Clp Protease Complex Research Articles

Streptococcus suis Deploys Multiple ATP-Dependent Proteases for Heat Stress Adaptation.

Streptococcus suis is an important zoonotic pathogen, causing cytokine storms of Streptococcal toxic shock-like syndrome amongst humans after a wound infection into the bloodstream. To overcome the challenges of fever and leukocyte recruitment, invasive S. suis must deploy multiple stress responses forming a network and utilize proteases to degrade short-lived regulatory and misfolded proteins induced by adverse stresses, thereby adapting and evading host immune responses. In this study, we found that S. suis encodes multiple ATP-dependent proteases, including single-chain FtsH and double-subunit Clp protease complexes ClpAP, ClpBP, ClpCP, and ClpXP, which were activated as the fever of infected mice in vivo. The expression of genes ftsH, clpA/B/C, and clpP, but not clpX, were significantly upregulated in S. suis in response to heat stress, while were not changed notably under the treatments with several other stresses, including oxidative, acidic, and cold stimulation. FtsH and ClpP were required for S. suis survival within host blood under heat stress in vitro and in vivo. Deletion of ftsH or clpP attenuated the tolerance of S. suis to heat, oxidative and acidic stresses, and significantly impaired the bacterial survival within macrophages. Further analysis identified that repressor CtsR directly binds and controls the clpA/B/C and clpP operons and is relieved by heat stress. In summary, the deployments of multiple ATP-dependent proteases form a flexible heat stress response network that appears to allow S. suis to fine-tune the degradation or refolding of the misfolded proteins to maintain cellular homeostasis and optimal survival during infection.

AmCBF1 activates the expression of GhClpR1 to mediate dark-green leaves in cotton (Gossypium hirsutum).

The transcription factor AmCBF1 deepens the leaf colour of transgenic cotton by binding to the promoter of the chloroplast development-related protein GhClpR1 to promote photosynthesis. The ATP-dependent caseinolytic protease (Clp protease) family plays a crucial role within chloroplasts, comprising several Clp proteins to maintain chloroplast homeostasis. At present, research on Clp proteins mainly focuses on Arabidopsis, leaving its function in other plants, particularly in crops, less explored. In this study, we overexpressed AmCBF1 from Ammopiptanthus mongolicus (A. mongolicus) in wild type (R15), and found a significant darkening of leaf colour in transgenic plants (L28 and L30). RNA-seq analysis showed an enrichment of pathways associated with photosynthesis. Subsequent screening of differentially expressed genes revealed a significant up-regulation of GhClpR1, a gene linked to chloroplast development, in the transgenic strain. In addition, GhClpR1 was consistently expressed in upland cotton, with the highest expression observed in leaves. Subcellular localization analysis revealed that the protein encoded by GhClpR1 was located in chloroplasts. Yeast one hybrid and dual luciferase experiments showed that the AmCBF1 transcription factor positively regulates the expression of GhClpR1. VIGs-mediated silencing of GhClpR1 led to a significant yellowing phenotype in the leaves. This was accompanied by a reduction in chlorophyll content, and microscopic examination of chloroplast ultrastructure revealed severe developmental impairment. Finally, yeast two-hybrid assays showed that GhClpR1 interacts with the Clp protease complex accessory protein GhClpT2. Our study provides a foundation for studying the function of the Clp protease complex and a new strategy for cultivating high-light-efficiency cotton resources.

Abstract 1823: Imipridones show pre-clinical efficacy in MYCN-amplified and MYCN non-amplified neuroblastoma cell lines

