Inhibitor Cystatin Research Articles

Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage

Asparaginyl endopeptidase (AEP) is an endo/lysosomal cysteine endopeptidase with a preference for an asparagine residue at the P1 site and plays an important role in the maturation of toll-like receptors 3/7/9. AEP is known to undergo autoproteolytic maturation at acidic pH for catalytic activation. Here, we describe crystal structures of the AEP proenzyme and the mature forms of AEP. Structural comparisons between AEP and caspases revealed similarities in the composition of key residues and in the catalytic mechanism. Mutagenesis studies identified N44, R46, H150, E189, C191, S217/S218 and D233 as residues that are essential for the cleavage of the peptide substrate. During maturation, autoproteolytic cleavage of AEP's cap domain opens up access to the active site on the core domain. Unexpectedly, an intermediate autoproteolytic maturation stage was discovered at approximately pH 4.5 in which the partially activated AEP could be reversed back to its proenzyme form. This unique feature was confirmed by the crystal structure of AEPpH4.5 (AEP was matured at pH 4.5 and crystallized at pH 8.5), in which the broken peptide bonds were religated and the structure was transformed back to its proenzyme form. Additionally, the AEP inhibitor cystatin C could be digested by the fully activated AEP, but could not be digested by activated cathepsins. Thus, we demonstrate for the first time that cystatins may regulate the activity of AEP through substrate competition for the active site.

Arabidopsis cysteine proteinase inhibitor AtCYSb interacts with a Ca2+-dependent nuclease, AtCaN2

Plant cysteine proteinase inhibitors (cystatins) play important roles in plant defense mechanisms. Some proteins that interact with cystatins may defend against abiotic stresses. Here, we showed that AtCaN2, a Ca2+-dependent nuclease in Arabidopsis, is transcribed in senescent leaves and stems and interacts with an Arabidopsis cystatin (AtCYSb) in a yeast two-hybrid screen. The interaction between AtCYSb and AtCaN2 was confirmed by in vitro pull-down assay and bimolecular fluorescence complementation. Agarose gel electrophoresis showed that the nuclease activity of AtCaN2 against λDNA was inhibited by AtCYSb, which suggests that AtCYSb regulates nucleic acid degradation in cells. Structured summary of protein interactionsAtCYSb and AtCaN2physically interact by bimolecular fluorescence complementation (View interaction)AtCYSbbinds to AtCaN2 by pull down (View interaction)AtCYSbphysically interacts with AtCaN2 by two hybrid (View interaction)

The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia) facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin) system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

Biochemical, Immunological and Kinetic Characterisation of Thiol Protease Inhibitor (Cystatin) from Liver

Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.

Cystatin C Properties Crucial for Uptake and Inhibition of Intracellular Target Enzymes

To elucidate the molecular requirements for cancer cell internalization of the extracellular cysteine protease inhibitor cystatin C, 12 variants of the protein were produced and used for uptake experiments in MCF-7 cells. Variants with alterations in the cysteine cathepsin binding region ((Δ1-10)-, K5A-, R8G-, (R8G,L9G,V10G)-, (R8G,L9G,V10G,W106G)-, and W106G-cystatin C) were internalized to a very low extent compared with the wild-type inhibitor. Substitutions of N39 in the legumain binding region (N39K- and N39A-cystatin C) decreased the internalization and (R24A,R25A)-cystatin C, with substitutions of charged residues not involved in enzyme inhibition, was not taken up at all. Two variants, W106F- and K75A-cystatin C, showed that the internalization can be positively affected by engineering of the cystatin molecule. Microscopy revealed vesicular co-localization of internalized cystatin C with the lysosomal marker proteins cathepsin D and legumain. Activities of both cysteine cathepsins and legumain, possible target enzymes associated with cancer cell invasion and metastasis, were down-regulated in cell homogenates following cystatin C uptake. A positive effect on regulation of intracellular enzyme activity by a cystatin variant selected from uptake properties was illustrated by incubating cells with W106F-cystatin C. This resulted in more efficient down-regulation of intracellular legumain activity than when cells were incubated with wild-type cystatin C. Uptake experiments in prostate cancer cells corroborated that the cystatin C internalization is generally relevant and confirmed an increased uptake of W106F-cystatin C, in PC3 cells. Thus, intracellular cysteine proteases involved in cancer-promoting processes might be controled by cystatin uptake.

