A previously unknown way in which cells mark proteins for destruction has been found in bacteria — phosphorylation of the amino acid arginine targets proteins for degradation by protease enzymes. See Article p.48
Bacteria are expert at adapting to changing lifestyles. Dealing with damaged or misfolded proteins is a key challenge during adaptation to stressful conditions such as high temperature. In such situations, chaperone proteins restore unfolded proteins to their functional, folded states and protease enzymes degrade damaged proteins1. But how are proteins that have been damaged beyond redemption recognized and sent for degradation? On page 48, Trentini et al.2 show that a surprisingly simple degradation tag — phosphorylation of the amino acid arginine — has a central role in the handling of heat-damaged proteins in the bacterium Bacillus subtilis.
Protein degradation in eukaryotes and prokaryotes (organisms with or without a cellular nucleus, respectively) is carried out by protease-enzyme complexes3, which require the nucleotide ATP for their activity. Access to the protease must be controlled to ensure that only damaged or unwanted proteins are degraded. The protease active site is in a chamber that is accessible only to proteins that have been unfolded and moved there by the ATPase-enzyme portion of the protease-enzyme complex (Fig. 1). Thus, binding and unfolding of proteins by the ATPase governs degradation selectivity.
The selection process for protein degradation often depends on the attachment of molecular 'tags' to the protein to be degraded. For example, eukaryotes might attach the polypeptide ubiquitin to proteins targeted for degradation by proteases4. Tagging is also found in prokaryotic archaea and mycobacteria5, which use a non-ubiquitin polypeptide tag (Fig. 1).
However, most bacteria select proteins for degradation using other mechanisms than tagging. One such mechanism is the exposure of an amino-acid motif known as a degron6, an intrinsic part of a protein. Changes in binding partners of proteins in multi-protein complexes can hide or reveal degrons. Proteins can also be targeted for degradation if they are bound and specifically delivered to the protease by an adaptor protein, which provides a supporting role in the degradation process, rather than a direct enzymatic one. Modifications or changes in the availability of adaptors and protein-binding partners can determine whether or not a specific protein is degraded at a particular time.
Although the mechanisms that target proteins for destruction are well understood, how damaged proteins are recognized is less clear. When proteins are misfolded as a result of heat or oxidation, they might expose an internal degron that is normally shielded1,7, resulting in the protein's destruction. However, little is known about other possible mechanisms, and Trentini and colleagues' study provides additional mechanistic insights.
McsB, a B. subtilis protein that is involved in the heat-shock response8, was identified as a protein-kinase enzyme that phosphorylates arginine9,10. McsB-kinase phosphorylation promotes degradation of the protein CtsR (refs 11,12), which represses expression of heat-shock genes. A previous study10 had found that more than 100 proteins underwent McsB-dependent phosphorylation, and Trentini and colleagues decided to test whether arginine phosphorylation might cause protein degradation.
The authors investigated degradation by the ClpCP-protease complex in B. subtilis. This complex consists of the ClpP protease and ClpC, which is an ATPase and unfoldase enzyme. Under heat-shock conditions, Trentini et al. used an inactive version of ClpP to trap proteins undergoing degradation, and, by means of proteomic techniques, identified 20 different trapped proteins that carried a phosphorylated arginine tag; they estimate that 25% of ClpP substrates are marked by arginine phosphorylation.
To demonstrate the role of arginine phosphorylation in degradation, the authors turned to in vitro biochemical studies involving purified proteins. Previous investigations of protein degradation by ClpCP found that the process involved adaptor proteins, and McsB was thought to be an adaptor protein12. Trentini et al. found that casein (an unfolded protein commonly used to study protein degradation) could be degraded by ClpCP, but that degradation required either the ClpC adaptor protein MecA or McsB. However, phosphorylated casein was degraded by ClpCP without the action of an adaptor protein, suggesting that the protease recognizes the phosphorylation tag directly. Consistent with a model in which the ATPase contains a binding site for phosphorylated arginine, the authors found that a free molecule of phosphorylated arginine bound to ClpC.
The amino-terminal domain of most members of the bacterial AAA family of unfoldases is involved in binding adaptor proteins or proteins to be degraded3. Trentini et al. obtained an X-ray crystal structure of the N-terminal domain of ClpC in the presence of a phosphorylated arginine. They identified a binding site for phosphorylated arginine in ClpC that could simultaneously accommodate the positively charged arginine and the negatively charged phosphoryl group of the phosphate.
The binding site in ClpC for phosphorylated arginine partially overlaps with the binding site for the MecA adaptor. Trentini and colleagues mutated two amino acids in the N terminus of ClpC, thereby abolishing its binding of phosphorylated arginine, but still enabling MecA-mediated protein binding. This allowed the authors to test directly whether protease degradation of proteins that have phosphorylated arginines is a necessary part of the heat-shock response. They found that cells lacking ClpC or containing a mutant form of ClpC that does not bind to phosphorylated arginine had a similarly reduced ability to recover from heat-shock stress. This suggests that, during heat shock, ClpC is required to recognize and degrade proteins marked by arginine phosphorylation.
Integrating these findings into our understanding of heat-shock regulation in bacteria will require more work. It is not clear what changes occur in proteins or McsB at high temperatures that lead to increased arginine phosphorylation. However, McsB and the phosphorylated-arginine binding site in ClpC are evolutionarily conserved across many members of a group known as Gram-positive bacteria, so perhaps arginine phosphorylation might be a widely used mechanism to tag proteins for destruction. Intriguingly, McsB is necessary for Staphylococcus aureus to act as a pathogen13.
Future experiments should try to unravel the relative importance of the degradation of misfolded proteins compared with the degradation of regulatory proteins during stress survival. Perhaps the machinery for adding and recognizing phosphorylated arginine on proteins will provide new drug-development targets, for example in S. aureus.
More broadly, the many biological roles of phosphorylation have now been expanded even further. The ability of this modification to mark proteins for degradation, coupled with the probable perturbation of protein function by the addition of a negatively charged phosphate group to a positively charged arginine, suggests that Trentini and colleagues' work might be relevant far beyond the B. subtilis bacteria they have studied.Footnote 1
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