Caseinolytic Protease Protease Families: Comparison
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Bacterial proteases participate in the proteolytic elimination of misfolded or aggregated proteins, carried out by members of the AAA+ protein superfamily such as Hsp100/Clp (heat shock protein-100/caseinolytic protease), Lon, and FtsH. It is estimated that the Clp and Lon families perform around 80% of cellular proteolysis in bacteria. The HSP100/Clp family of ATPases plays crucial roles in the folding, assembly, and degradation of proteins during normal growth and, mainly, under stress-inducing conditions. This family is formed by several ATPase chaperones and the peptidase ClpP (caseinolytic protease proteolytic subunit).

  • Clp proteases
  • Gram-positive
  • chaperone-protease complex

1. Introduction

Proteolysis mediates the selective renewal of several cellular proteins, eliminating those that are defective or unwanted, thus allowing quality control of proteins and the different cellular processes [1]. One of the functions of bacterial proteases is the proteolytic elimination of misfolded or aggregated proteins, carried out by members of the AAA+ protein superfamily (ATPase associated with various cellular activities), such as Hsp100/Clp (heat shock protein-100/caseinolytic protease), Lon, and FtsH [2]. It has been estimated that the Clp and Lon families perform around 80% of cellular proteolysis in bacteria [3,4][3][4]. In addition, they control the proteolysis of regulatory proteins, such as key transcription factors that control the cell cycle and bacterial development or adaptation. These two opposite functions are regulated, in part, through the spatial and/or temporal use of adapter proteins, which participate in the recognition and delivery of specific substrate proteins to proteases [5,6][5][6].
The HSP100/Clp family of ATPases plays crucial roles in the folding, assembly, and degradation of proteins during normal growth and, mainly, under stress-inducing conditions [7,8][7][8]. This family is formed by several ATPase chaperones and the peptidase ClpP (caseinolytic protease proteolytic subunit). Chaperones are divided into two classes: Class I, whose members are ClpA (caseinolytic protease subunit (A), ClpB (caseinolytic protease subunit (B), and ClpC (caseinolytic protease subunit (C), which have two ATP-binding domains separated by a spacer region; and Class II, which includes ClpX (caseinolytic protease subunit X) and ClpY (caseinolytic protease subunit Y), that present only one domain of binding to ATP. Most of the chaperones bind to ClpP peptidase to form a proteolytic complex, with the exception of ClpY which only interacts with ClpQ, forming ClpQY peptidase, also known as HsIUV [9]. ClpQ is part of the Clp family, and like ClpP, is an ATP-dependent peptidase. However, it is one of the least-studied and its biological function and regulation are still not very clear. In addition, it has been shown to exhibit differences in the active site between Gram-positive and Gram-negative organisms [10].
The chaperone–ClpP complex is capable of degrading proteins in a specific manner where the chaperones can use ATP to promote protein folding changes and direct protein degradation by ClpP [11,12][11][12]. Although ClpP is part of this family, it does not have the same functions, since, unlike the rest, it is an ATP-dependent peptidase that, when associated with one of the chaperones, has serine protease activity [13]. Most bacteria contain the ClpXP protease, which makes ClpXP the most ubiquitous of the Clp proteases. On the other hand, ClpA and ClpC are orthologous; ClpA is usually found in Gram-negative bacteria, while ClpC is found in Gram-positive bacteria and cyanobacteria. ClpYQ exists together with ClpAP in most Gram-negative bacteria, and is also found in certain Gram-positive bacteria. Protein degradation dependent on these proteases has been studied in detail in the Gram-negative bacterium Escherichia coli (E. coli), whereas ClpCP has been characterized in the Gram-positive, spore-forming bacterium, Bacillus subtilis (B. subtilis) [9,14][9][14].
At the end of the 1990s, ClpP began to attract attention due to its potential as an antibacterial target, demonstrating that it participates in the bacterial virulence of the Gram-positive pathogens Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes). Since then, its use as a pharmaceutical target has been described in both Gram-positive and Gram-negative bacteria, being effective mostly in Gram-positive bacteria, where it proved to be effective in eliminating S. aureus, Enterococcus faecalis (E. faecalis), Streptococcus pneumoniae (S. pneumoniae), B. subtilis, and L. monocytogenes [9,15][9][15].

