Activity Of Cysteine Cathepsins Research Articles

Investigating how CSTB affects the activity of cysteine cathepsin in the AD‐DS, EOAD, and human cellular models of trisomy Hsa21

AbstractBackgroundPeople with Down syndrome (DS) have a high genetic risk of developing Alzheimer’s disease (AD) ‐related dementia and pathology. Triplication of a human chromosome 21 (Hsa21)‐located gene APP play an important role in AD pathology. Wiseman et al. demonstrated that triplication of a gene or genes on Hsa21, other than APP exacerbates amyloid‐β pathology in an APP transgenic mouse model. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and Aβ accumulation. Cystine cathepsins are predominantly expressed in microglia in the brain and are involved in key neuroinflammation pathways. Hsa21‐located gene CYSTATIN B (CSTB) is an endogenous inhibitor of cathepsin proteases. We hypothesise that three copies of CSTB inhibit cathepsin activity and that this could contribute to altered neuroinflammation in people with DS.MethodHuman fibroblasts from individuals with DS and euploid controls, temporal cortex samples from individuals who had AD‐DS and Braak‐stage matched euploid cases of early‐onset AD (EOAD) or healthy aging and human iPSC‐derived microglia‐like cells (iMLCs: T21 and an isogenic control) were used. Western blot and immunocytochemistry were conducted to determine the abundance and subcellular localisation of CSTB. Cathepsin B (CatB) activity in human fibroblasts and temporal cortex samples was measured using an enzymatic assay (Abcam) and/or a pan‐cysteine cathepsin quenched activity‐based probe (BMV109).ResultWestern blotting confirmed that trisomy of Hsa21 resulted in an increase in the protein level of CSTB, that endogenous CSTB is predominantly localised in the cytoplasm and that its subcellular localisation is not altered by trisomy of Hsa21. Significantly lower CatB activity occurs in the temporal cortex of people who had AD‐DS compared with individuals who had EOAD. However, CatB enzymatic activity was not significantly altered in Hsa21 trisomic fibroblasts.ConclusionNext, we will determine in iMLCs, whether trisomy of chromosome 21 alters CatB activity and which genes have altered expression at baseline and after neuroinflammatory challenge.

Thiol Cathepsins and Oxidative Modification of Stallion’s Seminal Plasma Proteins with Normal and Low Percentage of Live Spermatozoa Post Cryopreservation

Background: Stallion sperms are very sensitive to cryopreservation. Free radicals generated during cryopreservation lead to spermatozoa membrane damage, decline in their viability and change the activity of cysteine cathepsins. On the other hand, cathepsins may involve in the utilization of oxidatively modified proteins. The present study was conducted to assess cysteine cathepsins activity and level of oxidatively modified proteins in seminal plasma of stallions with a low ( less than 30%) and normal ( greater than 30%) percentage of live spermatozoa post cryopreservation Methods: A total of 33 breeding stallions comprising 6 stallions of the Budennovskaya breed, 22 Arabian breed, 4 Soviet Heavy-Draft and 1 Bashkir breed. We assessed standard quality parameters in fresh and cryopreserved sperm, as well as cysteine cathepsins B,L,H activity, the level of oxidatively modified proteins in stallions seminal plasma with a low ( less than 30%) and normal ( greater than 30%) percentage of live spermatozoa post cryopreservation. Result: The group with a low percentage of live spermatozoa was characterized by decreased sperm quality, increased level of aldehyde dinitrophenylhydrazones (p=0.045) and ketone dinitrophenylhydrazones (p=0.049) of a neutral nature, as well as increased activity of cathepsin L (p=0.029). Evaluation of activity of cathepsin L and the concentration of oxidatively modified proteins in the seminal plasma can serve as a marker in the selection of stallions for sperm cryopreservation.

Investigations on Primary Cilia of Nthy-ori 3-1 Cells upon Cysteine Cathepsin Inhibition or Thyrotropin Stimulation.

