The key to unlock the Hsp100/Clp protein degradation machines of Mycobacterium

Hsp100/Clp protease complexes are molecular machines important for cellular protein homeostasis and are concurrently embedded in the control of various signal transduction networks by regulatory proteolysis. In Mycobacteria, the genes encoding the components of these Hsp100/Clp protease complexes are essential for growth and were identified as targets for antibiotics, with a new antimicrobial mechanism, that are active on slow growing or even dormant cells. Schmitz and Sauer (2014) report the biochemical characterization of mycobacterial Hsp100/Clp protease complexes actively degrading folded substrate proteins. Their results suggest an unusual activation mechanism for this protease complex and will set the stage for further mechanistic studies of antibiotics acting on this new cellular target.     

Mycobacterium tuberculosis ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability in vitro and During Infection

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy.     

Quantitative Analysis of the Chloroplast Molecular Chaperone ClpC/Hsp93 in Arabidopsis Reveals New Insights into Its Localization,Interaction with the Clp Proteolytic Core,and Functional Importance

The molecular chaperone ClpC/Hsp93 is essential for chloroplast function in vascular plants. ClpC has long been held to act both independently and as the regulatory partner for the ATP-dependent Clp protease, and yet this and many other important characteristics remain unclear. In this study, we reveal that of the two near-identical ClpC paralogs (ClpC1 and ClpC2) in Arabidopsis chloroplasts, along with the closely related ClpD, it is ClpC1 that is the most abundant throughout leaf maturation. An unexpectedly large proportion of both chloroplast ClpC proteins (30% of total ClpC content) associates to envelope membranes in addition to their stromal localization. The Clp proteolytic core is also bound to envelope membranes, the amount of which is sufficient to bind to all the similarly localized ClpC. The role of such an envelope membrane Clp protease remains unclear although it appears uninvolved in preprotein processing or Tic subunit protein turnover. Within the stroma, the amount of oligomeric ClpC protein is less than that of the Clp proteolytic core, suggesting most if not all stromal ClpC functions as part of the Clp protease; a proposal supported by the near abolition of Clp degradation activity in the clpC1 knock-out mutant. Overall, ClpC appears to function primarily within the Clp protease, as the principle stromal protease responsible for maintaining homeostasis, and also on the envelope membrane where it possibly confers a novel protein quality control mechanism for chloroplast preprotein import.     

Substrate delivery by the AAA+ ClpX and ClpC1 unfoldases activates the mycobacterial ClpP1P2 peptidase

Mycobacterial Clp‐family proteases function via collaboration of the heteromeric ClpP1P2 peptidase with a AAA+ partner, ClpX or ClpC1. These enzymes are essential for M. tuberculosis viability and are validated antibacterial drug targets, but the requirements for assembly and regulation of functional proteolytic complexes are poorly understood. Here, we report the reconstitution of protein degradation by mycobacterial Clp proteases in vitro and describe novel features of these enzymes that distinguish them from orthologues in other bacteria. Both ClpX and ClpC1 catalyse ATP‐dependent unfolding and degradation of native protein substrates in conjunction with ClpP1P2, but neither mediates protein degradation with just ClpP1 or ClpP2. ClpP1P2 alone has negligible peptidase activity, but is strongly stimulated by translocation of protein substrates into ClpP1P2 by either AAA+ partner. Interestingly, our results support a model in which both binding of a AAA+ partner and protein‐substrate delivery are required to stabilize active ClpP1P2. Our model has implications for therapeutically targeting ClpP1P2 in dormant M. tuberculosis, and our reconstituted systems should facilitate identification of novel Clp protease inhibitors and activators.     

Mycobacterium tuberculosis WhiB3 Maintains Redox Homeostasis by Regulating Virulence Lipid Anabolism to Modulate Macrophage Response

The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbΔwhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbΔwhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbΔwhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence.     

ClpS1 Is a Conserved Substrate Selector for the Chloroplast Clp Protease System in Arabidopsis

