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Austrian Team Discovers Bacterial Protein Degradation System

NEW YORK (GenomeWeb) -- Scientists at Austria's Research Institute of Molecular Pathology have identified arginine phosphorylation as a tag for protein degradation in bacteria.

The finding, described in a paper published this week in Nature, sheds new light on the regulation of proteasomal degradation in bacteria, presenting arginine phosphorylation as a general mark for degradation by bacterial Clp proteases, much like the ubiquitin-proteasome system in eukaryotes.

As the authors noted, protein degradation is a key cellular process, for example for the removal of damaged proteins, but researchers had not previously known what modifications marked bacterial proteins for degradation by Clp proteases.

Due to the positioning of many arginine residues at key points within proteins, arginine phosphorylation is generally thought to play important roles in protein stability. Additionally, McsB, the kinase responsible for arginine phosphorylation, is co-regulated with the bacterial protease ClpP, which, the study authors wrote, led them to hypothesize that arginine phosphorylation could be key to bacterial protein degradation.

To get at this question, the researchers performed a series of experiments in Bacillus subtilis, looking at interactions between Clp protease complexes and arginine- phosphorylated proteins. In the first, they used an inactive variant of the ClpP protease that would capture but not degrade target proteins to pull down proteins from heat-shocked bacteria, which they then analyzed via mass spec analysis on a Thermo Fisher Scientific LTQ Orbitrap Velos Pro ETD instrument.

While  arginine phosphorylation is difficult to detect, they identified 13 proteins with phosphorylated arginines among the 233 total proteins pulled down by the ClpP variant.

They then repeated this pulldown experiment in two different B. subtilis types, one wildtype and the other with the ClpC protein, a constituent of the Clp complex, knocked out. In that experiment, they found 14 phosphorylated arginine proteins in the wildtype but only one in the ClpC knock-out, indicating the relationship between Clp and phosphorylated arginine.

To establish the role of arginine phosphorylation in stimulating Clp-based degradation, the researchers performed in vitro experiments using the protein β-casein. They first demonstrated that McsB, which phosphorylates arginine, is required for degradation of the protein. They also established that addition of free phosphoarginine amino acid competed with phosphoarginine residues on the β-casein protein and slowed the rate of Clp-based degradation.

The phosphoarginine mark is the first known "general post-translational modification regulating proteolysis in bacteria," the researchers wrote. "Based on the described functional similarities, the discovered pArg-ClpCP system seems to represent a simple bacterial version of the eukaryotic ubiquitin-proteasome system."