In the thyroid gland, cysteine cathepsins are secreted upon thyrotropin stimulation for thyroglobulin processing, and they are present at the primary cilia of thyroid epithelial cells. Treatment with protease inhibitors resulted in the loss of cilia from rodent thyrocytes and caused redistribution of the thyroid co-regulating G protein-coupled receptor Taar1 to the endoplasmic reticulum. These findings suggest that ciliary cysteine cathepsins are important to maintain sensory and signaling properties for the proper regulation and homeostasis of thyroid follicles. Therefore, it is important to better understand how cilia structure and frequencies are maintained in human thyroid epithelial cells. Hence, we aimed to investigate the potential role of cysteine cathepsins for the maintenance of primary cilia in the normal human Nthy-ori 3-1 thyroid cell line. This was approached by determining cilia lengths and frequencies in cysteine peptidase inhibition conditions in Nthy-ori 3-1 cell cultures. Cilia lengths were shortened upon 5 h of cysteine peptidase inhibition with cell-impermeable E64. Likewise, cilia lengths and frequencies were decreased upon additional overnight treatment with the cysteine peptidase-targeting, activity-based probe DCG-04. The results suggest that cysteine cathepsin activity is required for the maintenance of the cellular protrusions not only in rodents, but also in human thyrocytes. Hence, thyrotropin stimulation was used to simulate physiological conditions that eventually lead to cathepsin-mediated thyroglobulin proteolysis, which is initiated in the thyroid follicle lumen. Immunoblotting revealed that thyrotropin stimulation conditions result in the secretion of little procathepsin L and some pro- and mature cathepsin S but no cathepsin B from the human Nthy-ori 3-1 cells. Unexpectedly, however, 24 h incubation periods with thyrotropin shortened the cilia although higher amounts of cysteine cathepsins were present in the conditioned media. These data point to the necessity of further studies to delineate which of the cysteine cathepsins plays the most prominent role in cilia shortening and/or elongation. Collectively, the results of our study provide corroboration for the hypothesis of thyroid autoregulation by local mechanisms that our group previously proposed.

Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I.

CD8+ T cell recognition of Mycobacterium tuberculosis (Mtb)-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of Mtb antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of Mtb-infected primary human macrophages reveals that substrates of Mtb's type VII secretion systems (T7SS) are overrepresented among Mtb-derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of Mtb peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization. Chemical inhibition of proteasome activity, lysosomal acidification, or cysteine cathepsin activity did not block presentation of Mtb antigens on MHC-I, suggesting involvement of other proteolytic pathways or redundancy among multiple pathways. Our study identifies Mtb antigens presented on MHC-I that could serve as targets for TB vaccines, and reveals how the activity of multiple T7SSs interacts to contribute to presentation of Mtb antigens on MHC-I.

Abstract 3591: Does the metastatic immunoenvironment determine poor immunotherapy responses

Abstract Despite new treatments, cancer remains the second leading cause of death worldwide with breast cancer being the most common type. Of these deaths, the majority are due to the formation of distant metastases. While new immunotherapies have shown some success in primary tumors, their efficacy in combating metastases has been lacking. Thus we aim to investigate the hypothesis that immune responses at the metastatic site contribute to limitations of current therapies.Within the last decade, new microscopy technologies now enable real-time visualization of immune responses at primary and secondary sites. Here, we present use of these microscope technologies, along with the development of novel probes to detect apoptotic cancer cells, to investigate the success of checkpoint inhibitors in the primary and secondary sites. We validate, in multiple in vitro and in vivo assays (including intravital imaging), that fluorescent cyclic amphipathic peptide probes specifically label cells undergoing therapy-induced cancer death. In addition, we introduce a newly developed fluorescent, enzyme-activatable, chemokine-conjugate capable of targeting defined macrophage subsets in live tumors. This chemokine-conjugate exploits the high expression of chemokine receptors (e.g. CCR2) and the activity of cysteine cathepsins in tumor associated macrophages to selectively target and eradicate them without affecting other macrophages or immune cells (e.g., neutrophils, T cells, B cells). This research will help to elucidate the role of combination therapy on the metastatic foci and will provide the first insights into the benefit and limitations of combination therapy on secondary site immune responses. Citation Format: Nicole D. Barth, Floris J. van Dalen, Camille Duran, Utsa Karmakar, Lorena Mendive-Tapia, Takanori Kitamura, Martjin Verdoes, John S. Condeelis, Maja Oktay, Marc Vendrell, Valerie Brunton, David Entenberg. Does the metastatic immunoenvironment determine poor immunotherapy responses. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3591.