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood, accounting for 15% of all pediatric cancer-related deaths, with around 63% survival for high-risk patients. Treatment remains limited for advanced disease, calling for the development of novel therapies. We investigated the anti-tumor effect of three imipridones (ONC201, ONC206, and ONC212), a promising new class of small molecules, in the treatment of neuroblastoma. Imipridones are potent anticancer drugs that have previously shown efficacy for a variety of adult and pediatric malignancies. ONC201 and ONC206 are antagonists of dopamine receptor D2 (DRD2), while ONC212 is an agonist of GPR132, both of which are overexpressed in many cancers. We performed drug treatment with ONC201, ONC206, and ONC212 on established MYCN-amplified SK-N-BE(2) and MYCN non-amplified SH-SY5Y pediatric neuroblastoma cell lines in vitro. Cell viability assays were performed 72 hours post-treatment to generate dose responses curves. The IC50s for ONC201, ONC206, and ONC212 were 17.82 uM, 547 nM, and 70 nM for SK-N-BE(2), and 997 nM, 314 nM, and 6 nM for SH-SY5Y. Imipridones demonstrated greater efficacy for the non-MYCN-amplified cell line. Using protein quantification studies and downstream target analysis on drug-treated cells, we investigated the mechanisms of how these therapies caused cell death. We showed that imipridones inactivate cell proliferation kinases Akt/ERK and induce cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). They also induce tumor cell apoptosis by modifying the mitochondrial Clp protease complex, decreasing expression of chaperone subunit ClpX and activating mitochondrial proteolysis. Histone deacetylases (HDACs) play a role in controlling MYCN function, which is amplified in more aggressive neuroblastoma; HDAC activity and MYCN are also upregulated in chemotherapy-resistant neuroblastoma. Cell viability assays with HDAC inhibitors Vorinostat and Panobinostat demonstrated single-agent efficacy in vitro. The IC50s for Vorinostat and Panobinostat were 609 nM and 5.51 nM for SK-N-BE(2), and 884 nM and 6.13 nM for SH-SY5Y. However, further investigation is needed to determine synergy and mechanisms of synergy when histone deacetylase (HDAC) inhibitors are used in novel combinations with imipridones. Overall, our data reveals promise in imipridone therapy for neuroblastoma, and future studies are proposed to explore potential novel therapeutic combinations in this difficult-to-treat pediatric cancer. Citation Format: Claire Lin, Wen-I Chang, Joshua N. Honeyman, Lanlan Zhou, Varun V. Prabhu, Joshua Allen, Wafik El-Deiry. Imipridones show pre-clinical efficacy in MYCN-amplified and MYCN non-amplified neuroblastoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1823.

Protein degradation control and regulation of bacterial survival and pathogenicity: the role of protein degradation systems in bacteria.

Protein degradation systems play crucial roles in all the kingdoms of life. Their natural function is to eliminate proteins that are improperly synthesized, damaged, aggregated, or short-lived, ensuring the timely and accurate regulation of the response to abrupt environmental changes. Thus, proteolysis plays an important role in protein homeostasis, quality control, and the control of regulatory processes, such as adaptation and cell development. Except for the lysosome, ATPases Associated with various cellular Activities (AAA+) ATPase-protease complex is another major protein degradation system in the cell. The AAA+ ATPase-protease complex is a giant energy-dependent protease complex found in almost all kinds of cells, including bacteria, archaea and eukarya. Based on sequence analysis of ClpQ (HslV) and 20S proteasome beta subunits, it was found that bacterial ClpQ possess multiple same highly conserved motifs with 20S proteasome beta subunits of archaea and eukaryote. In this review, we also discussed the structure and functional mechanism, protein degradation signals and pathogenic role of proteasome / Clp protease complex in prokaryotes. Bacterial protein degradation systems play important roles in stress tolerance, protein quality control, DNA protection, transcription and pathogenicity of bacteria. But our current knowledge of the bacterial protease system is incomplete, and further research into the Clp protease complex and associated protein degradation signals will extend our understanding of the metabolism, physiology, reproduction, and pathogenicity of bacteria.

