Cysteine cathepsins are normally found in the lysosomes where they are involved in intracellular protein turnover. Their ability to degrade the components of the extracellular matrix in vitro was ...first reported more than 25years ago. However, cathepsins were for a long time not considered to be among the major players in ECM degradation in vivo. During the last decade it has, however, become evident that abundant secretion of cysteine cathepsins into extracellular milieu is accompanying numerous physiological and disease conditions, enabling the cathepsins to degrade extracellular proteins.
In this review we will focus on cysteine cathepsins and their extracellular functions linked with ECM degradation, including regulation of their activity, which is often enhanced by acidification of the extracellular microenvironment, such as found in the bone resorption lacunae or tumor microenvironment. We will further discuss the ECM substrates of cathepsins with a focus on collagen and elastin, including the importance of that for pathologies. Finally, we will overview the current status of cathepsin inhibitors in clinical development for treatment of ECM-linked diseases, in particular osteoporosis.
Cysteine cathepsins are among the major proteases involved in ECM remodeling, and their role is not limited to degradation only. Deregulation of their activity is linked with numerous ECM-linked diseases and they are now validated targets in a number of them. Cathepsins S and K are the most attractive targets, especially cathepsin K as a major therapeutic target for osteoporosis with drugs targeting it in advanced clinical trials.
Due to their major role in ECM remodeling cysteine cathepsins have emerged as an important group of therapeutic targets for a number of ECM-related diseases, including, osteoporosis, cancer and cardiovascular diseases. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.
•Cysteine cathepsins are major proteases involved in ECM remodeling.•The role of cysteine cathepsins in ECM remodeling is not limited to degradation only.•Deregulation of cathepsin activity is linked with numerous ECM-linked diseases.•Cathepsin K is a major therapeutic target for osteoporosis.
Cysteine cathepsins have been for a long time considered to execute mainly nonspecific bulk proteolysis in the endolysosomal system. However, this view has been changing profoundly over the last ...decade as cathepsins were found in the cytoplasm, nucleus and in the extracellular milieu. Cathepsins are currently gaining increased attention largely because of their extracellular roles associated with disease development and progression. While kept under tight control under physiological conditions, their dysregulated and elevated activity in the extracellular milieu are distinctive hallmarks of numerous diseases such as various cancers, inflammatory disorders, rheumatoid arthritis, bone disorders and heart diseases. In this review, we discuss cysteine cathepsins with a major focus on their extracellular roles and extracellular proteolytic targets beyond degradation of the extracellular matrix. We further highlight the perspectives of cathepsin research and novel avenues in cathepsin-based diagnostic and therapeutic applications.
•Cysteine cathepsins are often highly upregulated in inflammation-associated diseases linked with ECM remodelling.•Proteomic studies led to identification of several new cathepsin substrates revealing novel roles of cathepsins in the ECM.•Cathepsin K inhibitor Odanacatib failed in advanced clinical trials, but alternatives seem to be on a way.•Diagnostic imaging of cysteine cathepsins in oncology has entered clinical trials•Cathepsins are largely used as activators of prodrugs and antibody-drug conjugates in oncology
Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel‐based label‐free proteomic approach (DIPPS—direct in‐gel profiling ...of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in‐gel digestion of the gel‐separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length‐limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase‐7, and legumain) to broadly specific (matrix‐metalloproteinase‐3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH‐dependent change in the specificity of this protease, further supporting its broad applicability.
Synopsis
Direct In‐gel Profiling of Protease Specificity (DIPPS) enables quick and reliable determination of protease cleavage specificities under a large variety of experimental conditions, offering an excellent starting point for protease characterization and substrate design.
Gel‐based, label‐free whole‐proteome degradomics provides an easy‐to‐use approach applicable under a broad range of assay conditions.
DIPPS validation on 10 distinct proteases from three major mechanistic classes shows an excellent correlation with published protease specificities, without observable experimental biases.