Whereas the plastid caseinolytic peptidase (Clp) P protease system is essential for plant development, substrates and substrate selection mechanisms are unknown. Bacterial ClpS is involved in N-degron substrate selection and delivery to the ClpAP protease. Through phylogenetic analysis, we show that all angiosperms contain ClpS1 and some species also contain ClpS1-like protein(s). In silico analysis suggests that ClpS1 is the functional homolog of bacterial ClpS. We show that Arabidopsis thaliana ClpS1 interacts with plastid ClpC1,2 chaperones. The Arabidopsis ClpS1 null mutant (clps1) lacks a visible phenotype, and no genetic interactions with ClpC/D chaperone or ClpPR core mutants were observed. However, clps1, but not clpc1-1, has increased sensitivity to the translational elongation inhibitor chloramphenicol suggesting a link between translational capacity and ClpS1. Moreover, ClpS1 was upregulated in clpc1-1, and quantitative proteomics of clps1, clpc1, and clps1 clpc1 showed specific molecular phenotypes attributed to loss of ClpC1 or ClpS1. In particular, clps1 showed alteration of the tetrapyrrole pathway. Affinity purification identified eight candidate ClpS1 substrates, including plastid DNA repair proteins and Glu tRNA reductase, which is a control point for tetrapyrrole synthesis. ClpS1 interaction with five substrates strictly depended on two conserved ClpS1 residues involved in N-degron recognition. ClpS1 function, substrates, and substrate recognition mechanisms are discussed.     

Phylogenetic analysis predicts structural divergence for proteobacterial ClpC proteins

Regulated proteolysis is required in all organisms for the removal of misfolded or degradation-tagged protein substrates in cellular quality control pathways. The molecular machines that catalyze this process are known as ATP-dependent proteases with examples that include ClpAP and ClpCP. Clp/Hsp100 subunits form ring-structures that couple the energy of ATP binding and hydrolysis to protein unfolding and subsequent translocation of denatured protein into the compartmentalized ClpP protease for degradation. Copies of the clpA, clpC, clpE, clpK, and clpL genes are present in all characterized bacteria and their gene products are highly conserved in structure and function. However, the evolutionary relationship between these proteins remains unclear. Here we report a comprehensive phylogenetic analysis that suggests divergent evolution yielded ClpA from an ancestral ClpC protein and that ClpE/ClpL represent intermediates between ClpA/ClpC. This analysis also identifies a group of proteobacterial ClpC proteins that are likely not functional in regulated proteolysis. Our results strongly suggest that bacterial ClpC proteins should not be assumed to all function identically due to the structural differences identified here.     

Role of the Clp system in stress tolerance, biofilm formation, and intracellular invasion in Porphyromonas gingivalis

Clp proteases and chaperones are ubiquitous among prokaryotes and eukaryotes, and in many pathogenic bacteria the Clp stress response system is also involved in regulation of virulence properties. In this study, the roles of ClpB, ClpC, and ClpXP in stress resistance, homotypic and heterotypic biofilm formation, and intracellular invasion in the oral opportunistic pathogen Porphyromonas gingivalis were investigated. Absence of ClpC and ClpXP, but not ClpB, resulted in diminished tolerance to high temperatures. Response to oxidative stress was not affected by the loss of any of the Clp proteins. The clpC and clpXP mutants demonstrated elevated monospecies biofilm formation, and the absence of ClpXP also enhanced heterotypic P. gingivalis-Streptococcus gordonii biofilm formation. All clp mutants adhered to gingival epithelial cells to the same level as the wild type; however, ClpC and ClpXP were found to be necessary for entry into host epithelial cells. ClpB did not play a role in entry but was required for intracellular replication and survival. ClpXP negatively regulated the surface exposure of the minor fimbrial (Mfa) protein subunit of P. gingivalis, which stimulates biofilm formation but interferes with epithelial cell entry. Collectively, these results show that the Clp protease complex and chaperones control several processes that are important for the colonization and survival of P. gingivalis in the oral cavity.     

Lon protease promotes survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> during anaerobic glucose starvation

In Escherichia coli, Lon is an ATP-dependent protease which degrades misfolded proteins and certain rapidly-degraded regulatory proteins. Given that oxidatively damaged proteins are generally degraded rather than repaired, we anticipated that Lon deficient cells would exhibit decreased viability during aerobic, but not anaerobic, carbon starvation. We found that the opposite actually occurs. Wild-type and Lon deficient cells survived equally well under aerobic conditions, but Lon deficient cells died more rapidly than the wild-type under anaerobiosis. Aerobic induction of the Clp family of ATP-dependent proteases could explain these results, but direct quantitation of Clp protein established that its level was not affected by Lon deficiency and overexpression of Clp did not rescue the cells under anaerobic conditions. We conclude that the Lon protease supports survival during anaerobic carbon starvation by a mechanism which does not depend on Clp. Shen Luo and Megan McNeill contributed equally to this research.     