Abstract 1060: Combinatorial therapy of imipridones and histone deacetylase inhibitors in Ewing sarcoma cell lines demonstrates synergistic cell death

Abstract Novel therapies are much needed in treating pediatric sarcomas, such as Ewing sarcoma. In the setting of metastatic Ewing sarcoma, survival expectancy ranges around 10%. A novel class of small molecules, imipridones, target G protein-coupled receptors and mitochondrial protease ClpP. Imipridones inactivate cell proliferation kinases Akt/ERK and induce cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5. In pre-clinical and clinical trials, imipridone ONC201 shows increased activity against tumors with mutations in histones, specifically histone H3 at the location of lysine 27 (H3K27). Changes in histone acetylation and the epigenetic effects of imipridones remain under investigation. Most commonly caused by a fusion of the EWS-FLI1 genes, Ewing sarcoma exhibits epigenetic modifications with the EWS-FLI1 protein acting as a transcription factor that causes covalent modifications of histone H3, including acetylation and activation of H3K27. Ewing sarcomas have also shown in vitro response to ERK pathway inhibition, leading to apoptosis and inhibition of metastasis formation. We hypothesized that Ewing sarcoma cell lines, with increased H3K27 acetylation due to EWS-FLI1 acting as an activating transcription factor, would respond to the combination of imipridones with histone deacetylase inhibitors. We performed combinatorial drug treatment on Ewing sarcoma cell lines SK-N-MC and RD-ES with 3 imipridones (ONC201, ONC206, and ONC212) and 3 HDAC inhibitors (vorinostat, entinostat, and panobinostat). Cell viability was measured after drug treatment in vitro. We investigated markers of cell death in established pediatric sarcoma cell lines via protein analysis and flow cytometry analysis. Using protein quantification studies and downstream target analysis on combination drug-treated cells, we investigated the mechanisms of how these two targeted therapies interact synergistically to cause cell death. We show that imipridones inactivate cellular proliferation through the Akt/ERK pathway and induce cell death through the TRAIL pathway, with ONC212 exhibiting the highest cell killing potency. Protein quantification by Western blotting revealed treatment with imipridone ONC201 affected the mitochondrial Clp protease complex by decreasing the chaperone subunit ClpX. Cell viability studies and analysis with Compusyn demonstrate potent synergistic effects causing tumor cell death when imipridones are combined with HDAC inhibitors. Our work presents a novel therapeutic combination for the treatment of Ewing sarcoma. Citation Format: Wen-i Chang, Lanlan Zhou, Attila Seyhan, Varun V. Prabhu, Wafik S. El-Deiry. Combinatorial therapy of imipridones and histone deacetylase inhibitors in Ewing sarcoma cell lines demonstrates synergistic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1060.

Abstract 3902: Novel therapeutic targeting of epigenetic aberrations in pediatric sarcomas through combination of ONC201 and HDAC inhibitors

Abstract Patients diagnosed with pediatric sarcomas have poor outcomes with current standard therapy in the setting of relapse, progressive disease, or upfront metastasis. Imipridones, a class of novel small molecules, specifically ONC201, have shown pre-clinical and clinical efficacy in adult malignancies and in brain tumors with aberrant methylation of histone H3 on lysine 27 (associated with H3K27M mutation). ONC201 is an antagonist of dopamine receptor D2 (DRD2), which is a G protein-coupled receptor that is overexpressed in several malignancies. ONC201 inactivates cell proliferation kinases Akt/ERK and induces cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5. Prior in vitro studies of pediatric sarcomas have produced promising data with ERK pathway inhibition, leading to apoptosis and inhibition of metastasis formation. Some pediatric sarcomas, such as malignant peripheral nerve sheath tumors and fusion-positive sarcomas, have been noted to have aberrant activation or modifications of H3K27. H3K27 can be methylated or acetylated and both modifications are abrogated in H3K27M mutated alleles. Changes in histone acetylation and the epigenetic effects of imipridones remain under investigation. We hypothesized that sarcomas with aberrant H3K27 modifications may respond to the novel small molecule similarly to H3K27-mutated brain tumors, and that the combination of ONC201 with histone deacetylase inhibitors (HDACi) could lead to a synergistic effect on cell death mechanisms, potentially through effects of histone acetylation. We performed drug treatment with ONC201 on established pediatric sarcoma cell lines and applied combinatory drug treatment with HDACi, including vorinostat (SAHA). Cell viability was measured after drug treatment in vitro. We investigated markers of cell death in established pediatric sarcoma cell lines via protein analysis and flow cytometry analysis. Using protein quantification studies and downstream target analysis on combination drug-treated cells, we investigated the mechanisms of how these two targeted therapies interact synergistically to cause cell death. We show that ONC201 inactivates cellular proliferation in pediatric sarcomas through the Akt/ERK pathway and induces cell death through the TRAIL pathway. We demonstrated that treatment of cells with ONC201 modified the mitochondrial Clp protease complex, by activating the ClpP protease and by decreasing the chaperone subunit ClpX. Our studies demonstrate that ONC201 induces cell death in pediatric sarcomas, and that ONC201 has potent synergistic effects causing tumor cell death when combined with HDACi. Further studies are underway in elucidating the combinatory epigenetic effects of HDACi and ONC201 in H3K27-aberrant sarcomas, leading to synergistic cell death mechanisms. Our studies present a novel therapeutic combination for pediatric sarcomas that have relapsed or are refractory to standard chemotherapy. Citation Format: Wen-I Chang, Lanlan Zhou, Attila A. Seyhan, Yiqun Zhang, Wafik S. El-Deiry. Novel therapeutic targeting of epigenetic aberrations in pediatric sarcomas through combination of ONC201 and HDAC inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3902.

