https://www.science.org/content/blog-post/reverse-proteolysis-will-you-look

Here’s a phenomenon - yet another one - that never crossed my mind before. It’s long been known that enzymes that catalyze proteolysis (cleavage of peptide bonds) can, under certain circumstances, catalyze the reverse reaction of peptide bond formation. Folks who have had to think about chemical kinetics will immediately realize that those conditions would include high concentrations of the two cleavage products and low concentrations of the longer protein substrate, an example of Le Chatlier’s principle in action. It’s also an example of the principle of Microscopic Reversibility in action, too: the chemical steps are the same whether you run things forwards or backwards. That doesn’t mean those steps are always thermodynamically feasible, of course - the energies involved (with both enthalpic and entropic contributions) might be too great a barrier to run backwards very easily, as in unburning a piece of wood back from a cloud of soot and hot gases. Fire is not a good example of an equilibrium process, but peptide bond breakage and formation is a lot closer to balancing on a knife edge than combustion is.

This recent preprint suggests, though, that this “reverse proteolysis” is happening under physiological conditions, particularly with cysteine-based cathepsin enzymes. And it’s not just re-formation of the proteins that have just been cleaved (although that must be happening, too). No, you get mix-and-match combinations of various proteins to generate species that were certainly never coded for in the genome. And on top of that, you can even spot chimeras between human proteins and bacterial or viral ones (!)

Now, some species of this sort have been reported before (in reports going back to at least 2004) but this new work suggests that it’s a much more common process than anyone realized, one with implications for immunity and perhaps other cellular processes as well. Recall that antigen proteins are displayed to the immune system via the major histocompatibility complex, and that these antigens are cleaved from larger proteins via degradation. Displaying weirdo newly assembled protein sequences from this chemical splicing route could cause some real effects downstream. This could, for example, be one of the links between prior infections and later autoimmune disease, through those human/pathogen hybrid proteins.

The authors here shore up that connection by showing that auto-antigenic peptides implicated in Type I diabetes can be produced by cathepsins running in reverse, and that proteins that have been modified by citrullination (on arginine residues) seem to undergo the process more readily. That sort of Arg modification is already known to be over-represented in autoimmune antigens. In addition, the cathepsin enzyme subtypes that are most dominant in immune tissues (such as inside macrophages) seem to be the best at producing such splicing hybrids. These reverse reactions are also more prevalent at closer to neutral pH, which suggests that lysosomal dysfunction (where cathepsins and other enzymes normally work in an acidic environment) might be a source of increased neo-peptides.

Overall, it seems that we’re going to have to learn to deal with these species, and to study them in the context of both normal conditions and in infectious disease. Acute viral infections might well be producing waves of human/viral protein hybrid species, and we can’t expect them all to be silent! 

https://www.science.org/content/blog-post/reverse-proteolysis-will-you-look