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.     

Distinctive types of ATP-dependent Clp proteases in cyanobacteria

Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis and are thought to be ancestors to plant chloroplasts. Like chloroplasts, cyanobacteria possess a diverse array of proteolytic enzymes, with one of the most prominent being the ATP-dependent Ser-type Clp protease. The model Clp protease in Escherichia coli consists of a single ClpP proteolytic core flanked on one or both ends by a HSP100 chaperone partner. In comparison, cyanobacteria have multiple ClpP paralogs plus a ClpP variant (ClpR), which lacks the catalytic triad typical of Ser-type proteases. In this study, we reveal that two distinct soluble Clp proteases exist in the unicellular cyanobacterium Synechococcus elongatus. Each protease consists of a unique proteolytic core comprised of two separate Clp subunits, one with ClpP1 and ClpP2, the other with ClpP3 and ClpR. Each core also associates with a particular HSP100 chaperone partner, ClpC in the case of the ClpP3/R core, and ClpX for the ClpP1/P2 core. The two adaptor proteins, ClpS1 and ClpS2 also interact with the ClpC chaperone protein, likely increasing the range of protein substrates targeted by the Clp protease in cyanobacteria. We also reveal the possible existence of a third Clp protease in Synechococcus, one which associates with the internal membrane network. Altogether, we show that presence of several distinctive Clp proteases in cyanobacteria, a feature which contrasts from that in most other organisms.     

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram‐positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl‐leucyl‐leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP‐activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP‐dependent protein degradation.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

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.     

Discovery of a Unique Clp Component,ClpF, in Chloroplasts: A Proposed Binary ClpF-ClpS1 Adaptor Complex Functions in Substrate Recognition and Delivery

Clp proteases are found in prokaryotes, mitochondria, and plastids where they play crucial roles in maintaining protein homeostasis (proteostasis). The plant plastid Clp machinery comprises a hetero-oligomeric ClpPRT proteolytic core, ATP-dependent chaperones ClpC and ClpD, and an adaptor protein, ClpS1. ClpS1 selects substrates to the ClpPR protease-ClpC chaperone complex for degradation, but the underlying substrate recognition and delivery mechanisms are currently unclear. Here, we characterize a ClpS1-interacting protein in Arabidopsis thaliana, ClpF, which can interact with the Clp substrate glutamyl-tRNA reductase. ClpF and ClpS1 mutually stimulate their association with ClpC. ClpF, which is only found in photosynthetic eukaryotes, contains bacterial uvrB/C and YccV protein domains and a unique N-terminal domain. We propose a testable model in which ClpS1 and ClpF form a binary adaptor for selective substrate recognition and delivery to ClpC, reflecting an evolutionary adaptation of the Clp system to the plastid proteome.     

The C-Terminus of ClpC1 of Mycobacterium tuberculosis Is Crucial for Its Oligomerization and Function

Mycobacterium tuberculosis ClpC1 is a member of the Hsp100/Clp AAA+ family of ATPases. The primary sequence of ClpC1 contains two N-terminal domains and two nucleotide binding domains (NBD). The second NBD has a long C-terminal sub-domain containing several motifs important for substrate interaction. Generally, ClpC proteins are highly conserved, however presence of C-terminal domains of variable lengths is a remarkable difference in ClpC from different species. In this study, we constructed deletion mutants at the C-terminus of M. tuberculosis ClpC1 to determine its role in the structure and function of the protein. In addition, a deletion mutant having the two conserved N-terminal domains deleted was also constructed to investigate the role of these domains in M. tuberculosis ClpC1 function. The N-terminal domains were found to be dispensable for the formation of oligomeric structure, and ATPase and chaperone activities. However, deletions beyond a specific region in the C-terminus of the ClpC1 resulted in oligomerization defects and loss of chaperonic activity of the protein without affecting its ATPase activity. The truncated mutants, defective in oligomerization were also found to have lost the chaperonic activity, showing the formation of oligomer to be required for the chaperonic activity of M. tuberculosis ClpC1. The current study has identified a region in the C-terminus of M. tuberculosis ClpC1 which is essential for its oligomerization and in turn its function.     

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.     

Interaction specificity between the chaperone and proteolytic components of the cyanobacterial 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. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.     

Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus

The Hsp100/Clp ATPases constitute a family of closely related proteins of which some members function solely as chaperones whereas others additionally can associate with the unrelated ClpP peptidase forming a Clp proteolytic complex. We have investigated the role of four Clp ATPases in the versatile pathogen, Staphylococcus aureus. Previously, we showed that ClpX is required for expression of major virulence factors and for virulence of S. aureus, but not for survival during heat shock. In the present study, we have inactivated clpC, clpB and clpL and, while none of these mutations affected toxin production, both ClpC and ClpB and to a minor extent ClpL were required for intracellular multiplication within bovine mammary epithelial cells. These defects were paralleled by an inability of the clpC mutant to grow at high temperature and of the clpB mutant to induce thermotolerance indicating that the protective functions of these proteins are required both at high temperature and during infection. By primer extension analysis and footprint studies, we show that expression of clpC and clpB is controlled by the negative heat-shock regulator, CtsR, and that ClpC is required for its repressor activity. Thus, ClpC is a likely sensor of stress encountered during both environmental stress and infection. In addition to virulence factor production the ability to form biofilms is of importance to S. aureus as a nosocomial pathogen. Interestingly, biofilm formation was reduced in the absence of ClpX or ClpC whereas it was enhanced in the absence of ClpP. Thus, our data show that Clp proteolytic complexes and the Clp ATPases control several key processes of importance to the success of S. aureus as a pathogen.     

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.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

ATP-dependent association between subunits of Clp protease in pea chloroplasts

The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of the proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both subunits are found in the stroma, the interaction between the two is dynamic. When immunoprecipitation with antibodies against ClpC was performed on stroma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-gamma-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma was fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma depleted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrated separately, whereas in the presence of ATP-gamma-S both subunits co-migrated. Similar results were observed in size-exclusion chromatography. To further characterize the precipitated enzyme, its proteolytic activity was assayed by testing its ability to degrade beta-casein. No degradation was observed in the absence of ATP, and degradation was inhibited in the presence of phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested using chimeric OE33 as a model substrate. This protein was also degraded in an ATP-dependent manner, supporting the suggested role of Clp protease as a major housekeeping protease in the stroma.     

耻垢分枝杆菌ClpC和ClpX敲低表达菌株的构建及表型分析*

蛋白质平衡稳定对细菌的生长繁殖以及应对宿主免疫压力十分重要。Clp蛋白酶复合体在结核分枝杆菌的蛋白质降解和平衡稳定中发挥重要作用。Clp蛋白酶中负责识别底物蛋白并将其解折叠的蛋白质有两种:ClpC和ClpX。为初步探究分枝杆菌中ClpC和ClpX各自的功能特点,运用CRISPRi的方法成功构建了耻垢分枝杆菌的ClpC和ClpX诱导型敲低表达菌株,并对其生长相关表型进行分析。结果显示:与野生菌株相比,ClpC和ClpX的低表达均能严重影响耻垢分枝杆菌的生长。ClpC低表达可导致菌株丧失生物膜的形成能力,而ClpX低表达则导致菌株无法维持正常细胞形态,电镜显示细胞壁不完整且细胞呈丝状化,提示ClpC和ClpX可能在分枝杆菌中具有不同的生理功能。可为后期深入开展ClpC和ClpX对分枝杆菌生理调控功能研究及新型抗结核药物筛选提供基础。     

The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins

The presence of the heat stress response-related ATPases ClpC and ClpX or the peptidase ClpP in the cell is crucial for tolerance of many forms of stress in Bacillus subtilis. Assays for detection of defects in protein degradation suggest that ClpC, ClpP, and ClpX participate directly in overall proteolysis of misfolded proteins. Turnover rates for abnormal puromycyl peptides are significantly decreased in clpC, clpP, and clpX mutant cells. Electron-dense aggregates, most likely due to the accumulation of misfolded proteins, were noticed in studies of ultrathin cryosections in clpC and clpP mutant cells even under nonstress conditions. In contrast, in the wild type or clpX mutants such aggregates could only be observed after heat shock. This phenomenon supports the assumption that clpC and clpP mutants are deficient in the ability to solubilize or degrade damaged and aggregated proteins, the accumulation of which is toxic for the cell. By using immunogold labeling with antibodies raised against ClpC, ClpP, and ClpX, the Clp proteins were localized in these aggregates, showing that the Clp proteins act at this level in vivo.     

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.     

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.