Antibiotic Acyldepsipeptides Stimulate the Streptomyces Clp-ATPase/ClpP Complex for Accelerated Proteolysis.

Clp proteases consist of a proteolytic, tetradecameric ClpP core and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs, and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the housekeeping Clp protease in Streptomyces, consisting of a heterotetradecameric core built of ClpP1, ClpP2, and the cognate Clp-ATPases ClpX, ClpC1, or ClpC2, all interacting with ClpP2 only. Antibiotic acyldepsipeptides (ADEP) dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP binds Streptomyces ClpP1, but not ClpP2, thereby not only triggering the degradation of nonnative protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease. IMPORTANCE Clp proteases are antibiotic and anticancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multiresistant and persistent bacterial infections. The molecular mechanism of ADEP is multilayered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides of Streptomyces ClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the major Streptomyces Clp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1, or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP in Streptomyces.

Contribution of the Clp Protease to Bacterial Survival and Mitochondrial Homoeostasis.

Fast adaptation to environmental changes ensures bacterial survival, and proteolysis represents a key cellular process in adaptation. The Clp protease system is a multi-component machinery responsible for protein homoeostasis, protein quality control, and targeted proteolysis of transcriptional regulators in prokaryotic cells and prokaryote-derived organelles of eukaryotic cells. A functional Clp protease complex consists of the tetradecameric proteolytic core ClpP and a hexameric ATP-consuming Clp-ATPase, several of which can associate with the same proteolytic core. Clp-ATPases confer substrate specificity by recognising specific degradation tags, and further selectivity is conferred by adaptor proteins, together allowing for a fine-tuned degradation process embedded in elaborate regulatory networks. This review focuses on the contribution of the Clp protease system to prokaryotic survival and summarises the current state of knowledge for exemplary bacteria in an increasing degree of interaction with eukaryotic cells. Starting from free-living bacteria as exemplified by a non-pathogenic and a pathogenic member of the Firmicutes, i.e., Bacillus subtilis and Staphylococcus aureus, respectively, we turn our attention to facultative and obligate intracellular bacterial pathogens, i.e., Mycobacterium tuberculosis, Listeria monocytogenes, and Chlamydia trachomatis, and conclude with mitochondria. Under stress conditions, the Clp protease system exerts its pivotal role in the degradation of damaged proteins and controls the timing and extent of the heat-shock response by regulatory proteolysis. Key regulators of developmental programmes like natural competence, motility, and sporulation are also under Clp proteolytic control. In many pathogenic species, the Clp system is required for the expression of virulence factors and essential for colonising the host. In accordance with its evolutionary origin, the human mitochondrial Clp protease strongly resembles its bacterial counterparts, taking a central role in protein quality control and homoeostasis, energy metabolism, and apoptosis in eukaryotic cells, and several cancer cell types depend on it for proliferation.

α-Amino Diphenyl Phosphonates as Novel Inhibitors of Escherichia coli ClpP Protease.

Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition, and a chemical exploration around the hit compounds was conducted. Altogether, 14 new potent inhibitors of E. coli ClpP were identified. Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.