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.

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.

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.

Subunits of the plastid ClpPR protease complex have differential contributions to embryogenesis, plastid biogenesis, and plant development in Arabidopsis.

The plastid ClpPR protease complex in Arabidopsis thaliana consists of five catalytic ClpP and four noncatalytic ClpR subunits. An extensive analysis of the CLPR family and CLPP5 is presented to address this complexity. Null alleles for CLPR2 and CLPR4 showed delayed embryogenesis and albino embryos, with seedling development blocked in the cotyledon stage; this developmental block was overcome under heterotrophic conditions, and seedlings developed into small albino to virescent seedlings. By contrast, null alleles for CLPP5 were embryo lethal. Thus, the ClpPR proteins make different functional contributions. To further test for redundancies and functional differences between the ClpR proteins, we overexpressed full-length cDNAs for ClpR1, R2, R3, R4 in clpr1, clpr2 and clpr4 mutants. This showed that overexpression of ClpR3 can complement for the loss of ClpR1, but not for the loss of ClpR2 or ClpR4, indicating that ClpR3 can functionally substitute ClpR1. By contrast, ClpR1, R2 and R4 could not substitute each other. Double mutants of weak CLPR1 and 2 alleles were seedling lethal, showing that a minimum concentration of different ClpR proteins is essential for Clp function. Microscopy and large-scale comparative leaf proteome analyses of a CLPR4 null allele demonstrate a central role of Clp protease in chloroplast biogenesis and protein homeostasis; substrates are discussed. Lack of transcriptional and translational feedback regulation within the CLPPR gene family indicates that regulation of Clp activity occurs through Clp complex assembly and substrate delivery.

A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes strain C3.

Lysobacter enzymogenes strain C3, a biological control agent for plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various fungal and oomycetous species. However, little is known about the regulation of these enzymes or their roles in antimicrobial activity and biocontrol. A study was undertaken to identify mutants of strain C3 affected in extracellular enzyme production and to evaluate their biocontrol efficacy. A single mini-Tn5-lacZ(1)-cat transposon mutant of L. enzymogenes strain C3 that was globally affected in a variety of phenotypes was isolated. In this mutant, 5E4, the activities of several extracellular lytic enzymes, gliding motility, and in vitro antimicrobial activity were reduced. Characterization of 5E4 indicated that the transposon inserted in a clp gene homologue belonging to the Crp gene family of regulators. Immediately downstream was a second open reading frame similar to that encoding acetyltransferases belonging to the Gcn5-related N-acetyltransferase superfamily, which reverse transcription-PCR confirmed was cotranscribed with clp. Chromosomal deletion mutants with mutations in clp and between clp and the acetyltransferase gene verified the 5E4 mutant phenotype. The clp gene was chromosomally inserted in mutant 5E4, resulting in complemented strain P1. All mutant phenotypes were restored in P1, although the gliding motility was observed to be excessive compared with that of the wild-type strain. clp mutant strains were significantly affected in biological control of pythium damping-off of sugar beet and bipolaris leaf spot of tall fescue, which was partially or fully restored in the complemented strain P1. These results indicate that clp is a global regulatory gene that controls biocontrol traits expressed by L. enzymogenes C3.