Clp Complex Research Articles

Metabolites from Streptomyces aureus (VTCC43181) and Their Inhibition of Mycobacterium tuberculosis ClpC1 Protein.

Tuberculosis is one of the most common infectious diseases in the world, caused by Mycobacterium tuberculosis. The outbreak of multiple drug-resistant tuberculosis has become a major challenge to prevent this disease worldwide. ClpC1 is a Clp ATPase protein of Mycobacterium tuberculosis, functioning as a chaperon when combined with the Clp complex. ClpC1 has emerged as a new target to discover anti-tuberculosis drugs. This study aimed to explore the ClpC1 inhibitors from actinomycetes, which have been known to provide abundant sources of antibiotics. Two cyclic peptides, including nocardamin (1), halolitoralin A (3), and a lactone pleurone (2), were isolated from the culture of Streptomyces aureus (VTCC43181). The structures of these compounds were determined based on the detailed analysis of their spectral data and comparison with references. This is the first time these compounds have been isolated from S. aureus. Compounds 1-3 were evaluated for their affection of ATPase activity of the recombinant ClpC1 protein. Of these compounds, halolitoralin A (1), a macrocyclic peptide, was effective for the ATPase hydrolysis of the ClpC1 protein.

Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (CLPP) As a Novel Mediator of, and Target in High Risk Multiple Myeloma

Background: Up to 30% of newly diagnosed myeloma (NDMM) patients have high-risk disease characterized by increased proliferation, resistance to apoptosis, focal and bone marrow-independent growth, and intraclonal heterogeneity. High-risk can be defined by fluorescence in situ hybridization (FISH) features including 17p deletion, translocation (t)(4;14), t(14;16), or t(14;20), and gain of 1q. However, some standard risk patients also exhibit high-risk characteristics, indicating that identifying novel targetable mediators of this phenotype is of interest. Methods: We conducted single cell RNA-Sequencing (scRNA-Seq) of CD138 + monoclonal/neoplastic and polyclonal/normal plasma cells (PCs) from 72 individuals across the plasma cell dyscrasia spectrum and 3 healthy controls to identify differentially expressed genes associated with myeloma pathobiology. Also, we leveraged publicly available gene expression profiling (GEP) databases, and performed pre-clinical studies both in vitro and in vivo. Results: CLPP was expressed with increased frequency in NDMM and relapsed/refractory myeloma patients compared to the precursors monoclonal gammopathy of undetermined significance and smoldering myeloma (GSE6477; p=4.03x10 -14). Notably, CLPP is the proteolytic component, along with CLPX, of the CLP complex which cleaves peptides and proteins in an ATP-dependent manner and is associated with the inner mitochondrial membrane. The GEP data were confirmed in our scRNA-Seq database, which showed CLPP over-expression occurred preferentially in each patient's monoclonal versus their own polyclonal PCs. Moreover, analysis of the Broad Institute Cancer Cell Line Encyclopedia revealed MM cells had the highest mean CLPP expression of any tumor cell type. In the NDMM CoMMpass database, high CLPP levels were associated with markers of increased disease burden (beta-2-microglobulin (p<0.001), calcium (p=0.0027), circulating plasma cells (p<0.001), marrow plasmacytosis (p<0.001), and lactate dehydrogenase (p=0.0025)). Moreover, these patients showed a significantly shorter progression-free and overall survival (both p<0.0001), and did not cluster in any of the Arkansas GEP70-defined myeloma subtypes. In MM.1S and H929 myeloma cells, specific CLPP knockdown with an inducible shRNA decreased cell proliferation and activated apoptosis as shown by increased Annexin V positivity. Similarly, pharmacologic CLPP inhibition produced a time- and concentration-dependent induction of apoptosis in a panel of cell lines representing myeloma molecular subtypes and drug resistant phenotypes. Ectopic CLPP overexpression promoted myeloma organoid growth, while its suppression had the opposite effect. Functional analysis revealed that CLPP knockdown or inhibition impaired cell proteolytic activity, mitochondrial transmembrane potential, and reduced adhesion to stromal cells while inducing reactive oxygen species and autophagy. To further pursue the underlying molecular mechanisms, we first examined the net downstream effects of CLPP knockdown and inhibition by mass spectrometry followed by mapping to the KEGG database. Recurrently up-regulated pathways included the mitochondrial unfolded protein response (UPR mt), lysosome transport, and autophagy utilizing and metabolic processes. In contrast, down-regulated pathways included oxidative phosphorylation, c-Myc signaling, RNA polymerase I-directed transcription, DNA replication and elongation, and the G2M checkpoint. Next, our metabolomics analysis revealed a significant attenuation of the Warburg effect in myeloma cells upon CLPP inhibition consistent with reduced glycolysis and pentose phosphate pathway activity. Notably, mitochondrial ATP production was significantly reduced, as confirmed by a seahorse XF Real-time ATP Rate assay. Finally, in vivo experiments demonstrated that pharmacologic CLPP inhibition impaired myeloma growth in an MM.1S-based xenograft model ( Figure). Conclusions: These pre-clinical data support the hypothesis that CLPP represents a novel mediator of high-risk behavior in multiple myeloma, demonstrate that its targeting results in mitochondrial dysfunction that attenuates myeloma growth and survival, and support development of pharmacologic CLPP inhibitors to target myeloma.

