An ATP-dependent degradation of 80% of cellular proteins is catalyzed by a macromolecular complex called the 26S proteasome that preferentially degrades these proteins that are soluble and poly-ubiquitinated (Hershko and Ciechanover, 1986). The 26S proteasome is composed of the 20S complex having the proteolytic core and the 19S, which is a regulatory complex that consists of heat-shock proteins, ATPases, and enzymes removing ubiquitin tags from the substrate proteins. The 19S proteasome thus provides the connection between the proteasome-mediated proteolysis and the ubiquitination process (Navon and Ciechanover, 2009). In high eukaryotes, the 20S proteasome is built of 4 rings (α-subunits form the outer rings and β-subunits the inner rings, respectively), with each one containing seven separate subunits forming together a cylinder-shaped structure (Hershko and Ciechanover, 1992).

The 20S proteasome is made up of threonine proteases that exhibit caspase-, trypsin-, and chymotrypsin-like activities (Finley et al., 2016); in effect, the product of proteasomal degradation is typically a short peptide, consisting of 8-12 residues. Interestingly, such peptides can be incorporated into other processes that include antigen presentation by the major histocompatibility complex (MHC) class I pathway (Galluzzi et al., 2017).

That proteasome complex is responsible for an enzymatic breakdown of oxidized, damaged, or misfolded proteins. It is also involved in the physiological and biochemical intracellular processes, including cell growth and differentiation, DNA replication and repair, or even cell metabolism and immune system response (Wang and Le, 2019).

For protein degradation by the 26S proteasome, the protein needs to be initially tagged with ubiquitin, a conserved 76 amino acids long protein with seven lysine residues in its chain. In the ubiquitination process, a covalent linkage of the ubiquitin is conducted by a group of enzymes in the following steps. The process begins with the activation of the ubiquitin by the E1 enzyme. The molecule that has been activated is then transferred to the member of the family of E2 enzymes, which play the role of ubiquitin-conjugating enzymes. In a final step, one of the widely distributed E3 ligases will bind to a ubiquitin-carrying E2 enzyme and catalyze the conjugation of ubiquitin to the lysine residues of the target protein. Cycles of ubiquitination can be repeated, resulting in manufacturing the polyubiquitin chain, which is further recognized by the elements of regulatory complex 19S of the proteasome (Ciechanover, 1993). Different levels of ubiquitination, starting from addition of just one molecule of ubiquitin to the target protein, as well as different lysine (K) residues of ubiquitin used for its conjugation with the substrate protein, allow for more precise control of the protein fate. For instance, mono-ubiquitination serves as signal for the regulation of DNA repair or gene expression. Polyubiquitination through K48 of ubiquitin leads to proteasomal degradation, and K63-linked ubiquitin plays a role in signaling, endocytosis, or even regulating kinase activation (Sadowski and Sarcevic, 2010). It was demonstrated that ubiquitination via K63 is important for the regulation of signal transduction by multiple receptors of both innate and adaptive immunity, including the TLRs, NLRs, TNFR, T- and B-cell antigen receptors and many other (Madiraju et al., 2022).

We can distinguish three ways of possible autophagic degradation of cytoplasmic material of exogenous or endogenous origin. Nevertheless, these autophagic responses are dependable on lysosomal activity but differ in the molecular mechanisms they operate on (Galluzzi et al., 2017). In microautophagy, cytoplasmic cargo is directly taken up by the lysosome. Macroautophagy is associated with morphological changes in vesicular compartments with the ability to ingest a sizeable part of the cytoplasm. The last possible mechanism concerns chaperone-mediated autophagy (CMA), where cytosolic proteins destined for degradation are delivered to the lysosomal lumen.

Microautophagy relies on the direct capture of cytosolic material through a lysosomal membrane invagination. It is still a poorly characterized mechanism in mammalian cells, unlike yeast or plants (Schuck, 2020). The lysosome, an organelle bound by a single lipid bilayer, supplies the cell with a variety of almost 60 hydrolases (including proteases, glycosidases, lipases, and nucleases). The highest activity of these enzymes occurs at low pH values of 4.5–5.5; the necessary highly acidic environment inside the lysosome is maintained by membrane-bound proton pumps (Mony et al., 2016). The significant difference between the ubiquitin-proteasome and the lysosomal systems is that the UPS degrades only proteins, whereas the latter can digest different macromolecules, aggregates, or even an extracellular material. The autophagy-lysosome system also plays a fundamental role in maintaining cellular homeostasis. Since the lysosomal hydrolases catabolize their substrates to the most elementary forms of compounds, there is an available recycled material for synthesizing biomolecules and performing metabolic reactions. This catabolic pathway, among others, initiates apoptosis, tissue remodeling, and cholesterol homeostasis (Yang and Klionsky, 2010).

