University of Cincinnati Cancer Center researchers looked billions of years into the past to learn more about the potential future of precision medicine.
Led by first author Bibek R. Karki and senior author Tom Cunningham, new research published July 8 in the journal Nature Communications traced the evolutionary origins of the PRPS enzyme complex and learned more about how this complex functions and influences cellular biochemistry.
Study background
The researchers focused on one of nature's most important and evolutionarily conserved metabolic enzymes, phosphoribosyl pyrophosphate synthetase (PRPS). Most mammalian cells produce four separate PRPS-related proteins: PRPS1, PRPS2, and PRPS-associated proteins 1 and 2 (AP1 and AP2).
Karki said the vast majority of research over the last 30 years has focused on either PRPS1 or PRPS2 as individual standalone enzymes, primarily because they can function that way in isolation, in turn facilitating biochemical and structural studies. Because AP1 and AP2 lack the features necessary to be a catalyst, meaning they cannot independently carry out chemical reactions, their roles remain poorly understood.
"In a way, AP1 and AP2 have been like 'zombie' enzymes," said Karki, a doctoral cancer biology student in Cunningham's lab. "They exist, but they don't seem to have catalytic properties."
The field has developed no conventional wisdom as to what role AP1 and AP2 seem to be playing in PRPS enzyme function, and the team was curious why the genes encoding these supposedly "dead" enzymes would be kept around over vast evolutionary timescales.
"If a gene is evolutionarily conserved, it means it's repeatedly favored by nature," Karki explained. "That indicates it must be doing something important; otherwise, nature would have let it go."
Tracing evolutionary origins
To learn more about AP1 and AP2's potential roles within cells, the team traced the evolutionary history of each PRPS-encoding gene found in mammal cells today.
PRPS1 is the ancestral version of the enzyme that made its way into eukaryotes by virtue of a bacterial donor billions of years ago. The first PRPS paralog, or gene copy, to emerge was AP2, which appeared in the lineage containing animals and fungi over 1 billion years ago. Millions of years later, AP1 and PRPS2 arose from AP2 and PRPS1 via a genome duplication event that helped give rise to jawed vertebrates.
"These paralogs are preserved throughout the various jawed vertebrate species: sharks, bony fishes, amphibians, reptiles, up through mammals. You can imagine how important those proteins might be," Karki said. "All four of these proteins have been kept intact over hundreds of millions of years of evolution, each possessing specific emergent properties."
In addition to observing their presence in the lineage containing animals and fungi, researchers noted that eukaryotic organisms as different from one another as plants, amoebozoa and other protists show a similar tendency to duplicate and retain copies of PRPS-encoding genes, highlighting just how critical these events may be for metabolic adaptability of more advanced lifeforms on this planet.
"Harboring multiple PRPS-encoding genes is nearly universal across eukaryotes and rarely occurs in bacteria, so that's a really interesting feature that distinguishes eukaryotes from prokaryotes," said Cunningham, PhD, a Cancer Center researcher and associate professor in the Department of Cancer Biology in UC's College of Medicine. "The discovery that duplicated versions of ancestral PRPS enzymes had lost key residues important for facilitating chemical catalysis in numerous eukaryotic lineages lent credence to the idea that the emergent properties of these PRPS-associated proteins were worthy of further investigation."
Defining enzyme functions
With evolution suggesting all four PRPS copies had an important role in cells, the team used CRISPR gene editing technology to knock out every different combination of the enzymes in mammalian cell lines to learn more about each of their functions.
"In all of the combinations we tried, the cells' overall fitness declined," Karki said. "This effect was most pronounced in cells that only had PRPS1. They simply weren't equipped to meet cellular demands, as they grew more slowly, produced fewer nucleotides and showed defects with mitochondrial functions."
The researchers used a filtering method called size-exclusion chromatography that found the four enzymes form a large structure, or complex, within cells that is approximately 10 times larger than the structures created when one of the enzymes is deleted from the cells.
"What was really striking was how the larger complex disassembled without AP1 — or to a degree, AP2," Karki said. "So in a way, AP1 and AP2 act as molecular scaffolds, helping build and stabilize the larger enzyme complex."
"We've obtained the first blueprint of how this enzyme is constructed," added Cunningham. "Not at the granular level, but the very basics of what interacts with what and what is driving the assembly of the complex. We now understand those fundamental details because of Bibek's work."
When cells were engineered to only express PRPS1, they formed large, aberrant assemblies that functioned poorly compared to the four-enzyme complex found in normal cells. Karki noted this means many previous research projects that isolate PRPS1 in vitro likely produced results that do not mimic how PRPS1 operates in normal cell environments.
Essentially, researchers in the past have been looking at one-quarter of a full picture. Looking at the full interaction of all four proteins will be vital for future research in the field.
"Studying these proteins in isolation only gives you part of the story," Karki said. "Going forward, it's clear we need to study these proteins together because the way the components coordinate with one another will determine the enzyme's active and regulatory properties."
Research application
Karki said several human diseases are linked to deregulation of PRPS enzymes, with many of them still not very well understood. Looking at how the entire PRPS complex is affected by mutations could open new doors of understanding and potential therapeutic opportunities.
Cunningham said because the PRPS enzymes are the only path for sugars to be converted to nucleotides to form the building blocks of RNA and DNA, there is great potential for developing personalized treatments aimed at enhancing or limiting PRPS enzyme activity.
"In the context of cancer, you may want to restrict PRPS activity so you can suppress nucleotide production and slow tumor growth," he said. "You may want to enhance activity in the case of a nucleotide deficiency syndrome. Uncovering the basics of the complex's architecture gives us a foothold for taking those next steps that translate our knowledge into new diagnostics and therapeutics that can improve patient health."
Other study coauthors include Austin C. Macmillan, Sara Vicente-Muñoz, Kenneth D. Greis and Lindsey E. Romick. This work received support by NIH grants (R01CA230904 and R35GM133561) to Cunningham and S10OD026717 to Greis for the mass spectrometer used in the proteomics studies.
Karki and Cunningham have filed patent applications on this work. The remaining authors declare no competing interests.