Genomic insight into the salt tolerance and proteolytic activity of Bacillus subtilis.

We assessed the salt tolerance and proteolytic activity of 40 genome-published Bacillus subtilis strains isolated from fermented Korean foods to illuminate the genomic background behind the functionality of B. subtilis in high-salt fermentation. On the basis of the salt tolerance and phenotypic proteolytic activity of the 40 strains, we selected five strains exhibiting different phenotypic characteristics. Comparative genomic analyses of these five strains provided genomic insight into the salt tolerance and proteolytic activity of B. subtilis. Two-component system (TCS) genes annotated as ybdGJK and laterally acquired authentic ATP-binding cassette (ABC) transporter system genes of tandem repeat structure might contribute to increase salt tolerance. The additional possession of gene homologs for CAAX protease family proteins and components of Clp (caseinolytic protease) complex, ATP-dependent Clp proteolytic subunit ClpP and AAA+ (ATPases Associated with diverse cellular Activities) family ATPases, might determine the proteolytic activity of B. subtilis. This study established the scientific foundation for the viability and functionality of B. subtilis in high-salt fermentation.

The Intron Retention Variant CsClpP3m Is Involved in Leaf Chlorosis in Some Tea Cultivars.

Tea products made from chlorotic or albino leaves are very popular for their unique flavor. Probing into the molecular mechanisms underlying the chlorotic leaf phenotype is required to better understand the formation of these tea cultivars and aid in future practical breeding. In this study, transcriptional alterations of multiple subunit genes of the caseinolytic protease complex (Clp) in the chlorotic tea cultivar ‘Yu-Jin-Xiang’ (YJX) were found. Cultivar YJX possessed the intron retention variant of ClpP3, named as CsClpP3m, in addition to the non-mutated ClpP3. The mutated variant results in a truncated protein containing only 166 amino acid residues and lacks the catalytic triad S182-H206-D255. Quantitative analysis of two CsClpP3 variants in different leaves with varying degrees of chlorosis in YJX and analyses of different chlorotic tea cultivars revealed that the transcript ratios of CsClpP3m over CsClpP3 were negatively correlated with leaf chlorophyll contents. The chlorotic young leaf phenotype was also generated in the transgenic tobacco by suppressing ClpP3 using the RNAi method; complementation with non-mutated CsClpP3 rescued the wild-type phenotype, whereas CsClpP3m failed to complement. Taken together, CsClpP3m is involved in leaf chlorosis in YJX and some other tea cultivars in a dose-dependent manner, likely resulting from the failure of Clp complex assembly due to the truncated sequence of CsClpP3m. Our data shed light on the mechanisms controlling leaf chlorosis in tea plants.