Structure and Function of a Novel Type of ATP-dependent Clp Protease

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. The main constitutive Clp protease in photosynthetic organisms has evolved into a functionally essential and structurally intricate enzyme. The model Clp protease from the cyanobacterium Synechococcus consists of the HSP100 molecular chaperone ClpC and a mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We have purified the ClpP3/R complex, the first for a Clp proteolytic core comprised of heterologous subunits. The ClpP3/R complex has unique functional and structural features, consisting of twin heptameric rings each with an identical ClpP3₃ClpR₄ configuration. As predicted by its lack of an obvious catalytic triad, the ClpR subunit is shown to be proteolytically inactive. Interestingly, extensive modification to ClpR to restore proteolytic activity to this subunit showed that its presence in the core complex is not rate-limiting for the overall proteolytic activity of the ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable similarities to the 20 S core of the proteasome, revealing a far greater degree of convergent evolution than previously thought between the development of the Clp protease in photosynthetic organisms and that of the eukaryotic 26 S proteasome.Proteases perform numerous tasks vital for cellular homeostasis in all organisms. Much of the selective proteolysis within living cells is performed by multisubunit chaperone-protease complexes. These proteases all share a common two-component architecture and mode of action, with one of the best known examples being the proteasome in archaebacteria, certain eubacteria, and eukaryotes (1).The 20 S proteasome is a highly conserved cylindrical structure composed of two distinct types of subunits, α and β. These are organized in four stacked heptameric rings, with two central β-rings sandwiched between two outer α-rings. Although the α- and β-protein sequences are similar, it is only the latter that is proteolytic active, with a single Thr active site at the N terminus. The barrel-shaped complex is traversed by a central channel that widens up into three cavities. The catalytic sites are positioned in the central chamber formed by the β-rings, adjacent to which are two antechambers conjointly built up by β- and α-subunits. In general, substrate entry into the core complex is essentially blocked by the α-rings, and thus relies on the associating regulatory partner, PAN and 19 S complexes in archaea and eukaryotes, respectively (1). Typically, the archaeal core structure is assembled from only one type of α- and β-subunit, so that the central proteolytic chamber contains 14 catalytic active sites (2). In contrast, each ring of the eukaryotic 20 S complex has seven distinct α- and β-subunits. Moreover, only three of the seven β-subunits in each ring are proteolytically active (3). Having a strictly conserved architecture, the main difference between the 20 S proteasomes is one of complexity. In mammalian cells, the three constitutive active subunits can even be replaced with related subunits upon induction by γ-interferon to generate antigenic peptides presented by the class 1 major histocompatibility complex (4).Two chambered proteases architecturally similar to the proteasome also exist in eubacteria, HslV and ClpP. HslV is commonly thought to be the prokaryotic counterpart to the 20 S proteasome mainly because both are Thr proteases. A single type of HslV protein, however, forms a proteolytic chamber consisting of twin hexameric rather than heptameric rings (5). Also displaying structural similarities to the proteasome is the unrelated ClpP protease. The model Clp protease from Escherichia coli consists of a proteolytic ClpP core flanked on one or both sides by the ATP-dependent chaperones ClpA or ClpX (6). The ClpP proteolytic chamber is comprised of two opposing homo-heptameric rings with the catalytic sites harbored within (7). ClpP alone displays only limited peptidase activity toward short unstructured peptides (8). Larger native protein substrates need to be recognized by ClpA or ClpX and then translocated in an unfolded state into the ClpP proteolytic chamber (9, 10). Inside, the unfolded substrate is bound in an extended manner to the catalytic triads (Ser-97, His-122, and Asp-171) and degraded into small peptide fragments that can readily diffuse out (11). Several adaptor proteins broaden the array of substrates degraded by a Clp protease by binding to the associated HSP100 partner and modifying its protein substrate specificity (12, 13). One example is the adaptor ClpS that interacts with ClpA (EcClpA) and targets N-end rule substrates for degradation by the ClpAP protease (14).Like the proteasome, the Clp protease is found in a wide variety of organisms. Besides in all eubacteria, the Clp protease also exist in mammalian and plant mitochondria, as well as in various plastids of algae and plants. It also occurs in the unusual plastid in Apicomplexan protozoan (15), a family of parasites responsible for many important medical and veterinary diseases such as malaria. Of all these organisms, photobionts have by far the most diverse array of Clp proteins. This was first apparent in cyanobacteria, with the model species Synechococcus elongatus having 10 distinct Clp proteins, four HSP100 chaperones (ClpB1–2, ClpC, and ClpX), three ClpP proteins (ClpP1–3), a ClpP-like protein termed ClpR, and two adaptor proteins (ClpS1–2) (16). Of particular interest is the ClpR variant, which has protein sequence similarity to ClpP but appears to lack the catalytic triad of Ser-type proteases (17). This diversity of Clp proteins is even more extreme in photosynthetic eukaryotes, with at least 23 different Clp proteins in the higher plant Arabidopsis thaliana, most of which are plastid-localized (18).We have recently shown that two distinct Clp proteases exist in Synechococcus, both of which contain mixed proteolytic cores. The first consists of ClpP1 and ClpP2 subunits, and associates with ClpX, whereas the other has a proteolytic core consisting of ClpP3 and ClpR that binds to ClpC, as do the two ClpS adaptors (19). Of these proteases, it is the more constitutively abundant ClpCP3/R that is essential for cell viability and growth (20, 21). It is also the ClpP3/R complex that is homologous to the single type in eukaryotic plastids, all of which also have ClpC as the chaperone partner (16). In algae and plants, however, the complexity of the plastidic Clp proteolytic core has evolved dramatically. In Arabidopsis, the core complex consists of five ClpP and four ClpR paralogs, along with two unrelated Clp proteins unique to higher plants (22). Like ClpP3/R, the plastid Clp protease in Arabidopsis is essential for normal growth and development, and appears to function primarily as a housekeeping protease (23, 24).One of the most striking developments in the Clp protease in photosynthetic organisms and Apicomplexan parasites is the inclusion of ClpR within the central proteolytic core. Although this type of Clp protease has evolved into a vital enzyme, little is known about its activity or the exact role of ClpR within the core complex. To address these points we have purified the intact Synechococcus ClpP3/R proteolytic core by co-expression in E. coli. The recombinant ClpP3/R forms a double heptameric ring complex, with each ring having a specific ClpP3/R stoichiometry and arrangement. Together with ClpC, the ClpP3/R complex degrades several polypeptide substrates, but at a rate considerably slower than that by the E. coli ClpAP protease. Interestingly, although ClpR is shown to be proteolytically inactive, its inclusion in the core complex is not rate-limiting to the overall activity of the ClpCP3/R protease. In general, the results reveal remarkable similarities between the evolutionary development of the Clp protease in photosynthetic organisms and the eukaryotic proteasome relative to their simpler prokaryotic counterparts.     