FRI0138 The importance of cystatin c as a predictor of accelerated atherosclerosis in premenopausal non-diabetic patients with rheumatoid patients

BackgroundLife expectancy in rheumatoid arthritis (RA) patients is reduced by 3-10 years mainly due to cardiovascular diseases. Cystatin C is a basic microprotein, widely distributed in human tissues and body...

Proteolytic activity in Plagiodera versicolora Laicharting (Coleoptera: Chrysomelidae): Characterization of digestive proteases and effect of host plants

Proteolytic profiles in the midgut of Plagiodera versicolora were studied using biochemical approaches, and the effects of host plants on possible changes in their activity were determined. Morphology of the alimentary canal revealed several areas of sections, namely bucca, pharynx, esophagus, crop, midgut, ileum, rectum and anus. A pH of 6 and 11 was found to be optimal for soluble and membrane-bound fractions, by using azocasein 2% as a substrate. Determination of specific proteases demonstrates the presence of trypsin-like, chymotrypsin-like, elastase, cathepsin B, cathepsin L and cathepsin D, as well as two exopeptidases. Regarding site of activity for each specific protease, it was found that the major activity of cathepsin B and cathepsin L was in the soluble fraction, chymotrypsin, cathepsin D and two exopeptidases in membrane-bound fraction. Additionally, trypsin-like and elastase activities had no significant differences between fractions. The presence of the above mentioned specific proteases was verified using the specific inhibitors PMSF, TLCK, TPCK, cystatin, phenanthroline and DTT. Feeding of the beetle on four host plants: including Salix aegyptica, S. alba, Populus alba and P. caspica, from the 1st larval instar to adult, revealed the highest trypsin-, chymotrypsin-like and elastase activities in the individuals fed on S. aegyptica and S. alba, respectively. Regarding cathepsins B and L, the highest activities were observed on S. alba and S. aegypticum but cathepsin D was higher in S. Alba and P. alba. Feeding on S. alba and S. aegypticum showed the highest activities of amino- and carboxy-peptidases, respectively.

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 μM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 μM), and E-64 (IC50 3 μM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14(+) human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK.

Deletion of Cysteine Cathepsins B or L Yields Differential Impacts on Murine Skin Proteome and Degradome

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.

Cysteine proteases: mode of action and role in epidermal differentiation

Desquamation or cell shedding in mammalian skin is known to involve serine proteases, aspartic proteases and glycosidases. In addition, evidence continues to accumulate that papain-like cysteine proteases and an inhibitor cystatin M/E largely confined to the cutaneous epithelia also play key roles in the process. This involves the complete proteolysis of cell adhesive structures of the stratum corneum, the corneodesmosomes and notably of the desmogleins. Continual cell replacement in the epidermis is the result of the balance between the loss of the outer squames and mitosis of the cells in the basal cell layer. This article provides a brief account of the salient features of the characteristics and catalytic mechanism of cysteine proteases, followed by a discussion of the relevant epidermal biology. The proteases include the asparaginyl endopeptidase legumain, which exerts a strict specificity for the hydrolysis of asparaginyl bonds, cathepsin-V and cathepsin-L. The control of these enzymes by cystatin M/E regulates the processing of transglutaminases and is crucial in the biochemical pathway responsible for regulating the cross-linking and desquamation of the stratum corneum. In addition, caspase-14 has now been shown to play a major part in epidermal maturation. Uncontrolled proteolytic activity leads to abnormal hair follicle formation and deleterious effects on the skin barrier function.