Co-Suppression of NbClpC1 and NbClpC2, Encoding Clp Protease Chaperons, Elicits Significant Changes in the Metabolic Profile of Nicotiana benthamiana.

Metabolites in plants are the products of cellular metabolic processes, and their differential amount can be regarded as the final responses of plants to genetic, epigenetic, or environmental stresses. The Clp protease complex, composed of the chaperonic parts and degradation proteases, is the major degradation system for proteins in plastids. ClpC1 and ClpC2 are the two chaperonic proteins for the Clp protease complex and share more than 90% nucleotide and amino acid sequence similarities. In this study, we employed virus-induced gene silencing to simultaneously suppress the expression of ClpC1 and ClpC2 in Nicotiana benthamiana (NbClpC1/C2). The co-suppression of NbClpC1/C2 in N. benthamiana resulted in aberrant development, with severely chlorotic leaves and stunted growth. A comparison of the control and NbClpC1/C2 co-suppressed N. benthamiana metabolomes revealed a total of 152 metabolites identified by capillary electrophoresis time-of-flight mass spectrometry. The co-suppression of NbClpC1/C2 significantly altered the levels of metabolites in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, and the purine biosynthetic pathway, as well as polyamine and antioxidant metabolites. Our results show that the simultaneous suppression of ClpC1 and ClpC2 leads to aberrant morphological changes in chloroplasts and that these changes are related to changes in the contents of major metabolites acting in cellular metabolism and biosynthetic pathways.

Manipulation of Plastidial Protein Quality Control Components as a New Strategy to Improve Carotenoid Contents in Tomato Fruit.

Carotenoids such as β-carotene (pro-vitamin A) and lycopene accumulate at high levels during tomato (Solanum lycopersicum L.) fruit ripening, contributing to the characteristic color and nutritional quality of ripe tomatoes. Besides their role as pigments in chromoplast-harboring tissues such as ripe fruits, carotenoids are important for photosynthesis and photoprotection in the chloroplasts of photosynthetic tissues. Interestingly, recent work in Arabidopsis thaliana (L.) Heynh. has unveiled a critical role of chloroplast protein quality control components in the regulation of carotenoid biosynthesis. The accumulation (i.e. degradation rate) and activity (i.e. folding status) of phytoene synthase (PSY) and other Arabidopsis biosynthetic enzymes is modulated by chaperones such as Orange (OR) and Hsp70 in coordination with the stromal Clp protease complex. OR and Clp protease were recently shown to also influence PSY stability and carotenoid accumulation in tomato. Here we show how manipulating the levels of plastid-localized Hsp70 in transgenic tomato plants can also impact the accumulation of carotenoids in ripe fruit. The resulting carotenoid profile and chromoplast ultrastructure, however, are different from those obtained in tomatoes from transgenic lines with increased OR activity. These results suggest that different chaperone families target different processes related to carotenoid metabolism and accumulation during tomato ripening. We further discuss other possible targets for future manipulation in tomato based on the knowledge acquired in Arabidopsis.