DIPPS profiling of legumain reveals a pH‐dependent specificity switch between Asn and Asn/Asp cleavage specificity.
Direct In‐gel Profiling of Protease Specificity (DIPPS) under a large variety of experimental conditions offers a quick and reliable starting point for protease functional characterization and substrate design.
Traditional proteomics methodology allows global analysis of protein abundance but does not provide information on the regulation of protein activity. Proteases, in particular, are known for their ...multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes. In this review, we present structural features of activity-based probes for proteases and discuss their applications in proteomic profiling of various catalytic classes of proteases.
Proteases are considered of major importance in biomedical research because of their crucial roles in health and disease. Their ability to hydrolyze their protein and peptide substrates at single or ...multiple sites, depending on their specificity, makes them unique among the enzymes. Understanding protease specificity is therefore crucial to understand their biology as well as to develop tools and drugs. Recent advancements in the fields of proteomics and chemical biology have improved our understanding of protease biology through extensive specificity profiling and identification of physiological protease substrates. There are growing efforts to transfer this knowledge into clinical modalities, but their success is often limited because of overlapping protease features, protease redundancy, and chemical tools lacking specificity. Herein, we discuss the current trends and challenges in protease research and how to exploit the growing information on protease specificities for understanding protease biology, as well as for development of selective substrates, cleavable linkers, and activity-based probes and for biomarker discovery.
The newly discovered roles of proteases in disease are resulting in major efforts towards protease-based therapeutics and diagnostics.
With the use of better chemical tools, protease research is constantly shifting from in vitro to more in vivo-like conditions.
Focused proteomic approaches are producing large datasets of protease cleavages and substrates from various physiologic and pathologic samples with potential for biomarker discovery.
Profiling of natural protease specificity and identification of natural protease substrates is opening new possibilities for assay development and protease-cleavable linkers for targeted drug release evolving towards clinical applications.
The first successful examples of activity-based probes are entering clinical trials. In the future we expect to see their use in applications such as whole-body imaging and image-guided surgery.
Circular RNAs (circRNAs) have been shown to play an important role in the pathogenesis of hepatocellular carcinoma (HCC). By implementing available transcriptomic analyses of HCC patients, we ...identified an upregulated circRNA hsa_circ_0062682. Stable perturbations of hsa_circ_0062682 in Huh-7 and SNU-449 cell lines influenced colony formation, migration, cell proliferation, sorafenib sensitivity, and additionally induced morphological changes in cell lines, indicating an important role of hsa_circ_0062682 in oncogenesis. Pathway enrichment analysis and gene set enrichment analysis of the transcriptome data from hsa_circ_0062682 knockdown explained the observed phenotypes and exposed transcription factors E2F1, Sp1, HIF-1α, and NFκB1 as potential downstream targets. Biotinylated oligonucleotide pulldown combined with proteomic analyses identified protein interaction partners of which YBX1, a known oncogene, was confirmed by RNA immunoprecipitation. Furthermore, we discovered a complex cell-type-specific phenotype in response to the oncogenic potential of hsa_circ_0062682. This finding is in line with different classes of HCC tumours, and more studies are needed to shed a light on the molecular complexity of liver cancer.
Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated ...by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC⁻ and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.
Recent advances in global genomic and proteomic methods have lead to a greater understanding of how genes and proteins function in complex networks within a cell. One of the major limitations in ...these methodologies is their inability to provide information on the dynamic, post-translational regulation of enzymatic proteins. In particular proteases are often synthesized as inactive zymogens that need to be activated in order to carry out specific biological processes. Thus, methods that allow direct monitoring of protease activity in the context of a living cell or whole animal will be required to begin to understand the systems-wide functional roles of proteases. In this review, we discuss the development and applications of activity based probes (ABPs) to study proteases and their role in pathological processes. Specifically we focus on application of this technique for biomarker discovery, in vivo imaging and drug screening.
Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we ...present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.
Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing ...MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.
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