Lysosomes are specialized membrane-enclosed cytoplasmic organelles responsible for the degradation and recycling of several macromolecules such as proteins, carbohydrates, lipids, and nucleic acids. Endocytic, autophagic, and phagocytic pathways are responsible for the transportation of the cargos that feed the lysosomes. Once in the lysosomes, more than 50 different hydrolases oversee the processing of the different macromolecules for their subsequent reutilization. Lysosomes have been classically considered the “garbage disposals” of the cell. However, this paradigm has been overturned in recent decades, and the participation of the lysosomes in several cellular processes such as cell adhesion and repair, immunity, metabolic signaling, gene regulation, and migration has been reported [1]. In addition, lysosomal hydrolases, which were considered strictly functional and restricted to the acidic lysosomal lumen, are now known to display activity in diverse cellular locations, even at neutral pH for a limited period [2], such as the cytosol [3], the pericellular surroundings (PCSs) [4] and even the nuclei [5].

Lysosomal hydrolases are proteolytic enzymes or proteases that display their greatest stability at the acidic pH present within these organelles. Previously considered random catalytic devices, proteases are currently recognized to cleave specific substrates in an efficient and highly selective manner through limited proteolysis. In addition, it is important to acknowledge that proteases do not operate alone but in regulatory circuits and signaling cascades [6]. As a result, targeted proteolytic processing of substrates finely tunes several cellular processes such as apoptosis, cell-cycle, cell migration, immune cell activation, and extracellular matrix (ECM) remodeling among others [7]. Proteolytic impairment or dysregulation is responsible for most lysosomal storage disorders (LSDs) [8] and contributes to other human pathologies including neurodegenerative disorders [9], cancer [6], arthritis [10], and liver disease [11]. Several excellent revisions have been published on the contribution of lysosomal hydrolases to the progression of several pathological conditions [12]. However, no specific revision has summarized the specific role of lysosomal hydrolases in the development, progression, and resolution of liver fibrosis, something this review aims to address.

Cathepsins are the most abundant lysosomal hydrolases present within the lysosome but other types such as naspin A and asparagine endopeptidase (AEP) can also be found. Naspin A is mainly expressed in kidney and lung and its expression has only been detected in liver during liver cancer [13]. AEP expression has been reported in liver but its role during liver disease progression has not yet been explored [14]. For this reason, this review will mainly focus on the role of cathepsins in liver fibrosis.

The name cathepsin derives from the combination of Greek verbs cata- (digest) and hepsein (boil), defining their degradative capacity. All cathepsins are synthesized as inactive precursors in the ribosome including an N-terminal signal peptide to route them to the endoplasmic reticulum (ER), where simultaneous N-linked glycosylation and cleavage of the N-terminal peptide occurs. Phosphorylation of the high-mannose residue glycans in the Golgi causes the generation of mannose-6-phosphate (M6P) residues, which are recognized by the M6P receptors present in the endo/lysosomes and directs them to these cellular compartments. Triggered by the acidic pH of the lysosomes, cathepsins undergo cleavage of the pro-region, thought to act as an auto-inhibitor. This processing results in the generation of a double or a single chain, which are both active forms of the enzyme [2]. Alternative routes to reach the lysosomes [15] and the extracellular space [16] can occur in the absence of M6P residues.

There are three main classification systems for proteases currently in use, which group them according to either the amino acid present in their catalytic site (metallo, serine, cysteine, aspartic, threonine, glutamic, and asparagine) [17]; their catalytic reaction (endopeptidases, omega-peptidases, exopeptidases, aminopeptidases, carboxypeptidases, dipeptidyl-peptidases, tripeptidyl-peptidases, peptidyl-dipeptidases, and dipeptidases; https://www.qmul.ac.uk/sbcs/iubmb/) or their sequence homology and molecular structure (http://merops.sanger.ac.uk) [18]. The most commonly used classification for cathepsins is based on their catalytic site: aspartate (cathepsin D and E), cysteine [cathepsin B, C/dipeptidyl peptidase 1 (DPP1), F, H, K, L, O, S, V, W, and Z/X] and serine (cathepsin A and G). Cathepsins exhibit endo and exopeptidase activity and are ubiquitously distributed across tissues, except for cathepsin K (mainly expressed in osteoclasts and epithelial cells) and cathepsin S, E, and W (immune cells). The proteolytic activity of cathepsins is subjected to several regulatory steps at transcriptional [19], epigenetic [20], translational [21], and post-translational [22] levels. In addition, besides the pH, lysosomal hydrolases are also regulated by endogenous inhibitors such as cystatins and stefins [23]. This is essential as cathepsins contribute to several homeostatic cellular events, such as apoptosis, autophagy, chemotaxis, and antigen presentation. Hence, dysregulation of cathepsin activity is linked to the development and progression of several human diseases, ranging from liver disease to cancer. The following sections will discuss the main contributions of cathepsins to the different cellular events responsible for the development, progression, and resolution of liver fibrosis.