Search for the Cellular Functions of Plant Hsp100/Clp Family Proteins

Referee: Dr. Peter Csermely, Department of Medical Chemistry, Semmeliweis Univ. School of Medicine, P.O. Box 260, H-1444 Budapest 8, Hungary Hsp100/Clp family of proteins is ubiquitously distributed in living systems. Detailed work carried out in bacterial and yeast cells has shown that regulatory members of the Clp family (mainly ClpA, ClpB, and ClpC), together with the catalytic subunit (mainly ClpP), comprise an ATP-dependent two-component proteolytic system. Members of the Hsp100/Clp protein family are not only involved in the regulation of energy-dependent protein hydrolysis but also function as molecular chaperones. However, the biochemical/physiological role(s) of the Hsp100/Clp protein family in higher plants has yet to be elucidated. Recently, this protein family has been implicated in plant stress responses: the hot1 mutant of Arabidopsis thaliana, which has mutation in hsp101 gene, and is defective in tolerance to high temperature (S.-W. Hong and E. Vierling, 2000, Proc Natl Acad Sci USA, 97 (8), 4392-4397) and the transgenic Arabidopsis thaliana plants overexpressing AtHsp101 gene exhibit high temperature tolerance (C. Quietsch et al., 2000, Plant Cell, 12, 479–492). Furthermore, the Hsp101 protein is involved in the translational regulation of cellular mRNAs and one such candidate has been identified as the photosynthetic electron transport gene Ferredoxin 1 mRNA (J. Ling et al., 2000, Plant Cell, 12, 1213–1227). We present what is known about the bacterial, yeast, and plant Hsp100/Clp proteins, discuss their possible relationship, and, more importantly, examine the cellular roles that this important family of proteins plays in plants.     

Characterization of bacterial proteases with a panel of fluorescent peptide substrates

Bacteria produce a range of proteolytic enzymes. In an attempt to detect and identify bacteria on the basis of their protease activity, a panel of protease substrates was investigated. Peptides conjugated to the fluorophore 7-amino-4-methylcoumarin (AMC) are well-established substrates for measuring protease activity. Although peptide-AMC substrates are generally not specific for a single protease, a unique pattern can be achieved for both highly specific enzymes and those with a broader substrate range by comparing different peptide substrates. The panel of 7 peptide-AMC substrates chosen exhibited a unique pattern for nine microbial proteases. The selected peptides were used to determine protease activity in cultured strains of Pseudomonas aeruginosa and Staphylococcus aureus. A signal pattern obtained with peptides with arginine, lysine, and tyrosine in the P1 position characterized the bacterial protease activities in these samples. The kinetic parameters for the three best substrates for the P. aeruginosa sample were calculated. Further information about substrate specificity was gained by the selective use of protease inhibitors. The results presented show that peptide-AMC substrates provide a simple and sensitive tool to characterize protease activity in microbiological samples and that they have the potential to identify and distinguish different bacterial species.