Rapid sequence evolution is associated with genetic incompatibilities in the plastid Clp complex.

Replacing the native clpP1 gene in the Nicotiana plastid genome with homologs from different donor species showed that the extent of genetic incompatibilities depended on the rate of sequence evolution. The plastid caseinolytic protease (Clp) complex plays essential roles in maintaining protein homeostasis and comprises both plastid-encoded and nuclear-encoded subunits. Despite the Clp complex being retained across green plants with highly conserved protein sequences in most species, examples of extremely accelerated amino acid substitution rates have been identified in numerous angiosperms. The causes of these accelerations have been the subject of extensive speculation but still remain unclear. To distinguish among prevailing hypotheses and begin to understand the functional consequences of rapid sequence divergence in Clp subunits, we used plastome transformation to replace the native clpP1 gene in tobacco (Nicotiana tabacum) with counterparts from another angiosperm genus (Silene) that exhibits a wide range in rates of Clp protein sequence evolution. We found that antibiotic-mediated selection could drive a transgenic clpP1 replacement from a slowly evolving donor species (S. latifolia) to homoplasmy but that clpP1 copies from Silene species with accelerated evolutionary rates remained heteroplasmic, meaning that they could not functionally replace the essential tobacco clpP1 gene. These results suggest that observed cases of rapid Clp sequence evolution are a source of epistatic incompatibilities that must be ameliorated by coevolutionary responses between plastid and nuclear subunits.

Gene duplication and rate variation in the evolution of plastid ACCase and Clp genes in angiosperms

While the chloroplast (plastid) is known for its role in photosynthesis, it is also involved in many other metabolic pathways essential for plant survival. As such, plastids contain an extensive suite of enzymes required for non-photosynthetic processes. The evolution of the associated genes has been especially dynamic in flowering plants (angiosperms), including examples of gene duplication and extensive rate variation. We examined the role of ongoing gene duplication in two key plastid enzymes, the acetyl-CoA carboxylase (ACCase) and the caseinolytic protease (Clp), responsible for fatty acid biosynthesis and protein turnover, respectively. In plants, there are two ACCase complexes—a homomeric version present in the cytosol and a heteromeric version present in the plastid. Duplications of the nuclear-encoded homomeric ACCase gene and retargeting of one resultant protein to the plastid have been previously reported in multiple species. We find that these retargeted homomeric ACCase proteins exhibit elevated rates of sequence evolution, consistent with neofunctionalization and/or relaxation of selection. The plastid Clp complex catalytic core is composed of nine paralogous proteins that arose via ancient gene duplication in the cyanobacterial/plastid lineage. We show that further gene duplication occurred more recently in the nuclear-encoded core subunits of this complex, yielding additional paralogs in many species of angiosperms. Moreover, in six of eight cases, subunits that have undergone recent duplication display increased rates of sequence evolution relative to those that have remained single copy. We also compared substitution patterns between pairs of Clp core paralogs to gain insight into post-duplication evolutionary routes. These results show that gene duplication and rate variation continue to shape the plastid proteome.

Plastid biogenesis in malaria parasites requires the interactions and catalytic activity of the Clp proteolytic system

The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the P. falciparum Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (PfClpC) variants revealed a functional chaperone-protease interaction. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.

Consequences of the loss of catalytic triads in chloroplast CLPPR protease core complexes invivo.

