Highlights d Activation of the mitochondrial ClpP induces p53independent cancer cell lethality d Imipridones are allosteric agonists of ClpP, being tested in human clinical trials d ClpP activation increases proteolysis of mitochondrial proteins d ClpP-mediated mitochondrial proteolysis impairs mitochondrial respiratory function

In prokaryotic cells and eukaryotic organelles, the ClpP protease plays an important role in proteostasis. The disruption of the ClpP function has been shown to influence the infectivity and virulence of a number of bacterial pathogens. More recently, ClpP has been found to be involved in various forms of carcinomas and in Perrault syndrome, which is an inherited condition characterized by hearing loss in males and females and by ovarian abnormalities in females. Hence, targeting ClpP is a potentially viable, attractive option for the treatment of different ailments. Herein, the biochemical and cellular activities of ClpP are discussed along with the mechanisms by which ClpP affects bacterial pathogenesis and various human diseases. In addition, a comprehensive overview is given of the new classes of compounds in development that target ClpP. Many of these compounds are currently primarily aimed at treating bacterial infections. Some of these compounds inhibit ClpP activity, while others activate the protease and lead to its dysregulation. The ClpP activators are remarkable examples of small molecules that inhibit protein-protein interactions but also result in a gain of function.

Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

Highlights1. RavA-ViaA form a chaperone-like complex interacting with respiratory chains. 2.RavA-ViaA are induced under oxygen-limiting conditions. 3.RavA-ViaA interact with the flavin-containing subunit of fumarate reductase. 4.RavA-ViaA modulate the activity of the fumarate reductase complex. AbstractRavA is a MoxR AAA+ protein that functions together with a partner protein that we termed [187 words]

The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms of action. Analogues of the A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class of antimicrobial agents that act through dysregulation of caseinolytic protease (ClpP). Previous studies have shown that ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there are currently few studies examining Gram-negative bacteria. In this study, the synthesis and biological evaluation of 14 novel ADEPs against a variety of pathogenic Gram-negative and Gram-positive organisms is outlined. Optimization of the macrocyclic core residues and N-acyl side chain culminated in the development of 26, which shows potent activity against the Gram-negative species Neisseria meningitidis and Neisseria gonorrheae and improved activity against the Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis in comparison with known analogues. In addition, the co-crystal structure of an ADEP-ClpP complex derived from N. meningitidis was solved.

The human ClpP proteolytic complex (HsClpP) is a serine protease located in the mitochondrial matrix and participates in the maintenance of the mitochondrial proteome among other cellular functions. HsClpP typically forms a multimeric complex with the AAA+ protein unfoldase HsClpX. Notably, compared to that of normal, healthy cells, the expression of HsClpP in many types of solid and nonsolid cancers is found to be upregulated. While the exact role of HsClpP in tumorigenesis is not clear, certain types of cancers are highly dependent on the protease for cell proliferation and metastasis. In light of these observations, recent research has focused on the discovery and characterization of small organic molecules that can target and modulate HsClpP activity. These include compounds that inhibit HsClpP's proteolytic activity via covalent modification of its catalytic Ser residue as well as those that activate and dysregulate HsClpP by displacing HsClpX to negate its regulatory role. Importantly, several of these compounds have been shown to induce HsClpP-dependent apoptotic cell death in a variety of cancerous cells. This review provides an overview of these research efforts and highlights the various types of small molecule modulators of HsClpP activity with respect to their potential use as cancer therapeutics.

The vertebrate proglucagon gene encodes three glucagon-like sequences (glucagon, glucagon-like peptide-1 [GLP-1], and glucagon-like peptide 2 [GLP-2]) that have distinct functions in regulating metabolism in mammals. In contrast, glucagon and GLP-1 have similar physiological actions in fish, that of mammalian glucagon. We have identified sequences similar to receptors for proglucagon-derived peptides from the genomes of two fish (pufferfish and zebrafish), a frog (Xenopus tropicalis), and a bird (chicken). Phylogenetic analysis of the receptor sequences suggested an explanation for the divergent function of GLP-1 in fish and mammals. The phylogeny of our predicted and characterized receptors for proglucagon-derived peptides demonstrate that receptors for glucagon, GLP-1, and GLP-2 have an origin before the divergence of fish and mammals; however, fish have lost the gene encoding the GLP-1 class of receptors, and likely the incretin action of GLP-1. Receptors that bind GLP-1, but yield glucagon-like action, have been characterized in goldfish and zebrafish, and these sequences are most closely related to glucagon receptors. Both pufferfish and zebrafish have a second glucagon receptor-like gene that is most closely related to the characterized goldfish glucagon receptor. The phylogeny of glucagon receptor-like genes in fish indicates that a duplication of the glucagon receptor gene occurred on the ancestral fish lineage, and could explain the shared action of glucagon and GLP-1. We suggest that the binding specificity of one of the duplicated glucagon receptors has diverged, yielding receptors for GLP-1 and glucagon, but that ancestral downstream signaling has been maintained, resulting in both receptors retaining glucagon-stimulated downstream effects.