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Understanding cellular function: are customized proteases the way forward?

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New research published in the journal Proceedings of the National Academy of Sciences has discovered a potential new enzymatic tool that may allow us to map the post-translational modification (PTM) of proteins. This new technique, using protease enzymes, may revolutionize current mass spectrometry technology, which allows the in-depth study of cellular function.

After a protein has been translated, these chemical changes known as PTMs occur. These changes may completely alter a protein’s structure and function, and if unregulated, may lead to the subsequent development of disease.

This new research carried out at the Scripps Research Institute (CA, USA) therefore offers new understanding that may not only allow us to identify and study protein structures, but prevent the onset of disease.

Currently, the identification of such PTMs relies on mass spectrometry, which measures the mass of peptides that have been digested by enzymes. If a significant change of mass is observed, then this may indicate that a PTM has occurred.

The study’s senior author, Brian Paegel, comments on these current techniques used: “We have to observe these protein modifications directly through chemical analysis; we can’t read them out of DNA sequence.”

Trypsin enzymes, which are commonly used to enzymatically break larger proteins into smaller peptides for mass spectrometry, are unable to cleave a protein at its specific modified site.

Due to this non-specificity, the scientists in this study investigated whether a new site-specific protease mutant could be developed as an alternative.

To do this, these researchers used a technique called directed evolution to create a large number of trypsin mutants, each of which were tested for PTM-directed specificity.

It was uncovered during the study that one particular mutant was able to cut proteins at citrulline, which is a specific type of PTM associated with epigenetic and immunological function.

The researchers of this study believe that these findings may lead the way to the discovery of more enzymes that may target specific PTMs after this directed evolution.

Paegel comments on the importance of these findings: “I think we’re on the brink of an explosion of new tools for mass spectrometry”.

In the future, these new approaches for creating customised, PTM-specific enzymes may allow us to develop a wide range of PTM site-mapping strategies, and therefore may completely alter our current understanding of cellular function.

Sources: Trana DT, Cavetta VJ, Danga VQ, Torresa HL, Paegel BM. Evolution of a mass spectrometry-grade protease with PTM-directed specificity. PNAS. 133, 14686–14691 (2017); www.scripps.edu/news/press/2017/20170104paegel.html

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