Cysteine cathepsins are the largest cathepsin family, it encompasses 11 members (B, C, F, H, K, L, O, S, V, W, and Z/X) and shares the characteristic of presenting a cysteine in their catalytic site. Most cysteine cathepsins are ubiquitously expressed in tissues (B, C, F, H, L, O, V, and X). Conversely, cathepsin K, W, and S have a more specific tissue distribution. While cathepsin K is highly expressed in osteoclasts, cathepsin W [CD8⁺ lymphocytes and natural killer (NK)] and S [B-cells, dendritic cells (DCs), antigen presenting cells (APCs)] are expressed in immune cells.

Among the lysosomal hydrolases, cysteine cathepsins are the most extensively studied family of proteases in liver fibrosis and have been described to contribute to several important biological processes fundamental for its development and progression, which will be described in more details in the following subsections.

Aspartic and serine cathepsins are smaller families than cysteine cathepsins and display an aspartate or serine amino acid in their catalytic site, respectively. Both families have two members, cathepsin D and E are aspartyl hydrolases and cathepsin A and G are serine hydrolases. As cathepsin E and G are mainly expressed in immune cells, for that reason this section will focus on the role of cathepsin D and A in liver fibrosis.

Both cathepsin D and A have been far less studied than other cathepsins, such as cathepsin B, and their participation in the different cellular processes that contribute to liver fibrosis development is unclear. Similarly to cathepsin B, cathepsin D has been reported to participate in the canonical apoptotic pathway, upstream mitochondria, after TNF-α stimulation. Evidence suggests that cathepsin D proteolytically cleaves Bid into truncated Bid (t-Bid), resulting in the release of cytochrome c from mitochondria and the activation of the effector caspases, culminating in apoptosis [46]. Cathepsin D has also been shown to activate cathepsin B in HSC in a paracrine way, contributing to HSC activation [30]. Furthermore, a positive correlation between cathepsin D and autophagy with the ductular reaction (DR) has been found in human cirrhotic patients, suggesting cathepsin D could play a role in autophagy in the DR associated with cirrhosis [47]. Finally, cathepsin A has also been related to autophagy, as in vitro evidence in isolated lysosomes proposes that cathepsin A degrades lysosome associated membrane protein 2a (LAMP2a), restricting the rate of chaperone-mediated autophagy [48].

Experimental therapeutic approaches to slow-down liver fibrosis progression using cathepsin inhibitors have been reported in the literature but none of them have moved forward into clinical trials yet. The deeper understanding of the biological roles that cysteine cathepsins play during liver fibrosis, in comparison with aspartyl or serine cathepsins, has translated into more effective experimental approaches to reduce fibrosis using pharmacological inhibitors in vivo. To that respect, the use of cathepsin B inhibitors has been particularly successful, resulting in a decrease of experimental liver fibrosis from different aetiologies: CCl₄ chronic administration [30, 33, 40], bile duct ligation [26, 33], and high-fat choline-deficient (HFCD) feeding [40] in mouse. In addition, cathepsin B inhibitor has also been reported to decrease TNF-α-induced liver damage, reducing hepatocyte apoptotic injury [28]. A reduction in hepatocyte apoptosis can potentially also have a positive effect during fibrosis by reducing liver damage and the inflammatory response that feeds into the activation of HSC. For all of these reasons, cathepsin B seems to be the best candidate within the lysosomal hydrolases for the development of cathepsin-based drugs for the treatment of liver fibrosis. However, as cathepsin B plays a multifaceted role in the development of liver fibrosis contributing to different biological processes in different cell types (Table 1 and Figure 1), therapeutic interventions need to be carefully considered to avoid undesirable side effects.

Despite cathepsin B apparent suitability for drug development for liver fibrosis, growing knowledge and understanding of the participation of other cathepsins and their inhibition in other liver diseases [11] might provide new insights into their potential use as therapeutics for liver fibrosis. Finally, as cathepsins also play important roles in other diseases such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and cancer, research done in these disciplines may render novel cathepsin inhibitors [49, 50], which could potentially be repurposed for their use as liver fibrosis treatments.

Our understanding of lysosomal hydrolases has shifted over the last decades from waste-bag effectors to multipurpose enzymes that play important roles in several cellular homeostatic and pathological processes. In the context of liver disease, the contribution of lysosomal hydrolases to the development, progression, and resolution of liver fibrosis is no longer contested. Lysosomal hydrolases are expressed in both liver resident cells and infiltrating cells, and participate in apoptosis, activation of HSC, inflammation, autophagy, and ECM remodeling. Considering the variety of cellular processes that lysosomal hydrolases influence, their expression in several cell types and their various cellular locations, potential therapies need to be carefully designed to ensure maximal effectiveness with minimal side effects. Despite our increased knowledge on the basic functions of lysosomal hydrolases in liver fibrosis, their specific substrates and proteolytic networks are still unknown. Thus, it is essential to unravel the proteolytic signature of liver fibrosis and its contribution to disease progression in order to design novel and targeted therapies based on lysosomal hydrolases for the treatment of liver fibrosis.