AGEs form when carbohydrates in the body become stably bound to proteins they encounter. The process is similar to the caramelization of meat, or the formation of rust on iron. The result; increased risk of chronic disease in older people, along with the visual changes associated with aging - greying hair, wrinkles etc. Since there are no drugs to reverse the formation of AGEs, they have long been viewed as an inevitable and irreversible consequence of aging.  The paper, published in Nature Communications, is a promising first step in challenging that paradigm. 

Reversing the Irreversible

Modern machine-learning technologies have allowed the development of new enzymes capable of going where no natural enzyme has gone before. The team used AlphaFold, machine-learning software, to predict the 3D structure of similar natural enzymes that, with modifications, might bind to the bulky AGE molecule and remove the carbohydrate portion from the protein. After scanning 4500 known enzymes, the team identified glycine oxidase (GO), involved in amino acid metabolism in the bacterium Bacillus subtilis, as a suitable candidate. The team focused on reengineering the GO enzyme to recognize a new target, CML - carboxymethyl-lysine, a well studied AGE found abundantly in older humans.

Having chosen a starting enzyme, the team turned to directed evolution, an accelerated mimic of natural selection, to create the new enzyme. To do this, they cloned over 500 million GO variants and selected individuals best able to use CML as an energy supply for bacterial growth. CML is usually a poor growth medium for bacteria, but if they have an enzyme that can split CML into its component parts, the nutrient content in the petri dish is boosted and the bacteria rapidly multiply.  By isolating clones that grew the quickest, the team had developed a powerful selection mechanism for identifying their new enzyme. 

After each round of selection, the best candidates were subjected to further genetic  randomisations to better optimize the CML cleaving activity. Five rounds of directed evolution produced several enzyme variants with the desired activity. Genetic analysis showed that the most effective protein, named CMLase, differed from the original GO enzyme by fifteen amino acid changes and an additional two amino acid deletions. 

Into Human Tissue

With the test tube experiments showing promise, the team turned to testing CMLase in human tissue from elderly donors. They reported that CML levels dropped by 78% in lens proteins of the eye, and by 70% in coronary arteries. CML levels in skin collagen, the cause of wrinkles in older people, dropped 55% to levels typically seen in thirty year olds. All told, CMLase removed between 52% and 97% of carbohydrate residues from “older” proteins.

The authors acknowledged several challenges that need to be addressed before the CMLase can move toward testing in humans. These include …

• investigating the enzymes ability to penetrate deeper into human tissue.

• showing that the functioning of other proteins in the body is not impaired by CMLase activity. 

• addressing the immunogenicity risk of a protein derived from a bacterium. 

Several dozen AGEs have been described in the literature and the authors are hopeful that this approach could be extended beyond CML to other AGE targets.

The team used shotgun metagenomics to sequence all DNA found in the coprolite samples. Arctic ground squirrels are not fussy eaters, happily foraging for grasses, seeds, insects, and frequently scavenging from carrion. The rodents deposit their droppings in discrete middens, which first freeze and eventually fossilize, allowing for the preservation of fecal DNA for far longer than in conventional environments. 

Study Findings

• Thirteen coprolite samples provided an enriched source of ancient environmental DNA - the quality of which often exceeded samples from bone or sediment of similar age.

• Researchers identified 200 distinct plant clades; older coprolites contained grass, plantain, sagebrush and sedge, whilst more recent samples included boreal trees like spruce, birch and alder. 

• All 13 samples contained DNA from large megafauna, including woolly mammoth, steppe bison, Yukon horse, and possibly the extinct American cheetah.

• The study described 404 bacterial and archaea genomes not present in surrounding soil controls, suggesting they could have been part of the squirrel's gut microbiome. 

• The team sequenced 18 mitochondrial genomes, including the ground squirrel, snow hare, bison, and Yukon horse. They will present their woolly mammoth data in a follow-up paper. 

• The modern Arctic ground squirrel U.parryii replaced the Pleistocene species around 420 000 years ago. The ancient species is more closely related to the modern Eurasian ground squirrel U.undulatus. 

The Bottom Line 

This study establishes permafrost-preserved coprolites as a high-quality source of ancient environmental DNA, providing a clue to a changing ecological environment over time. With hundreds of ancient middens being identified in the Yukon, more samples should yield a better view of how ground squirrels, the food they ate and the microbes in their guts adapted to an evolving world over nearly 700 000 years.