine aminopeptidase 1) to include zinc in the client protein’s native conformation. METAP1 removes the first amino acid from about half of all newborn proteins, and plays a major role in protein stability, maturation and localization.

“There’s been a huge gap in the field of metal biology, where we have been unable to identify metallochaperones. It’s remarkable because so many proteins require metal cofactors,” said Eric Skaar, PhD, a professor of pathology, microbiology and immunology at Vanderbilt University and co-corresponding senior author of the paper.

The study was published on May 17, 2022, in an article titled “Zn regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis” in the journal Cell. The findings shed light on the public health issue of zinc deficiency and open new avenues of investigation.

“This study identifies the first zinc metallochaperone, as well as a client protein (METAP1) that zinc is supplied to. The metallochaperone and the interaction with its client is conserved from yeast to humans,” said co-first authors of the study, Caitlin Murdoch, PhD, and Andy Weiss, PhD, both postdoctoral fellows in Skaar’s lab.

Using biochemical, structural, genetic, and pharmacological assays in models including zebrafish and mice, the investigators demonstrate a critical role for ZNG1 proteins in regulating zinc homeostasis in cells.

“This is the first identified protein that puts zinc into other proteins,” said Skaar. “We think it may be one of the most important regulatory strategies by which humans cope with severe zinc starvation, which is one of the most important public health issues in the world.”

In addition to yeast-two-hybrid screens where the team used human, mouse and zebrafish homologs of ZNG1 as “bait” to identify the client, METAP1, Skaar’s team collaborated with co-corresponding senior author David Giedroc, PhD, and his team at Indiana University on biochemical studies to characterize the interaction of ZNG1 and METAP1 that results in the client protein’s activation.

“In this study, novel genetic reagents were made using CRISPR mutagenesis in zebrafish and mice. In collaboration with Dr. David Giedroc and his research team, we utilized a biochemical assay to measure the release of free methionine from commercially synthesized peptides, allowing us to assess METAP1 activity in vitro. Using this assay, we were able to demonstrate that ZNG1 promotes METAP1 activity in a GTP-hydrolysis and zinc-dependent manner,” said Murdoch and Weiss.

When both alleles of Zng1 are lost in either zebrafish or mice they become sensitive to dietary zinc starvation and show signs of

mitochondrial malfunction and decreased cellular proliferation. These observations in animal models match growth defects observed in humans with zinc deficiency.

“We think that when the body is starved for zinc, ZNG1 ensures that zinc gets delivered to the most important zinc-containing proteins,” Skaar said. “This opens up an exciting new area of biology, where we have these regulatory factors controlling a number of different physiological processes through metal insertion.”

Ongoing experiments in the Skaar lab continue to search for other ZNG1 client proteins to understand how ZNG1-mediated zinc transfer affects client-proteins and overall cellular function. The team is also trying to identify other metallochaperones and accessory factors that help transfer zinc onto client proteins.

The study was funded by the National Institutes of Health (NIH) and the Ernest W. Goodpasture Chair in Pathology at Vanderbilt University.