If you see a shorter version of a protein, don’t be too quick to dismiss it as a nonfunctional degradation product. It might be a shiny, new proteoform, an alternative version of a larger protein. The proteoform might even be more stable than the full-length protein. Although proteoforms may arise naturally as cells respond to changing conditions, they may also occur accidentally when scientists try, and fail, to completely suppress the expression of a particular gene.
To distinguish between protein detritus and actual proteoforms, it helps to be aware of alternative translation start codons—as is a team of researchers at Vlaams Instituut voor Biotechnologie (VIB), a research institute located in Flanders, Belgium. These researchers, and others, have made use of mass spectrometry and ribosome profiling techniques to find alternative translation start codons in up to 20% of the human protein-coding genes.
In their most recent work, the VIB scientists, led by Professor Petra Van Damme, performed a proteome-wide study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. They also assessed the relative stabilities of full-length proteins and their truncated proteoforms. To do so, they considered the effect on protein turnover of removing the initiator methionine by methionine aminopeptidases.
The results of this work were reported February 18 in the journal Molecular Systems Biology, in an article entitled, “Positional Proteomics Reveals Differences in N-Terminal Proteoform Stability.” Essentially, the work points to a role for alternative translation initiation and co-translational initiator methionine removal, next to alternative splicing, in the overall regulation of proteome homeostasis.
“By combining pulsed SILAC with N-terminal COFRADIC, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine,” wrote the authors. “N-terminally truncated proteoforms were less abundant than canonical proteoforms and often displayed altered stabilities, likely attributed to individual protein characteristics, including intrinsic disorder, but independent of N-terminal amino acid identity or truncation length.”
The removal of the initiator methionine by methionine aminopeptidases, the authors added, generally reduces the stability of processed proteoforms. Also, the authors indicated that susceptibility for N-terminal acetylation did not seem to influence protein turnover rates.
Overall, the findings suggest that the diversity of human proteins may be fundamentally underestimated. The findings may also have important implications for gene editing. “To knock out a gene,” explained Daria Gawron, a study co-author and a Ph.D. student at VIB, “a point mutation in its DNA sequence can be very accurately introduced with modern gene-editing techniques. However, scientists should be aware that by doing so, they might actually induce the formation of a truncated, more stable version of the protein, provoking the exact opposite effect than desired.”
“In the past, researchers who observed shorter versions of certain proteins, quickly shelved them as nonfunctional byproducts of protein degradation,” concluded Professor Van Damme. “Our work shows that these protein variants are generally conserved. Not only are these proteoforms coded for in the genome, they are also tightly regulated and often display altered stability.”