Contrary to microautophagy, the autophagic process of macroautophagy has been investigated more thoroughly. In this case, the catabolic operation starts with incorporating cytosolic cargo by a de-novo generated vesicle with a continuous membrane called the phagophore. After that, the autophagosome formation is involved, a double-membrane vesicle that originated from the phagophore. Once the cytosolic cargo is sequestered, and the autophagosome is sealed, the content of the vesicle is intended for degradation via fusion with the lysosome, resulting in the formation of autolysosome (Pavel and Rubinsztein, 2017). The above-mentioned mechanism is typically induced by stress factors such as inefficient nutrients, malfunctioning mitochondria, protein aggregates, or pathogens. With the aim of autophagosome formation, a set of autophagy-related genes (ATG) is expressed, evolutionarily well-preserved from yeast to mammals. So-called core machinery includes approximately 20 ATG genes essential for most subtypes of autophagy (Ohsumi, 2006).

All the above-mentioned macroautophagy processes are present also in the cells of the adaptive immune system. Thus, macroautophagy is believed to be crucial for lymphocyte homeostasis, both for B and T cells, as well as for the survival of effector plasma cells. In all these cell types it seems to play an important role in glucose and lipid metabolism and in maintenance of functional mitochondria and endoplasmic reticulum. Finally, macroautophagy is implicated also in antigen presentation by B lymphocytes. Various autophagy-related gene (ATG) products, including beclin1; Atg 3, 5, 7, 12, 16L1; WIPI2, and LAMP2A are engaged in the development, activation, and apoptosis of either B or T or NK lymphocytes (Arbogast and Gros, 2018).

Many studies have shown that there are also signal transduction pathways involved in regulating autophagy. The mechanistic target of rapamycin (mTOR) belongs to one of the regulatory serine/threonine-protein kinases from the phosphoinositide 3 kinase-related kinase (PKK) family. It performs the function of central regulatory protein converging signals (nutrition level or energy resources) from inside and outside of the cellular environment, thereby managing cell growth, proliferation, and protein synthesis (Wang and Zhang, 2019). In that manner, the induction of the autophagy process is negatively regulated by mTOR. In the case of a cell being provided with nutrients, mTOR subsequently induces phosphorylation of proteins responsible for the autophagy induction step (Atg1 and Atg13). Such interaction finally results in the suppression of the autophagy process. As described in several publications (Harrison et al., 2009; Sutton et al., 2018; Green et al., 2022), in order to increase the life span and because improper nutrient sensing is another of the nine pillars of cellular aging, macroautophagy is activated with the use of mTOR inhibitors or caloric restriction. A commonly used inhibitor is rapamycin, a pharmacological agent proven to effectively block the growth of eukaryotic cells (Selvarani et al., 2021).

Chaperone-mediated autophagy classifies as the third type of autophagy. As opposed to micro- and macroautophagy, it is targeted against single proteins removed selectively. The recognition of unfolded protein can be executed by the cytosolic heat-shock cognate chaperone of 70 kDa (Hsc70) (Kaushik and Cuervo, 2018). Owing to the presence of a unique KFERQ motif in the sequence of cytosolic substrate protein, hsc70 specifically binds to the substrate and forms a chaperone-substrate complex along with other heat-shock proteins (such as Hsp90 or Hsp40). This complex can bind then to lysosome-associated membrane protein type 2A (LAMP2A), and the assistance of a chaperone allows for unfolding the substrate protein. Subsequently, LAMP2A undergoes multimerization and enables direct translocation of the targeted protein across the lysosomal membrane (Juste and Cuervo, 2019). The most noticeable activity of CMA is noted under the conditions that result in protein damage or when the cellular environment faces oxidative stress or deficiency of nutrients. Thus selectivity of CMA enables the degradation of only modified molecules without disturbing the protein balance nearby.