2. Clp Protease Families

To date, ATP-dependent proteases Lon, FtsH, and Clp have been characterized [1,2,16][1][2][16]. These complexes are responsible for maintaining a proper balance between protein synthesis and degradation at the cellular level, helping to maintain homeostasis. The peptidases of the Hsp100/Clp family are part of the quality control system of proteins both in normal growth and under stress conditions, playing an important role [17] (Figure 1 and Table 1).
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Figure 1. ClpP models in different bacteria. In green, it shows single ClpP peptidases forming 2 rings (B. subtilis and S. aureus). ClpP1 subunit in blue and ClpP2 in yellow.
The ClpP peptidase is well-conserved and characterized in different bacterial species. It has a structure formed by two rings of heptamers. The axial pore of each ring acts as an entrance to the proteolytic chamber, where there are 14 active sites formed by the catalytic triad serine-histidine-aspartate. In most organisms, the ClpP is formed by 14 identical monomers [1,15][1][15]. However, it can not only be found as a homodimer but also as a heterodimer, as in the case of ClpP1P2 from Mycobacterium tuberculosis (M. tuberculosis), where the active peptidase is composed of two isoforms of ClpP [18]. Pathogenic bacteria such as Pseudomonas aeruginosa (P. aeruginosa) and Clostridioides difficile (C. difficile) also have two isoforms of ClpP; nevertheless, each isoform can produce an active homotetradecamer [19,20][19][20] (Figure 1). This protein acts as a peptidase capable of degrading short peptides that can enter its proteolytic chamber through its narrow axial pores, so for more efficient proteolysis and ability to degrade larger proteins, it forms complexes with any of the chaperones described above [1] (Figure 2A). These chaperones recognize, unfolded through hydrolysis, and introduce in the proteolytic chamber of ClpP a range of substrates for their degradations. Although it has been seen that proteins that are already deployed can enter the ClpP proteolytic chamber without the help of chaperones, this occurs much slower than in the presence of chaperones [21,22][21][22].
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Figure 2. Composition and mechanism of the chaperone–peptidase complex. Substrates are recognized and unfolded by the hexameric chaperone (orange) using an adapter (brown). The unfolded substrate (red) is transferred into the proteolytic chamber of the ClpP (green), where proteolysis is carried out. (A) General composition of the chaperone protease complex. (B) Composition of the ClpCP chaperone–protease complex in B. subtilis.
On the one hand, ClpX and ClpC chaperones are highly conserved ATP-dependent proteins in Gram-positive bacteria, in addition to their classic chaperone functions, and can associate with ClpP to form the proteolytic complexes ClpXP and ClpCP [23], while it is believed that ClpB do not interact with ClpP [5] (Table 1). The proteolysis carried out by the Clp complexes plays an important role in maintaining and controlling protein quality, avoiding the accumulation and aggregation of unfolded proteins, and eliminating proteins that have already completed their life cycle or are truncated. This is not their only function, since they play an important role in bacterial pathogenesis, participating in the formation of virulent phenotypes and in the response to the different types of stress faced when entering the host or that occur in the environment [1,15][1][15]. Considering this, Clp proteases appear as a promising target against bacteria due to their participation in different essential cellular processes and phenotypes related to virulence. It has been observed that ClpXP peptidase is necessary for the degradation of proteins whose translation is stalled by a labeling system. ClpXP can recognize the SsrA-tag at the C-terminal end of the unfinished protein, degrading it and thus preventing its aggregation [24,25][24][25]. Meanwhile, the ClpCP peptidase acts in the control of protein quality by degrading unfolded, misfolded, or aggregated proteins, which accumulate under stress conditions such as heat shock [17,26][17][26]. In addition, it has been seen that they are participating in the controlled degradation of transcription factors such as the master regulator of competition ComK [27], the anti-sigma factor SpoIIAB involved in sporulation [28], and the oxidative stress transcription factor Spx [29]. In the case of ClpEP, its expression is tightly controlled and is only induced after a strong heat shock, suggesting that it could act as an additional system in other severe stress conditions [30].
Table 1.
Different proteases and chaperones in Gram-positive bacteria.
Organism Protease Chaperone Reference
B. subtilis ClpP–ClpQ ClpC–ClpE–ClpX [17]
S. aureus ClpP ClpC–ClpX [23]
M. tuberculosis ClpP1P2 ClpX–ClpC–ClpB [31]
C. difficile ClpP1–ClpP2 ClpB–ClpC–ClpX [20]

