CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Molecular insights into the dynamic modulation of bacterial ClpP function and oligomerization by peptidomimetic boronate compounds.

Bacterial caseinolytic protease P subunit (ClpP) is important and vital for cell survival and infectivity. Recent publications describe and discuss the complex structure-function relationship of ClpP and its processive activity mediated by 14 catalytic sites. Even so, there are several aspects yet to be further elucidated, such as the paradoxical allosteric modulation of ClpP by peptidomimetic boronates. These compounds bind to all catalytic sites, and in specific conditions, they stimulate a dysregulated degradation of peptides and globular proteins, instead of inhibiting the enzymatic activity, as expected for serine proteases in general. Aiming to explore and explain this paradoxical effect, we solved and refined the crystal structure of native ClpP from Staphylococcus epidermidis (Se), an opportunistic pathogen involved in nosocomial infections, as well as ClpP in complex with ixazomib at 1.90 Å and 2.33 Å resolution, respectively. The interpretation of the crystal structures, in combination with complementary biochemical and biophysical data, shed light on how ixazomib affects the ClpP conformational state and activity. Moreover, SEC-SAXS and DLS measurements show, for the first time, that a peptidomimetic boronate compound also induces the assembly of the tetradecameric structure from isolated homomeric heptameric rings of a gram-positive organism.

Anti-infective therapy using species-specific activators of Staphylococcus aureus ClpP.

The emergence of methicillin-resistant Staphylococcus aureus isolates highlights the urgent need to develop more antibiotics. ClpP is a highly conserved protease regulated by ATPases in bacteria and in mitochondria. Aberrant activation of  bacterial ClpP is an alternative method of discovering antibiotics, while it remains difficult to develop selective  Staphylococcus aureus ClpP activators that can avoid disturbing Homo sapiens ClpP functions. Here, we use a structure-based design to identify (R)- and (S)-ZG197 as highly selective Staphylococcus aureus ClpP activators. The key structural elements in Homo sapiens ClpP, particularly W146 and its joint action with the C-terminal motif, significantly contribute to the discrimination of the activators. Our selective activators display wide antibiotic properties towards an array of multidrug-resistant staphylococcal strains in vitro, and demonstrate promising antibiotic efficacy in zebrafish and murine skin infection models. Our findings indicate that the species-specific activators of Staphylococcus aureus ClpP are exciting therapeutic agents to treat staphylococcal infections.

Nepetin reduces virulence factors expression by targeting ClpP against MRSA-induced pneumonia infection.

The resistance of Staphylococcus aureus (S. aureus) to various antibiotics has increased dramatically due to the misuse of antibiotics, and thus the development of new anti-infective drugs with new targets is urgently needed to combat resistance. Caseinolytic peptidase P is a case in hydrolase that regulates the virulence level of S. aureus. Here, we found that nepetin, a small-molecule compound from traditional Chinese herbal flavonoids, effectively inhibits ClpP activity. Nepetin suppressed the virulence of S. aureus and effectively combated the lethal pneumonia caused by MRSA. The results of cellular thermal shift assay showed that nepetin could bind to ClpP and reduce the thermal stability of ClpP, and the KD value of 602 nM between them was determined using localized surface plasmon resonance. The binding mode of nepetin and ClpP was further investigated by molecular docking, and it was found that Ser-22 and Gln-47 of ClpP residues were found to be involved in the binding of nepetin to ClpP. In conclusion, we determined that nepetin is a ClpP inhibitor and an effective lead compound for the development of a virulence factor-based treatment for MRSA infection.

Pantoea ananatis carotenoid production confers toxoflavin tolerance and is regulated by Hfq-controlled quorum sensing.

Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterized carotenoid biosynthetic genes of the plant-pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE-B, crtE-B-I, and crtE-B-I-Y. High-performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE-B, crtE-B-I, and crtE-B-I-Y operons produced phytoene, lycopene, and ß-carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that carotenoid production by P. ananatis depends on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (HfqArcZ →RpoS Í° ClpXP). We also demonstrated that Hfq-controlled quorum signaling de-represses EanR to activate RpoS, thereby initiating carotenoid production. Survival genes such as those responsible for the production of carotenoids of the plant-pathogenic P. ananatis must be expressed promptly to overcome stressful environments and compete with other microorganisms. This mechanism is likely maintained by a brake with excellent performance, such as EanR.

ONC201 and imipridones: Anti-cancer compounds with clinical efficacy.

ONC201 was originally discovered as TNF-Related Apoptosis Inducing Ligand (TRAIL)-inducing compound TIC10. ONC201 appears to act as a selective antagonist of the G protein coupled receptor (GPCR) dopamine receptor D2 (DRD2), and as an allosteric agonist of mitochondrial protease caseinolytic protease P (ClpP). Downstream of target engagement, ONC201 activates the ATF4/CHOP-mediated integrated stress response leading to TRAIL/Death Receptor 5 (DR5) activation, inhibits oxidative phosphorylation via c-myc, and inactivates Akt/ERK signaling in tumor cells. This typically results in DR5/TRAIL-mediated apoptosis of tumor cells; however, DR5/TRAIL-independent apoptosis, cell cycle arrest, or antiproliferative effects also occur. The effects of ONC201 extend beyond bulk tumor cells to include cancer stem cells, cancer associated fibroblasts and immune cells within the tumor microenvironment that can contribute to its efficacy. ONC201 is orally administered, crosses the intact blood brain barrier, and is under evaluation in clinical trials in patients with advanced solid tumors and hematological malignancies. ONC201 has single agent clinical activity in tumor types that are enriched for DRD2 and/or ClpP expression including specific subtypes of high-grade glioma, endometrial cancer, prostate cancer, mantle cell lymphoma, and adrenal tumors. Synergy with radiation, chemotherapy, targeted therapy and immune-checkpoint agents has been identified in preclinical models and is being evaluated in clinical trials. Structure-activity relationships based on the core pharmacophore of ONC201, termed the imipridone scaffold, revealed novel potent compounds that are being developed. Imipridones represent a novel approach to therapeutically target previously undruggable GPCRs, ClpP, and innate immune pathways in oncology.

The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis.

Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5' and 3' regulatory sequences.

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15-18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5'-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5'-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5'-UTR construct, described above, and another containing the same terminator, but with the promoter and 5'-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.

Regulation of the pehA gene encoding the major polygalacturonase of Xanthomonas campestris by Clp and RpfF.

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence factors. In this study, sequence and mutational analysis has demonstrated that Xcc pehA encodes the major polygalacturonase, a member of family 28 of the glycosyl hydrolases. Using the 5' RACE (rapid amplification of cDNA ends) method, the pehA transcription initiation site was mapped at 102 nt downstream of a Clp (cAMP receptor protein-like protein)-binding site. Transcriptional fusion assays showed that pehA transcription is greatly induced by polygalacturonic acid, positively regulated by Clp and RpfF (an enoyl-CoA hydratase homologue which is required for the synthesis of cis-11-methyl-2-dodecenoic acid, a low-molecular-mass diffusible signal factor), subjected to catabolite repression, which is independent of Clp or RpfF, and repressed under conditions of oxygen limitation or nitrogen starvation. Our findings extend previous work on Clp and RpfF regulation to show that they both influence the expression of pehA in Xcc.