The essential chloroplast CLP protease system consists of a tetradecameric proteolytic core with catalytic P (P1, 3–6) and non‐catalytic R (R1–4) subunits, CLP chaperones and adaptors. The chloroplast CLP complex has a total of ten catalytic sites,but it is not known how many of these catalytic sites can be inactivated before plants lose viability. Here we show that CLPP3 and the catalytically inactive variant CLPP3S164A fully complement the developmental arrest of the clpp3‐1 null mutant, even under environmental stress. In contrast, whereas the inactive variant CLPP5S193A assembled into the CLP core, it cannot rescue the embryo lethal phenotype of the clpp5‐1 null mutant. This shows that CLPP3 makes a unique structural contribution but its catalytic site is dispensable, whereas the catalytic activity of CLPP5 is essential. Mass spectrometry of affinity‐purified CLP cores of the complemented lines showed highly enriched CLP cores. Other chloroplast proteins were co‐purified with the CLP cores and are candidate substrates. A strong overlap of co‐purified proteins between the CLP core complexes with active and inactive subunits indicates that CLP cores with reduced number of catalytic sites do not over‐accumulate substrates, suggesting that the bottle‐neck for degradation is likely substrate recognition and unfolding by CLP adaptors and chaperones, upstream of the CLP core.

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.

PfClpC Is an Essential Clp Chaperone Required for Plastid Integrity and Clp Protease Stability in Plasmodium falciparum

The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp) genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.

Degradation of SsrA-tagged proteins in streptococci.

In prokaryotes, a conserved small RNA molecule, called tmRNA, rescues ribosomes from proteins that are abnormally truncated due to the presence of rare codons or degraded mRNA. During the rescue process, a peptide tag (SsrA) encoded by tmRNA is cotranslationally added to the truncated polypeptides, thereby targeting these proteins for proteolytic degradation. In Escherichia coli, ClpXP and ClpAP proteases primarily degrade SsrA-tagged proteins. Other proteases such as Lon and FtsH also participate in the degradation in E. coli. However, in Bacillus subtilis, ClpXP is the major protease that degrades the SsrA-tagged proteins. Degradation of SsrA-tagged protein in streptococci is not well understood except that ClpXP is responsible for the majority of the degradation. Here we show that in Streptococcus mutans, in addition to ClpXP, two other Clp complexes, ClpCP and ClpEP, are also involved in the degradation. We also found that ClpCP- and ClpEP-mediated proteolysis of SsrA-tagged substrates is induced by heat stress. As ClpCP and ClpEP proteins are highly conserved in streptococci, we predicted that ClpEP- and ClpCP-mediated degradation of SsrA-tagged proteins might be operational in other streptococci.

Bacterial caseinolytic proteases as novel targets for antibacterial treatment

Bacterial Clp proteases are important for protein turnover and homeostasis in order to maintain vital cellular functions particularly under stress conditions. Apart from their crucial role in general protein quality control by degrading abnormally folded or otherwise aberrant or malfunctioning proteins, their temporally and spatially precise proteolysis of key regulatory proteins additionally guides several developmental processes like cell motility, genetic competence, cell differentiation, sporulation as well as important aspects of virulence. Due to their apparent relevance for many physiological processes and their conservation among diverse bacterial species including human pathogens, Clp proteases have attracted considerable attention as targets for antibacterial action in recent years. Particularly a novel class of potent acyldepsipeptide antibiotics unleashes ClpP, the uniform proteolytic core unit of the degradative Clp complexes, to bring about bacterial death via uncontrolled proteolysis of proteins that are essential for bacterial viability. In addition, covalent inhibition of the catalytic center of ClpP by another class of small molecule inhibitors is investigated in the context of virulence inhibition. Both antibacterial mechanisms constitute innovative approaches with the potential to control infections caused by multi-resistant bacterial pathogens due to the lack of cross-resistance to established antibiotic classes.

Sequence analysis of a salt tolerant metagenomic clone.

