Trafficking of Full-Length and N-Terminally Truncated Cathepsin B in Human Colorectal Carcinoma Cells

Cathepsin B is an endo-lysosomal cysteine protease. However, its increased expression and altered localization to the extracellular space, to mitochondria, or to the nucleus has been linked to tumor progression. In the present study, we show enhanced levels of cathepsin B in adenocarcinoma tissue in comparison to adjacent normal colon. Additionally, cathepsin B was observed in the nuclear compartment of mucosal cells in adenocarcinoma tissue samples and in the nuclei of the colorectal carcinoma cell line HCT116. Accordingly, a distinct 40-kDa form of cathepsin B was detected in HCT116 cells, which is proposed to represent a specific form lacking the signal peptide and parts of the propeptide. Trafficking studies with an EGFP-tagged N-terminally truncated form, mimicking the 40-kDa form, demonstrated accumulation in aggresome-like inclusion bodies, while EGFP-tagged full-length cathepsin B revealed regular sorting to endo-lysosomes. We conclude that the identity of nuclear cathepsin B in colorectal adenocarcinoma (in situ) and in carcinoma cells (in vitro) cannot be attributed to either full-length or 40-kDa N-terminally truncated cathepsin B forms. Hence, future studies are needed to demonstrate which form/s of cathepsin B may be sorted to the nuclei of colorectal carcinoma cells, and whether redundant regulation of related cathepsin expression occurs.

Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

Cathepsin Release from Lysosomes Promotes Endocytosis of Clostridium perfringens Iota-Toxin

Iota-toxin from Clostridium perfringens type E is a binary toxin composed of two independent proteins: actin-ADP-ribosylating enzyme component, iota-a (Ia), and binding component, iota-b (Ib). Ib binds to target cell receptors and mediates the internalization of Ia into the cytoplasm. Extracellular lysosomal enzyme acid sphingomyelinase (ASMase) was previously shown to facilitate the internalization of iota-toxin. In this study, we investigated how lysosomal cathepsin promotes the internalization of iota-toxin into target cells. Cysteine protease inhibitor E64 prevented the cytotoxicity caused by iota-toxin, but aspartate protease inhibitor pepstatin-A and serine protease inhibitor AEBSF did not. Knockdown of lysosomal cysteine protease cathepsins B and L decreased the toxin-induced cytotoxicity. E64 suppressed the Ib-induced ASMase activity in extracellular fluid, showing that the proteases play a role in ASMase activation. These results indicate that cathepsin B and L facilitate entry of iota-toxin via activation of ASMase.

Calpain-2 participates in the process of calpain-1 inactivation by Shinkai-Ouchi et al.

Calpain belongs to the calcium-dependent non-lysosomal cysteine protease. Calpain-1 and calpain-2 expression are ubiquitous in mammals and an important mediator of the action of calcium. Specific substrate cleavage by calpain-1 and calpain-2 is critical for several calcium-dependent cellular pathways including neuronal function, muscle contraction, signal transduction, cell differentiation, proliferation, and apoptosis.Research suggests that calpain-1 and calpain-2 perform similar functions due to their structurally highly similar isoforms. Increasing evidence suggests that calpain-1 and calpain-2 carry out their specific function in vivo. A recent paper published by Shinkai-Ouchi et al. (Bioscience Reports (2020) 40, https://doi.org/10.1042/BSR20200552) elucidated the mechanism to differentiate the function of each calpain in respect to the efficiency and longevity for proteolysis after activation. Further, the study represented that calpain-1 and calpain-2 do not synergistically perform their workin vitro. On the other hand, the activity of calpain-1 is reduced in presence of calpain-2. This insight establishes the platform for future studies to examine how calpain-2 regulates the calpain-1 for substrate proteolysis.

Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.

Inhibition of Cathepsin S Induces Mitochondrial Apoptosis in Glioblastoma Cell Lines Through Mitochondrial Stress and Autophagosome Accumulation

Cathepsin S (CTSS), a lysosomal cysteine protease, is overexpressed in various cancers, including glioblastoma (GB). A high level of CTSS is associated with tumor progression and poor outcome in GB. However, the underlying mechanisms of its role in the biological characteristics of G5B remain to be elucidated. Here, we uncovered a potential role of CTSS in the lysosomes and mitochondria of GB cells (GBCs). Downregulation of CTSS in GBCs could increase the expression of autophagy-related proteins; however, there was no significant change in p62, suggesting autophagy blockade. Moreover, inhibition of CTSS increased the expression of mitochondrial calcium uniporter (MCU) and enhanced mitochondrial Ca2+ uptake ability, causing mitochondrial Ca2+ overload, the generation of copious reactive oxygen species (ROS) and eventual mitochondrial apoptosis. Additionally, elevated damage to mitochondria exacerbated the burden of autophagy. Finally, we found that silence of MCU could alleviate the inhibition of CTSS-induced autophagosome accumulation and mitochondrial stress. Collectively, these results demonstrate that CTSS plays an important role in the process of autophagic flux and mitochondrial functions in GBCs.

Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

Rhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases.

Fibrillar α-synuclein toxicity depends on functional lysosomes

Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability was not recovered by these inhibitors except for liproxstatin-1, a best-in-class ferroptosis inhibitor, when used at high doses. High-dose liproxstatin-1 visibly enlarged the area of a cell that contained acidic vesicles and elevated the expression of several proteins associated with the autophagy-lysosomal pathway similarly to the known lysosomal inhibitors, chloroquine and bafilomycin A1. Consistent with high-dose liproxstatin-1 protecting via a lysosomal mechanism, we further de-monstrated that loss of viability induced by α-synuclein PFFs was attenuated by chloroquine and bafilomycin A1 as well as the lysosomal cysteine protease inhibitors, leupeptin, E-64D, and Ca-074-Me, but not other autophagy or lysosomal enzyme inhibitors. We confirmed using immunofluorescence microscopy that heparin prevented uptake of α-synuclein PFFs into cells but that chloroquine did not stop α-synuclein uptake into lysosomes despite impairing lysosomal function and inhibiting α-synuclein toxicity. Together, these data suggested that α-synuclein PFFs are toxic in functional lysosomes in vitro. Therapeutic strategies that prevent α-synuclein fibril uptake into lysosomes may be of benefit in PD.