Inhibitory prodrug mechanism for cysteine cathepsin-targeted self-controlled drug release

Tumour-associated macrophages (TAMs) support tumour development and have emerged as important regulators of therapeutic response to cytostatic agents. To target TAMs, we have developed a novel drug delivery approach which induces drug release as it inhibits cysteine cathepsin activity. This inhibitory prodrug (IPD) approach establishes a self-regulated system where drug release stops after all cysteine cathepsins are inhibited. This could improve the therapeutic window for drugs with severe side effects. We demonstrate and characterise this self-regulation concept with a fluorogenic IPD model. Next, we applied this IPD strategy to deliver cytotoxic drugs, as doxorubicin and monomethyl auristatin E, which are efficiently released and dose-dependently eliminate RAW264.7 macrophages. Lastly, by exploiting the increased cathepsin activity in TAM-like M2-polarised primary macrophages, we show that IPD-Dox selectively eliminates M2 over M1 macrophages. This demonstrates the potential of our IPD strategy for selective drug delivery and modulation of the tumour microenvironment.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

After one year of the COVID-19 pandemic and hundreds of suggested drugs, will cathepsin L inhibitors be the solution?

Cysteine cathepsins are defined as lysosomal enzymes that are members of the papain family. Cysteine cathepsins (Cts) prevalently exist in whole organisms, varying from prokaryotes to mammals, and possess greatly conserved cysteine residues in their active sites. Cts are engaged in the digestion of cellular proteins, activation of zymogens, and remodeling of the extracellular matrix (ECM). Host cells are entered by SARS-CoV-2 via endocytosis. Cathepsin L and phosphatidylinositol 3-phosphate 5-kinase are crucial in endocytosis by cleaving the spike protein, which permits viral membrane fusion with the endosomal membrane and succeeds in the release of the viral genome to the host cell. Therefore, inhibition of cathepsin L may be advantageous in terms of decreasing infection caused by SARS-CoV-2. Coordinate inhibition of multiple Cts and lysosomal function by different drugs and biological agents might be of value for some purposes, such as a parasite or viral infections and antineoplastic applications. Zn2+ deficiency or dysregulation leads to exaggerated cysteine cathepsin activity, increasing the autoimmune/inflammatory response. For this purpose, Zn2+ metal can be safely combined with a drug that increases the anti-proteolytic effect of endogenous Zn2+, lowering the excessive activity of some CysCts. Biguanide derivative complexes with Zn2+ have been found to be promising inhibitors of CysCts protease reactions. Molecular docking studies of cathepsin L inhibited by the metformin-Zn+2 complex have been performed, showing two strong key interactions (Cys-25&His-163) and an extra H-bond with Asp-163 compared to cocrystallized Zn+2 (PDB ID 4axl).

Upregulation of gut cathepsin L during Eimeria tenella infection

Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial β-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian β-defensins that are key molecules of innate immunity.

Cystatin C Deficiency Increases LPS-Induced Sepsis and NLRP3 Inflammasome Activation in Mice.

Cystatin C is a potent cysteine protease inhibitor that plays an important role in various biological processes including cancer, cardiovascular diseases and neurodegenerative diseases. However, the role of CstC in inflammation is still unclear. In this study we demonstrated that cystatin C-deficient mice were significantly more sensitive to the lethal LPS-induced sepsis. We further showed increased caspase-11 gene expression and enhanced processing of pro-inflammatory cytokines IL-1β and IL-18 in CstC KO bone marrow-derived macrophages (BMDM) upon LPS and ATP stimulation. Pre-treatment of BMDMs with the cysteine cathepsin inhibitor E-64d did not reverse the effect of CstC deficiency on IL-1β processing and secretion, suggesting that the increased cysteine cathepsin activity determined in CstC KO BMDMs is not essential for NLRP3 inflammasome activation. The CstC deficiency had no effect on (mitochondrial) reactive oxygen species (ROS) generation, the MAPK signaling pathway or the secretion of anti-inflammatory cytokine IL-10. However, CstC-deficient BMDMs showed dysfunctional autophagy, as autophagy induction via mTOR and AMPK signaling pathways was suppressed and accumulation of SQSTM1/p62 indicated a reduced autophagic flux. Collectively, our study demonstrates that the excessive inflammatory response to the LPS-induced sepsis in CstC KO mice is dependent on increased caspase-11 expression and impaired autophagy, but is not associated with increased cysteine cathepsin activity.

