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No stress with pArg: Mechanisms of a distinct phospho-mark to coordinate stress response and protein quality control

Periodic Reporting for period 3 - pArg_deg_signal (No stress with pArg: Mechanisms of a distinct phospho-mark to coordinate stress response and protein quality control)

Reporting period: 2019-10-01 to 2021-03-31

Cellular proteins are prone to misfolding and aggregation, particularly under harsh environmental conditions. To counteract this danger, all organisms from bacteria to humans evolved sophisticated “protein quality control” networks. The mechanisms employed in them tend to represent some of the most exciting biochemistry occurring in living cells. In Gram-positive bacteria, the key factors combating protein damage include a specialized protein kinase phosphorylating arginine residues (McsB), the central housekeeping protease (ClpP), as well as a AAA chaperone targeting aggregated proteins (ClpC). The respective quality-control system, which is organized around a distinct protein phospho mark (phosphoarginine, pArg), represents a fascinating model to investigate novel principles of dealing with proteotoxic stress. Using an integrative approach, we started to address the precise role of protein arginine phosphorylation in the bacterial stress response. To this end, we analyzed how the pArg modification influences the stability and function of targeted proteins in vitro and in vivo. We are particularly interested in the possibility of pArg serving as a bacterial, ubiquitin-like degradation signal. Moreover, we addressed the mechanism and regulation of the protein arginine kinase McsB. This analysis will uncover the specificity of the pArg-tagging system. Additionally, these studies will reveal enzymatic innovations connected with the pArg chemistry that, due to the dependence of bacterial virulence on McsB, are of pharmaceutical interest. To address the further processing of pArg-modified proteins, we started to perform an in-depth structural characterization of ClpC and related AAA disaggregases. A better understanding of the mechanism and regulation of these HSP100 molecular machines is also highly relevant to uncover general principles of how cells deal with toxic protein aggregates and, in parallel, keep control over their potentially dangerous shredding devices.

The proposed experimental program is of high medical interest, particularly in terms of antibiotics development. New avenues in anti-bacterial therapy are badly needed as bacterial infections are still a leading cause of death worldwide, while the resistance to antibiotics is on the rise. The most worrying gram-positive pathogens including Staphylococcus aureus are characterized by their ability to deal with stress situations imposed by the host defense system. Components of the stress-response regulon such as McsB, ClpC and ClpP are essential for this capability, with ClpP being the target of the powerful acyldepsipeptide antibiotic. Thus, the mechanistic and structural peculiarities of McsB and ClpC that will be revealed by our work, as well as the expected insights into the bacterial version of the ubiquitin-proteasome system, open exciting possibilities in combating some of the most dangerous bacterial pathogens.
Cells never forget to take out the trash. It has long been known that cells tag proteins for degradation by labelling them with ubiquitin, a signal described as “the molecular kiss of death”. Now, our team identified an analogous system in gram-positive bacteria, where the role of a degradation tag is fulfilled by a little known post-translational modification: arginine phosphorylation. Although research into protein degradation has lagged behind research into protein synthesis, it is now well appreciated that, when it comes to proteins, death is just as important as birth – and as tightly regulated. Each protein is removed at the right moment and special care is taken to eliminate those that are either damaged or perform functions no longer necessary. The degradation process, which consists of chopping the protein substrate into small fragments, takes place inside compartmentalized, ATP-powered proteases – specialized molecular machines often compared to paper shredders. If, as in the eukaryotic ubiquitin-based system, the access to the protease depends on a specific tag that must be carried by the substrate, then the important decision who and when should be eliminated boils down to the timely and selective attachment of tags. The mechanism is elegant, yet scientists working with bacteria have been puzzled to find it. Our work changed the picture, when studying how bacteria cope with adverse conditions. As indicated before, McsB has the unique ability to phosphorylate other proteins on arginine residues. Although the modification was observed to be regulated in response to environmental conditions, the function has remained elusive. We could show that McsB functions as a “labeler”, tagging many diverse proteins with a “phospho-kiss of death”. Moreover, we could show that proteins marked with pArg are directly captured by the ClpC:ClpP (ClpCP) protease and shredded to pieces. As the discovered system is widely conserved across gram-positive bacteria – a large class that includes the notorious human pathogen Staphylococcus aureus – the study explains previous reports that identified McsB and ClpC as virulence factors. To infect, bacteria must overcome stresses encountered within the host and this in turn relies on efficient degradation of stress-damaged proteins. The present discovery therefore opens a new perspective on how bacteria-mediated disease could be combated. Apparently, an Achilles Heel mechanism has been identified. The next step is to block it.

One possible site of intervention is the “death-marking” kinase McsB. Fitting to the Latin phrase "Quis custodiet ipsos custodes?" ("Who watches the watchmen?"), most cellular processes are controlled by proteins, but what controls proteins themselves? This task is in part performed by protein kinases, which attach a small chemical unit called phosphoryl group to other proteins. Most protein kinases, promoting this “phosphorylation”, are similar to each other and stem from the same ancestral gene. Our group, however, unraveled the atomic structure of the protein kinase McsB that differs from all other protein kinases in structure and function. As such and owing to the critical role of McsB for bacterial infection, the reported findings may aid developing novel antibiotics. What is special about McsB is that, whereas other protein kinases modify the amino acids serine, threonine, and tyrosine, McsB acts specifically on the amino acid arginine, changing it to pArg. This seemingly small difference results in completely novel signaling pathways connected with a phospho mark. Importantly, pArg residues can be recognized by specific protein domains, three of which are located on ClpC, McsB and CtsR, respectively. The various pArg-reader domains identified by us point to a remarkably complex signaling system – which challenges simpler views of bacterial protein phosphorylation that was formerly believed to be based on isolated kinase modules rather than on phospho-protein networking.
Despite differing in size and complexity, the bacterial pArg modifier shares several features with the eukaryotic poly-Ub degradation tag: (i) Both modifiers are post-translationally attached to their substrates, allowing for dynamic regulation of degradation that is not available to mechanisms relying on sequence-encoded degrons. (ii) The pArg mark is recognized by highly specific receptor sites on the NTD of ClpC, as is ubiquitin by special receptors of the 19S Regulatory Particle. (iii) The pArg tag is attached reversibly, as the activity of the McsB kinase is counteracted by the pArg-specific phosphatase YwlE. Similarly, enzymes attaching poly-Ub chains are opposed by de-ubiquitinases. Considering the functional similarity of pArg to poly-Ub and ClpCP to the 26S-proteasome, the pArg-ClpCP system can be considered as a simple bacterial version of the eukaryotic ubiquitin-proteasome system. In fact, the pArg-ClpCP system is the first bacterial degradation pathway that allows the targeted elimination of aberrant proteins during proteotoxic stress conditions. Following the recent advances in hitchhiking the proteasome system to develop next-generation pharmaceutical agents, the so-called PROTACs (proteolysis targeting chimeras), we plan to transfer this knowledge to the prokaryotic world by designing “bac-PROTAC” compounds inducing the degradation of a protein-of-interest (POI) by the ClpCP system. While an approach more directly analogous to PROTAC would consist in targeting a POI to the McsB kinase (which is the counterpart of the E3 ligases in the bacterial system), the simplicity of the pArg degradation tag suggests that it might be possible to instead pharmaceutically target proteins to the ClpCP degradation machinery itself. Having such bac-PROTAC compounds available would be highly interesting for basic and medical research, as it would - in principle - allow to target any essential POI in Gram-positive bacteria, thus overcoming the major obstacle in identifying novel classes of antibiotics. In case our approach will work, it may revolutionize the development of antibiotics.