Metagenome represent an unlimited resource for discovery of novel genes. Here we report, sequence analysis of a salt tolerant metagenomic clone (6B4) from a pond water metagenomic library. Clone 6B4 had an insert of 2254 bp with G+C composition of 64.06%. DNA sequence from 6B4 showed homology to DNA sequences from pro-teobacteria indicating origin of 6B4 metagenomic insert from a yet uncharacterized proteobacteria. Two encoded proteins from clone 6B4 showed match with ATP-depen-dent Clp protease adaptor protein (ClpS) and phasin, while two truncated encoded proteins showed match with poly-3-hydroxybutyrate synthase and permease. Clp complex is known to play a role in stress tolerance. Expression of ClpS from metagenomic clone is proposed to be responsible for salt tolerance of the metagenomic clone 6B4.

Senescence of staphylococci: using functional genomics to unravel the roles of ClpC ATPase during late stationary phase

Disease caused by Staphylococcus aureus frequently takes a chronic persistent course, and such infections are difficult to treat. S. aureus has developed various stress response systems allowing for coordinated expression of virulence factors and adaptation to environmental conditions. Clp ATPase/protease complexes for protein reactivation and degradation are highly conserved systems with a primary function in stress response. In various bacterial species, the role of Clp complexes has been associated with competence, cell wall synthesis, virulence and other physiologic properties. More recently, in S. aureus various Clp ATPases have been found to influence global regulator functions resulting in complex phenotypic changes. In this review, we briefly outline current knowledge including our own work with ClpC ATPase. We could highlight an important role of ClpC that allows for post-stationary regrowth and entry into the bacterial death phase through a functional tricarboxylic acid (TCA) cycle metabolism. We have concluded that ClpC may play a major regulatory role for long-term survival. Furthermore, using functional genomics data, we could extend the global characterization of the functions of ClpC in S. aureus with respect to late-phase phenomena such as S. aureus carbon metabolism, ion homeostasis, oxidative stress response, survival, and programmed cell death. These studies will thus help to further unravel the putative role of Clp ATPases in the chronic-persistent course of disease.

Subunits of the Plastid ClpPR Protease Complex Have Differential Contributions to Embryogenesis, Plastid Biogenesis, and Plant Development inArabidopsis

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.

Molecular cloning of an actinobacterial-type ClpS gene from Celosia cristata expression library

In proteobacterial cytosol, ClpS protein is known as a molecular adaptor for substrate selectivity and proteolytic activity of the ATP-dependent chaperone-protease complex, ClpAP. ClpA-related ClpS is a small protein usually encoded immediately upstream of ClpA in the genome of proteobacteria. Recent bioinformatics analysis has revealed the presence of cyanobacterial-type ClpS or ClpC-related ClpS in organisms lacking ClpA, including all the plant species sequenced to date. Here we report the identification of an actinobacterial homologue of the ClpS (possibly Clp-related) gene from a plant system. A cDNA, spanning 566 bp with a complete coding region corresponding to 132 amino acids, was isolated from a Celosia cristata expression library constructed on a λ TriplEX2 vector. This cDNA product was considered to be an ATP-dependent Clp protease adaptor and was designated as Celosia actinobacterial-type ClpS, since it contains a highly conserved domain belonging to the ClpS family of proteins from actinobacteria. Celosia ClpS is about 80% identical to actinobacterial ClpS proteins in its overall deduced amino acid sequence. Based on this finding, we may define a novel target of ATP-dependent Clp complex in a plant system or speculate the presence of a second type of molecular chaperone besides ClpC in plants, as predicted for actinobacteria.

Post-translational control of the Streptomyces lividans ClgR regulon by ClpP

It has been shown previously that expression of the Streptomyces lividans clpP1P2 operon, encoding proteolytic subunits of the Clp complex, the clpC1 gene, encoding the ATPase subunit, and the lon gene, encoding another ATP-dependent protease, are all activated by ClgR. The ClgR regulon also includes the clgR gene itself. It is shown here that the degradation of ClgR and Lon is ClpP1/P2-dependent and that the two C-terminal alanines of these new substrates are involved in their stability. The ClpC1 protein, which does not end with two alanines, is also accumulated in a clpP1P2 mutant. The results presented here support the idea that ClpP1/P2 ensure post-translational control of ClgR regulon members, including ClgR itself.