The effects of Cstb duplication on APP/amyloid-β pathology and cathepsin B activity in a mouse model.

People with Down syndrome (DS), caused by trisomy of chromosome 21 have a greatly increased risk of developing Alzheimer's disease (AD). This is in part because of triplication of a chromosome 21 gene, APP. This gene encodes amyloid precursor protein, which is cleaved to form amyloid-β that accumulates in the brains of people who have AD. Recent experimental results demonstrate that a gene or genes on chromosome 21, other than APP, when triplicated significantly accelerate amyloid-β pathology in a transgenic mouse model of amyloid-β deposition. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and amyloid-β accumulation. Located on human chromosome 21 (Hsa21) is an endogenous inhibitor of cathepsin proteases, CYSTATIN B (CSTB) which is proposed to regulate cysteine cathepsin activity in vivo. Here we determined if three copies of the mouse gene Cstb is sufficient to modulate amyloid-β accumulation and cathepsin activity in a transgenic APP mouse model. Duplication of Cstb resulted in an increase in transcriptional and translational levels of Cstb in the mouse cortex but had no effect on the deposition of insoluble amyloid-β plaques or the levels of soluble or insoluble amyloid-β42, amyloid-β40, or amyloid-β38 in 6-month old mice. In addition, the increased CSTB did not alter the activity of cathepsin B enzyme in the cortex of 3-month or 6-month old mice. These results indicate that the single-gene duplication of Cstb is insufficient to elicit a disease-modifying phenotype in the dupCstb x tgAPP mice, underscoring the complexity of the genetic basis of AD-DS and the importance of multiple gene interactions in disease.

The Role of Cysteine Peptidases in Hematopoietic Stem Cell Differentiation and Modulation of Immune System Function.

Cysteine cathepsins are primarily involved in the degradation and recycling of proteins in endo-lysosomal compartments but are also gaining recognition as pivotal proteolytic contributors to various immune functions. Through their extracellular proteolytic activities within the hematopoietic stem cell niche, they are involved in progenitor cell mobilization and differentiation. Cysteine cathepsins, such as cathepsins L and S contribute to antigen-induced adaptive immunity through major histocompatibility complex class II antigen presentation whereas cathepsin X regulates T-cell migration. By regulating toll-like receptor signaling and cytokine secretion cysteine cathepsins activate innate immune cells and affect their functional differentiation. Cathepsins C and H are expressed in cytotoxic T lymphocytes and natural killer cells and are involved in processing of pro-granzymes into proteolytically active forms. Cytoplasmic activities of cathepsins B and L contribute to the maintenance of homeostasis of the adaptive immune response by regulating cell death of T and B lymphocytes. The expression pattern, localization, and activity of cysteine cathepsins is tightly connected to their function in immune cells. Furthermore, cysteine cathepsins together with their endogenous inhibitors, serve as mediators in the interplay between cancer and immune cells that results in immune cell anergy. The aim of the present article is to review the mechanisms of dysregulation of cysteine cathepsins and their inhibitors in relation to immune dysfunction to address new possibilities for regulation of their function.

Structure-guided protein engineering of human cathepsin L for efficient collagenolytic activity.

Engineering precise substrate specificity of proteases advances the potential to use them in biotechnological and therapeutic applications. Collagen degradation, a physiological process mediated by collagenases, is an integral part of extracellular matrix remodeling and when uncontrolled, implicated in different pathological conditions. Lysosomal cathepsin-K cleaves triple helical collagen fiber, whereas cathepsin-L cannot do so. In this study, we have imparted collagenolytic property to cathepsin-L, by systematically engineering proline-specificity and glycosaminoglycans (GAG)-binding surface in the protease. The proline-specific mutant shows high specificity for prolyl-peptidic substrate but is incapable of cleaving collagen. Engineering a GAG-binding surface on the proline-specific mutant enabled it to degrade type-I collagen in the presence of chondroitin-4-sulfate (C4-S). We also present the crystal structures of proline-specific (1.4Å) and collagen-specific (1.8Å) mutants. Finally docking studies with prolyl-peptidic substrate (Ala-Gly-Pro-Arg-Ala) at the active site and a C4-S molecule at the GAG-binding site enable us to identify key structural features responsible for collagenolytic activity of cysteine cathepsins.