Co-suppression of NbClpC1 and NbClpC2 alters plant morphology with changed hormone levels in Nicotiana benthamiana.

Co-suppression of chaperonic ClpC1 and ClpC2 in Nicotiana benthamiana significantly affect the development and exogenous application of gibberellin partially rescue the developmental defects. Over the past decade, the Clp protease complex has been identified as being implicated in plastid protein quality control in plant cells. CLPC1 and CLPC2 proteins form the chaperone subunits of the Clp protease complex and unfold protein substrates to thread them into the ClpP complex. Here, using the technique of virus-induced gene silencing (VIGS), we suppressed both Nicotiana benthamiana ClpC1 and ClpC2 (NbClpC1/C2) functioning as chaperone subunits in the protease complex. Co-suppression of NbClpC1/C2 caused chlorosis and retarded-growth phenotype with no seed formation and significantly reduced root length. We found that co-suppression of NbClpC1/C2 also affected stomata and trichome formation and vascular bundle differentiation and patterning. Analysis of phytohormones revealed significant alteration and imbalance of major hormones in the leaves of NbClpC1/C2 co-suppressed plant. We also found that application of gibberellin (GA3) partially rescued the developmental defects. Co-suppression of NbClpC1/C2 significantly affected the development of N. benthamiana and exogenous application of GA3 partially rescued the developmental defects. Overall, our findings demonstrate that CLPC1 and CLPC2 proteins have a pivotal role in plant growth and development.

Co-suppression of NbClpC1 and NbClpC2, chaperone subunits in the Clp protease complex, accelerates hypersensitive response and increases disease susceptibility in Nicotiana benthamiana

The Clp protease complex, which is the central protein degradation machinery in plastids, plays a role similar to proteasome. ClpC1 and ClpC2 comprise the chaperonic part of the Clp protease and share more than 90% nucleotide sequence similarity with each other. In a previous study, the co-suppression of Nicotiana benthamiana ClpC1 and ClpC2 (NbClpC1/C2) resulted in leaf chlorosis, growth retardation phenotype, disappearance of apical dominance and coarsely packed mesophyll cells. Quantitative real-time PCR analysis revealed that the co-suppression of NbClpC1/C2 upregulated the expression levels of defense-related genes including pathogenesis-related protein 1b (PR1b), glutathione-S-transferase, catalase, ascorbate peroxidase and systemic acquired resistance gene 8.2 (SAR8.2). NbClpC1/C2 co-suppressed leaves accelerated hypersensitive response by the infection with an incompatible pathogen, Pseudomonas syringae pv. tomato T1, but increased susceptibility via the infiltration of a compatible pathogen, P. syringae pv. tabaci. These findings indicate that disruption of the chaperones of ClpC1 and ClpC2 increases the growth of bacterial pathogens and therefore increases hypersensitive response and disease susceptibility.

Extreme variation in rates of evolution in the plastid Clp protease complex.

Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi-subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid-encoded gene (clpP1) and multiple nuclear genes. TheclpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid-encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history ofclpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find thatclpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns andRNA-editing sites) within seed plants. AlthoughclpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genusSilene, which has highly elevated and heterogeneous rates ofclpP1 evolution. We confirmed thatclpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear-encoded Clp subunits, even in one of the most divergentSilene species. Additionally, there is a tight correlation between amino acid substitution rates inclpP1 and the nuclear-encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex.

Control of plastidial metabolism by the Clp protease complex.

