# Cellular Self-Digestion and Persistence in Bacteria
## 4 Intracellular Degradation Mechanisms
### Protein Degradation  
Proteases play a vital role in maintaining basal levels of regulatory proteins and removing misfolded and abnormal proteins from bacteria. These proteolytic enzymes can be divided into two groups, based on whether the cleavage position is inside the protein (endopeptidases or proteinases) or at the terminus (exopeptidases or peptidases) [184]. Depending on their cellular location, proteases can also be classified as cytoplasmic (e.g., Lon, ClpAP, ClpXP, HsIUV), periplasmic (e.g., Tsp, HtrA, protease III), or membrane proteases (e.g., FtsH, OmpT) [185; 186; 187; 188; 189; 190]. Although there are a few examples of energy-independent proteases, including protease III, VII, HtrA, Mi, and Tsp, the majority of intracellular proteolytic processes operate at the post-translational level and are powered by ATP hydrolysis [100; 71]. Specifically, ATP hydrolysis is required to change the conformation of the protease, unfold the substrate, and pass the substrate through the protease active site [191]. Energy-dependent proteases are highly significant in _E. coli_ and are responsible for more than 90% of the proteolytic activity taking place in the cytoplasm [71]. This model organism encodes five different AAA+ (ATPases associated with diverse cellular activities) proteases, Lon, ClpXP, ClpAP, HsIUV, and ClpYQ, as well as the essential protease FtsH [192; 193; 194]. The ATPase and proteolytic domains of these proteins are located at the bacterial cytoplasm; the former is responsible for initiation of substrate degradation by the ATP-dependent unfoldase and translocation of the unfolded protein to the proteolytic domain. Here, it is further broken down into smaller peptides, five to 25 amino acids in length, with the help of peptidase [193; 195]. In some cases, protein degradation can occur through a multistep process, with initial cleavage mediated by an ATP-dependent protease (rate-limiting step), followed by digestion via ATP-independent proteases and peptidases, ultimately leading to formation of free amino acids [71; 196]. Peptides display very high levels of activity, and only a small number of intermediate products of proteolysis are found in cells [71]. Proteases, particularly the ATP-dependent proteases, are extremely substrate-specific due to their structural features. They are also much larger in size (up to 750 kDa) than peptidases active in the extracellular medium, such as trypsin, which has a size of less than 50 kDa [196; 188]. From a thermodynamic point of view, protein degradation is spontaneous, even for ATP-dependent proteases such as Lon and ClpAP, which can degrade a trace amount of small peptides without the need of ATP [197; 198; 199]. Lon, the first and most widely studied ATP-dependent protease, is a cytoplasmic serine protease, which is considered to be the primary protease for quality control in _E. coli_[200]. It is involved in the degradation of misfolded proteins, along with certain major regulatory proteins, such as the cell division regulator, SulA, and the capsule synthesis regulator, RcsA [201; 202; 203; 204; 205; 206; 207]. Lon can play an active role in persister formation, as it degrades several labile antitoxins of type II TA modules, releasing intra-bacterial toxins that cause growth inhibition. For example, the antitoxin RelB is degraded by Lon, decreasing intracellular toxin-antitoxin levels and leading to the accumulation of free RelE toxin, which induces global inhibition of translation [208]. Other antitoxins degraded by Lon include CcdA, HipB, and MazE [208; 209; 210]. Intriguingly, reduced levels of fluoroquinone-tolerant persisters were observed in _Lon_-deficient cells [13; 211], although the question of whether this phenotype is dependent on the activity of TA modules is highly debatable [13; 212; 213]. As part of the DNA-damage response, the cell division inhibitor, SulA, is upregulated when the cells are treated with fluoroquinone antibiotics. Thus, in the absence of Lon, SulA accumulation may also affect persister cell survival [13; 211; 213; 214].