The abnormal accumulation of heparan sulfate in patients with mucopolysaccharidosis prevents the elastolytic activity of cathepsin V

Mucopolysaccharidosis (MPS) are rare inherited diseases characterized by accumulation of lysosomal glycosaminoglycans, including heparan sulfate (HS). Patients exhibit progressive multi-visceral dysfunction and shortened lifespan mainly due to a severe cardiac/respiratory decline. Cathepsin V (CatV) is a potent elastolytic protease implicated in extracellular matrix (ECM) remodeling. Whether CatV is inactivated by HS in lungs from MPS patients remained unknown. Herein, CatV colocalized with HS in MPS bronchial epithelial cells. HS level correlated positively with the severity of respiratory symptoms and negatively to the overall endopeptidase activity of cysteine cathepsins. HS bound tightly to CatV and impaired its activity. Withdrawal of HS by glycosidases preserved exogenous CatV activity, while addition of Surfen, a HS antagonist, restored elastolytic CatV-like activity in MPS samples. Our data suggest that the pathophysiological accumulation of HS may be deleterious for CatV-mediated ECM remodeling and for lung tissue homeostasis, thus contributing to respiratory disorders associated to MPS diseases.

Changes in the activity of lysosomal cysteine proteases of plasma mononuclear and polymorphonuclear blood leukocytes in Alzheimer’s disease

Aim. To study the level of activity of lysosomal cysteine proteases (cathepsins H, B, L) in blood plasma and fractionated leukocytes (polymorphonuclear and mononuclear) in patients with Alzheimer’s disease in comparison with similar indicators in persons without signs of neurodegeneration as a possible marker of Alzheimer’s disease development and diagnosis. Materials and methods. The spectrofluorimetric study of cathepsins B, L, H activity level in plasma and fractionated leukocytes was conducted in 22 patients diagnosed with Alzheimer’s disease in comparison with the same indicators in 22 patients matched by sex, age and associated diseases with patients of the observation group, but having no signs of neurodegeneration. Results. The activity of all three enzymes, and especially cathepsin H, increased significantly in blood plasma. A significant increase is also noted in the activity of cathepsins H, B, and L in homogenates of fractionated leukocytes. At the same time, in both polymorphonuclear and mononuclear leukocytes the greatest degree of changes is demonstrated by the activity of cathepsin B, and the least is the activity of cathepsin L. Given the available data on an increased cathepsin B activity in the cerebrospinal fluid of patients with Alzheimer’s disease, we can assume a correlation between the state of lysosomal proteases activity in the Central nervous system and in the peripheral blood cells. Conclusion. Alzheimer’s disease is associated with increased activity of cysteine cathepsins in plasma, polymorphonuclear and mononuclear leukocytes of peripheral blood, which can be considered as one of the possible markers of development and diagnosis of the disease.

Discovery of small molecules that normalize the transcriptome and enhance cysteine cathepsin activity in progranulin-deficient microglia

Patients with frontotemporal dementia (FTD) resulting from granulin (GRN) haploinsufficiency have reduced levels of progranulin and exhibit dysregulation in inflammatory and lysosomal networks. Microglia produce high levels of progranulin, and reduction of progranulin in microglia alone is sufficient to recapitulate inflammation, lysosomal dysfunction, and hyperproliferation in a cell-autonomous manner. Therefore, targeting microglial dysfunction caused by progranulin insufficiency represents a potential therapeutic strategy to manage neurodegeneration in FTD. Limitations of current progranulin-enhancing strategies necessitate the discovery of new targets. To identify compounds that can reverse microglial defects in Grn-deficient mouse microglia, we performed a compound screen coupled with high throughput sequencing to assess key transcriptional changes in inflammatory and lysosomal pathways. Positive hits from this initial screen were then further narrowed down based on their ability to rescue cathepsin activity, a critical biochemical readout of lysosomal capacity. The screen identified nor-binaltorphimine dihydrochloride (nor-BNI) and dibutyryl-cAMP, sodium salt (DB-cAMP) as two phenotypic modulators of progranulin deficiency. In addition, nor-BNI and DB-cAMP also rescued cell cycle abnormalities in progranulin-deficient cells. These data highlight the potential of a transcription-based platform for drug screening, and advance two novel lead compounds for FTD.