Antibodies against homologous microbial caseinolytic proteases P characterise primary biliary cirrhosis

Background / Aims : Antibodies to caseinolytic protease P 177–194 (ClpP 177–194 ) of the proteolytic subunit of the Clp complex of Escherichia coli ( E. coli) are uniquely present in primary biliary cirrhosis (PBC). Molecular mimicry between the regulatory subunit ClpX and the principal T-cell epitope of pyruvate dehydrogenase complex (PDC-E2) in PBC, has been proposed to account for this. Since ClpP is highly conserved among bacteria we investigated whether the micro-organisms triggering these antibodies may be other than E. coli. Methods / Results : E. coli ClpP 177–194 is homologous with ClpP peptides of Yersinia enterocolitica (YEREN) and Haemophilus influenzae (HAEIN). Enzyme linked immunosorbent assay (ELISA) reactivity to these peptides was tested in 45 patients with PBC, 44 pathological and 32 healthy controls. Reactivity to at least one of the ClpP peptides was observed in 21 (47%) PBC patients, 5.8% pathological and 3.1% healthy controls ( P <0.01 for all). Among these 21 seropositive PBC patients, 15 (71%) reacted to ECOLI ClpP 177–194 , alone or in association with YEREN and/or HAEIN peptides, compared to three (14.2%) reactive with YEREN, two (9.5%) with YEREN/HAEIN and one (4.7%) with HAEIN peptide. Simultaneous reactivity to homologous sequences was due to cross-reactivity as confirmed by competition ELISAs. Conclusions : The PBC-specificity of anti-microbial ClpP reactivity is confirmed: the questions as to primary trigger(s) and relevance to PBC pathogenesis remain open.

Identification of a 350-kDa ClpP Protease Complex with 10 Different Clp Isoforms in Chloroplasts of Arabidopsis thaliana

A 350-kDa ClpP protease complex with 10 different subunits was identified in chloroplast of Arabidopsis thaliana, using Blue-Native gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight and nano-electrospray tandem mass spectrometry. The complex was copurified with the thylakoid membranes, and all identified Clp subunits show chloroplast targeting signals, supporting that this complex is indeed localized in the chloroplast. The complex contains chloroplast-encoded pClpP and six nuclear-encoded proteins nCpP1-6, as well as two unassigned Clp homologues (nClpP7, nClpP8). An additional Clp protein was identified in this complex; it does not belong to any of the known Clp genes families and is here assigned ClpS1. Expression and accumulation of several of these Clp proteins have never been shown earlier. Sequence and phylogenetic tree analysis suggests that nClpP5, nClpP2, and nClpP8 are not catalytically active and form a new group of Clp higher plant proteins, orthologous to the cyanobacterial ClpR protein, and are renamed ClpR1, -2, and -3, respectively. We speculate that ClpR1, -2, and -3 are part of the heptameric rings, whereas ClpS1 is a regulatory subunit positioned at the axial opening of the ClpP/R core. Several truncations and errors in intron and exon prediction of the annotated Clp genes were corrected using mass spectrometry data and by matching genomic sequences with cDNA sequences. This strategy will be widely applicable for the much needed verification of protein prediction from genomic sequence. The extreme complexity of the chloroplast Clp complex is discussed.

Clp protease complexes and their diversity in chloroplasts.

The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts. The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin. It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease. An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95-00). We demonstrate that in both tomato Lycopersicon esculentum Mill. and Arabidopsis thaliana, (L.) Heynh. the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein. Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass. The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli. In addition, various complexes between 160 and 560 kDa are detectable with the individual components. Both the processing "intermediates" and the mature nClpP are found in assembled form.