pArg deg signal

pArg - a degradation mark for damaged proteins

The ubiquitin-proteasome pathway allowing for regulated proteolysis.

Energy-dependent proteases are essential for all living organisms to carry out protein quality control and degrade short-lived regulatory proteins. Despite employing distinct components, the eukaryotic and prokaryotic protease machines evolved a common bi-partite architecture. They are composed of a proteolytic core (20S proteasome, ClpP) for protein shredding and an associated AAA complex (19S regulatory particle, various Clp ATPases) that mediates substrate recognition, unfolding, and translocation. In contrast to eukaryotes, which universally employ polyubiquitin chains for marking client proteins, a general post-translational modification regulating proteolysis in bacteria was not known. Our study characterized such a tagging system. It is the little known modification phospho-arginine (pArg) serving as degradation tag thus directly linking protein phosphorylation and protein degradation.

Our findings: 2016 pArg, a phospho-kiss of death

2016 Protein arginine phosphorylation marks protein for degradation by the ClpC:ClpP protease

Comparison of ubiquitin- and pArg-controlled protein degradation.

Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases had not been known. In our 2016 study we addressed the targeting mechanism of the ClpC:ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP trapping mutant showed that proteins phosphorylated on arginine residues are selectively targeted to ClpC:ClpP. In vitro reconstitution experiments revealed that the McsB-mediated arginine phosphorylation is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the N-terminal domain of the ClpC ATPase as resolved at high resolution in a co-crystal structure. Together, our data demonstrated that pArg functions as a bona fide degradation tag for the ClpC:ClpP protease. Several lines of evidence suggest that this system, widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin-proteasome system: (i) Both pArg and polyubiquitin are post-translationally attached to substrates, allowing for dynamic regulation of degradation of a particular substrate. (ii) The pArg mark is recognized by highly specific receptor sites on the NTD of ClpC, as is ubiquitin by special receptor proteins of the 19S regulatory particle. These interactions position substrate proteins properly for the translocation into the proteolytic chamber. (iii) Owing to charge inversion, the phosphorylation of arginine residues is predicted to destabilize the native structure of its substrates, priming them for subsequent catalyzed unfolding. Similarly, the polyubiquitin tag is capable of influencing the structure and stability of marked proteins. (iv) Both pArg and ubiquitin tags can be reversibly attached to substrate proteins, thus allowing for regulation by counteracting arginine phosphatases and de-ubiquitinases, respectively.