Cysteine cathepsins L and X differentially modulate interactions between myeloid-derived suppressor cells and tumor cells.

Increased proteolytic activity of cysteine cathepsins has long been known to facilitate malignant progression, and it has also been associated with tumor-promoting roles of myeloid-derived suppressor cells (MDSCs). Consequently, cysteine cathepsins have gained much attention as potential targets for cancer therapies. However, cross-talk between tumor cells and MDSCs needs to be taken into account when studying the efficacy of cathepsin inhibitors as anti-cancer agents. Here, we demonstrate the potential of the MDA-MB-231 breast cancer cell line to generate functional MDSCs from CD14+ cells of healthy human donors. During this transition to MDSCs, the overall levels of cysteine cathepsins increased, with the largest responses for cathepsins L and X. We used small-molecule inhibitors of cathepsins L and X (i.e., CLIK-148, Z9, respectively) to investigate their functional impact on tumor cells and immune cells in this co-culture system. Interactions with peripheral blood mononuclear cells reduced MDA-MB-231 cell invasion, while inhibition of cathepsin X activity by Z9 restored invasion. Inhibition of cathepsin L activity using CLIK-148 resulted in significantly increased CD8+ cytotoxicity. Of note, inhibition of cathepsins L and X in separate immune or tumor cells did not promote these functional changes. Together, our findings underlie the importance of tumor cell-immune cell interactions in the evaluation of the anti-cancer potential of cysteine cathepsin inhibitors.

Regulation of the Proteolytic Activity of Cysteine Cathepsins by Oxidants.

Besides their primary involvement in the recycling and degradation of proteins in endo-lysosomal compartments and also in specialized biological functions, cysteine cathepsins are pivotal proteolytic contributors of various deleterious diseases. While the molecular mechanisms of regulation via their natural inhibitors have been exhaustively studied, less is currently known about how their enzymatic activity is modulated during the redox imbalance associated with oxidative stress and their exposure resistance to oxidants. More specifically, there is only patchy information on the regulation of lung cysteine cathepsins, while the respiratory system is directly exposed to countless exogenous oxidants contained in dust, tobacco, combustion fumes, and industrial or domestic particles. Papain-like enzymes (clan CA, family C1, subfamily C1A) encompass a conserved catalytic thiolate-imidazolium pair (Cys25-His159) in their active site. Although the sulfhydryl group (with a low acidic pKa) is a potent nucleophile highly susceptible to chemical modifications, some cysteine cathepsins reveal an unanticipated resistance to oxidative stress. Besides an introductory chapter and peculiar attention to lung cysteine cathepsins, the purpose of this review is to afford a concise update of the current knowledge on molecular mechanisms associated with the regulation of cysteine cathepsins by redox balance and by oxidants (e.g., Michael acceptors, reactive oxygen, and nitrogen species).

Cysteine Cathepsins in Tumor-Associated Immune Cells

Cysteine cathepsins are key regulators of the innate and adaptive arms of the immune system. Their expression, activity, and subcellular localization are associated with the distinct development and differentiation stages of immune cells. They promote the activation of innate myeloid immune cells since they contribute to toll-like receptor signaling and to cytokine secretion. Furthermore, they control lysosomal biogenesis and autophagic flux, thus affecting innate immune cell survival and polarization. They also regulate bidirectional communication between the cell exterior and the cytoskeleton, thus influencing cell interactions, morphology, and motility. Importantly, cysteine cathepsins contribute to the priming of adaptive immune cells by controlling antigen presentation and are involved in cytotoxic granule mediated killing in cytotoxic T lymphocytes and natural killer cells. Cathepins'aberrant activity can be prevented by their endogenous inhibitors, cystatins. However, dysregulated proteolysis contributes significantly to tumor progression also by modulation of the antitumor immune response. Especially tumor-associated myeloid cells, such as tumor-associated macrophages and myeloid-derived suppressor cells, which are known for their tumor promoting and immunosuppressive functions, constitute the major source of excessive cysteine cathepsin activity in cancer. Since they are enriched in the tumor microenvironment, cysteine cathepsins represent exciting targets for development of new diagnostic and therapeutic moieties.