Calpains comprise the evolutionarily conserved family of neutral cytosolic cysteine proteases (Sorimachi et al., 2011). Their proteolytic activity strictly depends on a high concentration of calcium ions that greatly exceed a normal resting concentration of Ca²⁺ in the cytosol of resting cells, estimated as around 100 nM. The two first experimentally documented calpains (out of 14 described so far) are called conventional due to their ubiquitousness, similarity in sequence, and similar time of discovery (Yoshimura et al., 1983). In nomenclature, they come with the names: calpain I (μ-calpain) and calpain II (m-calpain) and are recognized by the concentration of Ca²⁺ required for their full proteolytic activation in vitro; the term μ-calpain reflects the concentrations of 1-100 μM Ca²⁺, whereas m-calpain 0,1-1 mM Ca²⁺ respectively (Ono and Sorimachi, 2012).

Calpains’ presence is reported in almost all eukaryotes and some bacteria, except for archaebacteria. They are proven to be expressed in all mammalian tissues with limited tissue-specificity. Only a minor fraction of calpain proteases become tissue-specific. Calpain III (historically known also as p94) can be used as an example of non-ubiquitous calpain, with its expression mainly restricted to the stomach (Sorimachi et al., 1993). Calpain-mediated proteolysis differs significantly from previously described proteolytic systems in a mode of action. Calpains act through proteolytic processing instead of completely degrading the protein. They perform somewhat limited hydrolysis of only one or a few sites in the substrate protein, modulating its structure and, ultimately, its activity or cellular function. For that reason, the calpain family is referred to as modulator proteases (Sorimachi et al., 2011). Still, the activity of calpain itself is tightly regulated by the other component, calpastatin, which represents the only endogenous specific inhibitor for conventional calpains.

The expression of calpains and calpastatin in human and murine lymphocytes is studied since the late 80’s of the XXth century (Takano et al., 1988; Murachi et al., 1990; Deshpande et al., 1993). Their function in the immune cells varies from regulation of protein kinase C (Shenoy and Brahmi, 1991), participation in activation and activation-induced cell death (AICD) of lymphocytes (Sarin et al., 1994; Deshpande et al., 1995), modification of T cell cytoskeleton and movement (Selliah et al., 1996; Stewart et al., 1998; Rock et al., 2000; Babich and Burkhardt, 2013), and intracellular signaling (Rock et al., 1997; Penna et al., 1999; Li and Iyengar, 2002; Hendry and John, 2004). Inhibition of calpains in activated peripheral blood mononuclear cells (PBMC) resulted in decreased secretion of IL-2 and reduced expression of CD25 on the surface of T cells (Schaecher et al., 2001). Its effect on intracellular signaling and migration of human monocytes was also documented (Noma et al., 2009). We have demonstrated that calpains are activated in stimulated human T cells (Mikosik et al., 2007), and that calpain inhibition in resting human T cells greatly reduces their proliferative response to polyclonal activation, secretion of multiple cytokines, as well as activation-induced phosphorylation of p56Lck, NFkB, and PLCgamma (Mikosik et al., 2016). Calpains and calpastatin constitute the calpain-calpastatin system (CCS), where both proteins are in stoichiometric balance in the cytoplasm and influence each other’s activity. We have shown this to be true for human T and B lymphocytes, simultaneously demonstrating that the amounts of both calpain 1 and 2 and calpastatin vary between CD4⁺ and CD8⁺ T cells and B lymphocytes (Mikosik et al., 2013). Like the aforementioned protein quality control systems, the calpain family is no exception to being physiologically relevant. It has been proven that calpains can regulate at least over 300 substrate proteins that are components of the signal transduction pathway, apoptosis, or immune system response (Ono and Sorimachi, 2012; Witkowski et al., 2021).