Plant metabolism is strongly dependent on plastids. Besides hosting the photosynthetic machinery, these endosymbiotic organelles synthesize starch, fatty acids, amino acids, nucleotides, tetrapyrroles, and isoprenoids. Virtually all enzymes involved in plastid-localized metabolic pathways are encoded by the nuclear genome and imported into plastids. Once there, protein quality control systems ensure proper folding of the mature forms and remove irreversibly damaged proteins. The Clp protease is the main machinery for protein degradation in the plastid stroma. Recent work has unveiled an increasing number of client proteins of this proteolytic complex in plants. Notably, a substantial proportion of these substrates are required for normal chloroplast metabolism, including enzymes involved in the production of essential tetrapyrroles and isoprenoids such as chlorophylls and carotenoids. The Clp protease complex acts in coordination with nuclear-encoded plastidial chaperones for the control of both enzyme levels and proper folding (i.e. activity). This communication involves a retrograde signaling pathway, similarly to the unfolded protein response previously characterized in mitochondria and endoplasmic reticulum. Coordinated Clp protease and chaperone activities appear to further influence other plastid processes, such as the differentiation of chloroplasts into carotenoid-accumulating chromoplasts during fruit ripening.

Temporal Proteomics of Inducible RNAi Lines of Clp Protease Subunits Identifies Putative Protease Substrates.

The Clp protease in the chloroplasts of plant cells is a large complex composed of at least 13 nucleus-encoded subunits and one plastid-encoded subunit, which are arranged in several ring-like structures. The proteolytic P-ring and the structurally similar R-ring form the core complex that contains the proteolytic chamber. Chaperones of the HSP100 family help with substrate unfolding, and additional accessory proteins are believed to assist with Clp complex assembly and/or to promote complex stability. Although the structure and function of the Clp protease have been studied in great detail in both bacteria and chloroplasts, the identification of bona fide protease substrates has been very challenging. Knockout mutants of genes for protease subunits are of limited value, due to their often pleiotropic phenotypes and the difficulties with distinguishing primary effects (i.e. overaccumulation of proteins that represent genuine protease substrates) from secondary effects (proteins overaccumulating for other reasons). Here, we have developed a new strategy for the identification of candidate substrates of plant proteases. By combining ethanol-inducible knockdown of protease subunits with time-resolved analysis of changes in the proteome, proteins that respond immediately to reduced protease activity can be identified. In this way, secondary effects are minimized and putative protease substrates can be identified. We have applied this strategy to the Clp protease complex of tobacco (Nicotiana tabacum) and identified a set of chloroplast proteins that are likely degraded by Clp. These include several metabolic enzymes but also a small number of proteins involved in photosynthesis.

Generation and characterization of a collection of knock-down lines for the chloroplast Clp protease complex in tobacco.

Protein degradation in chloroplasts is carried out by a set of proteases that eliminate misfolded, damaged, or superfluous proteins. The ATP-dependent caseinolytic protease (Clp) is the most complex protease in plastids and has been implicated mainly in stromal protein degradation. In contrast, FtsH, a thylakoid membrane-associated metalloprotease, is believed to participate mainly in the degradation of thylakoidal proteins. To determine the role of specific Clp and FtsH subunits in plant growth and development, RNAi lines targeting at least one subunit of each Clp ring and FtsH were generated in tobacco. In addition, mutation of the translation initiation codon was employed to down-regulate expression of the plastid-encoded ClpP1 subunit. These protease lines cover a broad range of reductions at the transcript and protein levels of the targeted genes. A wide spectrum of phenotypes was obtained, including pigment deficiency, alterations in leaf development, leaf variegations, and impaired photosynthesis. When knock-down lines for the different protease subunits were compared, both common and specific phenotypes were observed, suggesting distinct functions of at least some subunits. Our work provides a well-characterized collection of knock-down lines for plastid proteases in tobacco and reveals the importance of the Clp protease in physiology and plant development.

The Complete Sequence of the Acacia ligulata Chloroplast Genome Reveals a Highly Divergent clpP1 Gene.

Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 158,724 bp in size, comprising inverted repeats of 25,925 bp and single-copy regions of 88,576 bp and 18,298 bp. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease complex.

AAA+ chaperones and acyldepsipeptides activate the ClpP protease via conformational control.

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.

A pair of homoeolog ClpP5 genes underlies a virescent yellow-like mutant and its modifier in maize.

Gene-background interaction is a commonly observed phenomenon in many species, but the molecular mechanisms of such an interaction is less well understood. Here we report the cloning of a maize mutant gene and its modifier. A recessive mutant with a virescent yellow-like (vyl) phenotype was identified in an ethyl methanesulfonate-mutagenized population derived from the maize inbred line B73. Homozygous mutant maize plants exhibited a yellow leaf phenotype after emergence but gradually recovered and became indistinguishable from wild-type plants after approximately 2 weeks. Taking the positional cloning approach, the Chr.9_ClpP5 gene, one of the proteolytic subunits of the chloroplast Clp protease complex, was identified and validated as the candidate gene for vyl. When introgressed by backcross into the maize inbred line PH09B, the mutant phenotype of vyl lasted much longer in the greenhouse and was lethal in the field, implying the presence of a modifier(s) for vyl. A major modifier locus was identified on chromosome 1, and a paralogous ClpP5 gene was isolated and confirmed as the candidate for the vyl-modifier. Expression of Chr.1_ClpP5 is induced significantly in B73 by the vyl mutation, while the expression of Chr.1_ClpP5 in PH09B is not responsive to the vyl mutation. Moreover, expression and sequence analysis suggests that the PH09B Chr.1_ClpP5 allele is functionally weaker than the B73 allele. We propose that functional redundancy between duplicated paralogous genes is the molecular mechanism for the interaction between vyl and its modifier.

Regulation of Host Hemoglobin Binding by the Staphylococcus aureus Clp Proteolytic System

Protein turnover is a key process for bacterial survival mediated by intracellular proteases. Proteolytic degradation reduces the levels of unfolded and misfolded peptides that accumulate in the cell during stress conditions. Three intracellular proteases, ClpP, HslV, and FtsH, have been identified in the Gram-positive bacterium Staphylococcus aureus, a pathogen responsible for significant morbidity and mortality worldwide. Consistent with their crucial role in protein turnover, ClpP, HslV, and FtsH affect a number of cellular processes, including metabolism, stress responses, and virulence. The ClpP protease is believed to be the principal degradation machinery in S. aureus. This study sought to identify the effect of the Clp protease on the iron-regulated surface determinant (Isd) system, which extracts heme-iron from host hemoglobin during infection and is critical to S. aureus pathogenesis. Inactivation of components of the Clp protease alters abundance of several Isd proteins, including the hemoglobin receptor IsdB. Furthermore, the observed changes in IsdB abundance are the result of transcriptional regulation, since transcription of isdB is decreased by clpP or clpX inactivation. In contrast, inactivation of clpC enhances isdB transcription and protein abundance. Loss of clpP or clpX impairs host hemoglobin binding and utilization and results in severe virulence defects in a systemic mouse model of infection. These findings suggest that the Clp proteolytic system is important for regulating nutrient iron acquisition in S. aureus. The Clp protease and Isd complex are widely conserved in bacteria; therefore, these data reveal a novel Clp-dependent regulation pathway that may be present in other bacterial pathogens.

Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis

The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. Here, we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function in Arabidopsis (Arabidopsis thaliana). ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 (clpp3-1) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising, since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed, albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/ClpP5/ClpP6 and ClpR3. Remarkably, the association of ClpT1 to the modified Clp core was unchanged. Large-scale quantitative proteomics assays of clpp3-1 showed a 50% loss of photosynthetic capacity and the up-regulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated, whereas the major thylakoid protease (FtsH1/FtsH2/FtsH5/FtsH8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotypes between the CLPP3, CLPP4, and CLPP5 null mutants are discussed.

Validation of the Essential ClpP Protease in Mycobacterium tuberculosis as a Novel Drug Target

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.