Regulation of Complex

As mentioned above, chaperones can recognize the substrates that will be degraded by ClpP. The best-described substrate class comprises proteins tagged with the ssrA tag, a short peptide sequence C-terminally added to proteins by the tmRNA system to rescue stalled ribosomes. However, for this, the presence of specific substrate adapter proteins, such as YjbH, TrfA, and McsB is required [32]. For example, ClpC requires adapter proteins for all its functions since it is only capable of forming a hexameric ring in the presence of the adapter protein MecA [33] (Figure 2B). These adapter proteins are also regulated through anti-adapter proteins or their phosphorylation of adapters mediated by different signals, for example, the anti-adapter protein ComS and the phosphorylation of the adapter proteins RssB and McsB. This series of regulations, in conjunction with the different Clp ATPases, allows an extensive regulation of the selection of the substrate that will be degraded by the ClpP peptidase. Other findings showed that the ClpP1P2 complex of M. tuberculosis requires the presence of an activating peptide; this peptide can be N-blocked dipeptide, usually Z-Leu-Leu (Benzyloxycarbonyl-L-Leucyl-L-Leucine), Z-Leu-Leu-H (Benzyloxycarbonyl-L-Leucyl-L-Leucinal), or a similar molecule, that binds near the active sites of the proteolytic particle and stabilizes the active conformation of the ClpP1P2 double-ring [34]. This functional conformation of the complex is also stabilized by the presence of the chaperone ClpX and protein substrate, acting synergistically with the activator peptide [35,36][35][36]. The requirement for activators seems to be a unique feature of Actinobacteria, as other species that contain two ClpP proteoforms, such as Listeria monocytogenes [37,38][37][38] and Chlamydia trachomatis [39], are functional in absence of activators. In the absence of proteolytic degradation via ClpP1P2, the levels of misfolded proteins can reach toxic levels, leading to cell death. Interestingly, E. coli ClpX rings can interact with M. tuberculosis ClpP1P2 complex and even promote substrate degradation more than ten-fold faster compared to the M. tuberculosis chaperones [36]. Another microorganism that possesses two isoforms of ClpP (ClpP1 and ClpP2) is C. difficile, but their functions are not yet known in detail. ClpP2 was only reported in hypervirulent strains, while ClpP1 has been reported in strains of ribotype 630 and hypervirulent strains, and it has been suggested that the ClpP isoforms act in an independent manner and possess different functions [40]. ClpP1 and ClpP2 have 74% and 63% identity, respectively, with ClpP from B. subtilis, an evolutionarily related organism, and sequence alignment showed that key regions including the catalytic triad are conserved. The evaluation of the proteolytic activity of each isoform was studied; however, only ClpP1 has proteolytic activity. On the other hand, it was revealed that both isoforms can form a complex with ClpX and degrade substrates labeled with the SsrA-tag, suggesting that the binding of the chaperone produces a change at the level of the ClpP2 peptidase that produces its activation [20].

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