Caspases belong to the evolutionarily conserved family of cysteine-dependent endoproteases and hydrolyze their protein substrates sequence-specifically. When remaining inactive, caspases occur in the zymogen form. They undergo proximity-induced autoactivation triggered by dimerization-induced conformational changes where a linker between small and large subunits is proteolytically processed. Caspases are mainly responsible for inducing apoptosis, a homeostatic caspases process of programmed cell death, where either aged or damaged cells are eliminated. In apoptosis, caspases, depending on their function, fall into two distinct subtypes: initiator caspases (caspases 8, 9, and 10) or effector caspases (caspases 3, 6, and 7) that execute the ultimate cleavage of cellular components (Horvitz, 1999). Signaling events propagated by apoptotic caspases result in characteristic morphological changes in the cells undergoing apoptosis, and these typically include membrane blebbing, chromosomal DNA fragmentation, forming of apoptotic bodies, and cell death as the last step (Ramirez and Salvesen, 2018). What is more, an extensive body of evidence from past decades implicates caspases’ participation in another form of regulated cell death, pyroptosis, where pro-inflammatory caspases (such as caspase 1, caspase 5, and probably also caspase 4) execute cell termination. By definition, pyroptosis is a pro-inflammatory strategy for the regulated cell death and involves the secretion of the inflammatory cytokines interleukin (IL)-1β and IL-18 (Vande Walle and Lamkanfi, 2016). The innate immunity system is provided with pattern recognition receptors (PRRs) that scan the cellular environment for the presence of pathogen-associated molecular patterns (PAMPs) or host-derived molecules suggesting damage of host cells (DAMPs). Activated PRRs initiate an inflammasome assembly that subsequently recruits caspase 1 in the form of cytosolic zymogen to its active form. Later on, active inflammatory caspase 1 cleaves the inactive prointerleukins 1β and 18 to active IL-1β and IL-18 and gasdermin D (GSDMD) that oligomerizes to form membrane pores. It disrupts cell membrane integrity, which finally causes cell death and leakage of the pro-inflammatory cytokines and GSDMD itself (Liu and Lieberman, 2017). The important role of different caspases in T lymphocyte development and control of the immune reactivity (including proliferation, cell cycle progression, and the AICD/apoptosis) is well documented since decades (Denis et al., 1998; Los et al., 2001; Lakhani and Flavell, 2003; Schwerk and Schulze-Osthoff, 2003; Sehra and Dent, 2006; Brenner et al., 2008; Walsh and Edinger, 2010). Interestingly, it was recently shown that T lymphocytes, in addition to innate immune cells, harbor the components of classical and non-classical inflammasomes, which may get activated and, via various activation of caspase-1 and caspase 8, may be directed towards differentiated Th1, Th2 or Th17 and finally, similarly to innate immune cells, undergo pyroptosis upon GSDMD activation (Linder and Hornung, 2022).

A disintegrin and metalloprotease (ADAM) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) belong to zinc-dependent endopeptidases. They play an essential role in maintaining organ and tissue homeostasis. At the same time, they are responsible for the disruption of extracellular matrix proteins in disorders such as cancer or rheumatoid arthritis (RA). The ADAMs are multidomain transmembrane proteins, and their activity is implicated in proteolysis, cell proliferation, and adhesion (Mullooly et al., 2016). They also constitute major proteases associated with the shedding of transmembrane protein ectodomains. Here, a notable example is a soluble form of “anti-aging hormone,” Klotho, the ectodomain of which is shed (mainly from the surface of kidney cells with the ADAM10 and ADAM17 proteases). The reduced levels of soluble Klotho are associated with shorter life and certain aging-related diseases, including chronic kidney disease and vascular calcification leading to cardiovascular complications (Saar-Kovrov et al., 2021). The ADAMTSs are secreted and multidomain proteases and in control of the extracellular matrix structure. Unlike ADAMs, members of the ADAMTS family show a rather narrow substrate specificity (Kelwick et al., 2015). For instance, ADAMTS4 and ADAMTS5 can degrade aggrecan and therefore contribute to cartilage erosion (Rose et al., 2021). In human lymphocytes (Lambrecht et al., 2018), the ADAM proteases (mainly ADAM10 and ADAM17) are involved in their communication with structural cells by cleaving Notch, CD23, and CD44. ADAM10 and ADAM17 cleavage is necessary for activation, differentiation, and migration of helper T cells and their interaction with B lymphocytes, by affecting multiple target proteins, including CD40L, CD25, CD137, and CD154. In addition, ADAM17 reduces the expression of IFN-γ (Lambrecht et al., 2018; Sezin et al., 2022). On the other hand, the ADAMTS proteases were as yet not extensively studied in the human immune system and if, then only in pathological settings. Thus, their role in the development of atherosclerotic lesions is being suggested, via modulation of macrophage functions in arterial intima (Solanki et al., 2018). Specific ADAMTS protease, the ADAMTSL5 is considered an autoantigen in psoriasis (which however has nothing to do with its proteolytic activity; the same protein is apparently increasing the frequency of IL-17A and IFN-γ-expressing CD8⁺ T-cells, but only in psoriasis patients and not in healthy individuals. There are so far no mechanistic explanations for this phenomenon (Furue and Kadono, 2019).