Studies on interaction of buffalo brain cystatin with donepezil: an Alzheimer's drug.

When drugs bind to a protein, the intramolecular structures can be altered, resulting in conformational change of the protein. Donepezil, an Acetyl Cholinesterase inhibitor (AChE), is commonly prescribed to patients with Alzheimer's disease (AD) to enhance cholinergic neurotransmission. It is the “first-line” agents in the treatment of Alzheimer's disease used to improve cognitive function in the disease. In the present study, a cysteine protease inhibitor (cystatin) has been isolated from buffalo brain using alkaline treatment, 40 to 60% ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 with % yield of 64.13 and fold purification of 384.7. The purified inhibitor (Buffalo Brain Cystatin, (BBC)) was eluted as a single papain inhibitory peak which migrated as single band on native PAGE; however, on SDS-PAGE with and without beta mercaptoethanol (βME) BBC gave two bands of M W 31.6 and 12.4 KDa, respectively. The molecular weight determined by gel filtration came out to be 43.6 KDa. The UV spectra of cystatin on interaction with donepezil suggested a conformational change in the protein. The fluorescence spectra of BC-donepezil composite show structural changes indicating 40 nm red shift with significant increase in fluorescence intensity of cystatin in the presence of donepezil representing an unfolding of cystatin on interaction, which is an indication of side effect of donepezil during the use of this drug.

Cystatin M/E knockdown by lentiviral delivery of shRNA impairs epidermal morphogenesis of human skin equivalents

The protease inhibitor cystatin M/E (CST6) regulates a biochemical pathway involved in stratum corneum homeostasis, and its deficiency in mice causes ichthyosis and neonatal lethality. Cystatin M/E deficiency has not been described in humans so far, and we did not detect disease-causing mutations in the CST6 gene in a large number of patients with autosomal recessive congenital ichthyosis, who were negative for mutations in known ichthyosis-associated genes. To investigate the phenotype of CST6 deficiency in human epidermis, we used lentiviral delivery of short hairpin RNAs that target CST6 in a 3D reconstructed skin model. Surprisingly, CST6 deficiency did not cause an ichthyosis-like phenotype, but prevented the development of a multilayered epidermis. From this study, we conclude that CST6 deficiency may be incompatible with normal human foetal development.

Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation

There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39–102). We also developed a targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC–MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.

Effects of abscisic acid treatment on the expression of cysteine proteinase gene and enzyme inhibitor during wheat cold adaptation

The effects of exogenous ABA on the expression of the cysteine proteinase (CP) gene and its inhibitor cystatin (WC3) in the leaves of winter wheat (Triticum aestivum L.) seedlings that were hardened for 7 days at 5°C were studied. Seedling cold-hardiness was judged by the temperature causing death of 50% of palisade cells (LT50) following 5-min freezing at the temperatures from −5 to −10°C. In the initial period of cooling, when seedling cold tolerance increased, the CP and WC3 gene expression increased considerably, but at the highest cold-hardiness the transcript levels of these genes returned to the control level. When ABA was applied exogenously, the CP gene expression grew by the order of magnitude already in 1–5 h from the onset of low temperature treatment and then abruptly dropped, but it was further maintained at a high level. In cold- and ABA-treated seedlings, the WC3 gene expression initially decreased, but it grew considerably in 3–7 days. It is concluded that the development of wheat cold-hardiness is related to ABA-regulated changes in the CP and WC3 gene expression.

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.

Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

Myocilin (MYOC) is a 504 aa secreted glycoprotein induced by stress factors in the trabecular meshwork tissue of the eye, where it was discovered. Mutations in MYOC are linked to glaucoma. The glaucoma phenotype of each of the different MYOC mutation varies, but all of them cause elevated intraocular pressure (IOP). In cells, forty percent of wild-type MYOC is cleaved by calpain II, a cysteine protease. This proteolytic process is inhibited by MYOC mutants. In this study, we investigated the molecular mechanisms by which MYOC mutants cause glaucoma. We constructed adenoviral vectors with variants Q368X, R342K, D380N, K423E, and overexpressed them in human trabecular meshwork cells. We analyzed expression profiles with Affymetrix U133Plus2 GeneChips using wild-type and null viruses as controls. Analysis of trabecular meshwork relevant mechanisms showed that the unfolded protein response (UPR) was the most affected. Search for individual candidate genes revealed that genes that have been historically connected to trabecular meshwork physiology and pathology were altered by the MYOC mutants. Some of those had known MYOC associations (MMP1, PDIA4, CALR, SFPR1) while others did not (EDN1, MGP, IGF1, TAC1). Some, were top-changed in only one mutant (LOXL1, CYP1B1, FBN1), others followed a mutant group pattern. Some of the genes were new (RAB39B, STC1, CXCL12, CSTA). In particular, one selected gene, the cysteine protease inhibitor cystatin A (CSTA), was commonly induced by all mutants and not by the wild-type. Subsequent functional analysis of the selected gene showed that CSTA was able to reduce wild-type MYOC cleavage in primary trabecular meshwork cells while an inactive mutated CSTA was not. These findings provide a new molecular understanding of the mechanisms of MYOC-causative glaucoma and reveal CSTA, a serum biomarker for cancer, as a potential biomarker and drug for the treatment of MYOC-induced glaucoma.

Levels of cathepsins in acute myocardial infarction

Aims/objectiveOver expression of matrix degrading enzymes have been implicated in plaque destabilisation and rupture. Cathepsins associated with extracellular matrix breakdown make them intriguing suspects. The aim of the study was to analyse peripheral levels of cathepsin B and cathepsin K and their inhibitor cystatin C during acute myocardial infarction (AMI). Materials and methodsStudy population included AMI patients at acute event (AMI group, n=48), stable angina patients (stable angina group n = 17), and healthy individuals (Control group, n=31). Cathepsin B, cathepsin K, cystatin C, and matrix metalloproteinases (MMP)-9 were analysed by enzyme-linked immunosorbent assay (ELISA) method. ResultsCathepsin B (45.9%) and cathepsin K (92.31%) at acute event of myocardial infarction (AMI group) increased (P=0.001) while cystatin C decreased marginally (12.5%) as compared to controls. Stable angina group, demonstrated only marginal reduction in all the parameters studied as compared to controls. ConclusionCathepsin B and cathepsin K can be further evaluated as biomarkers in identifying high-risk individuals for AMI.

Cysteine proteinase inhibitors decrease rankl and lps induced differentiation of human and mouse osteoclast progenitor cells

Backgroundand objectivesThe authors have previously reported that the cysteine proteinase inhibitor (CPI) cystatin C (CysC) reduces osteoclast formation induced by signal pathways activated by either PTH receptor, vitamin D receptor...

Transcriptome analysis of resistant soybean roots infected by Meloidogyne javanica.

Soybean is an important crop for Brazilian agribusiness. However, many factors can limit its production, especially root-knot nematode infection. Studies on the mechanisms employed by the resistant soybean genotypes to prevent infection by these nematodes are of great interest for breeders. For these reasons, the aim of this work is to characterize the transcriptome of soybean line PI 595099-Meloidogyne javanica interaction through expression analysis. Two cDNA libraries were obtained using a pool of RNA from PI 595099 uninfected and M. javanica (J2) infected roots, collected at 6, 12, 24, 48, 96, 144 and 192 h after inoculation. Around 800 ESTs (Expressed Sequence Tags) were sequenced and clustered into 195 clusters. In silico subtraction analysis identified eleven differentially expressed genes encoding putative proteins sharing amino acid sequence similarities by using BlastX: metallothionein, SLAH4 (SLAC1 Homologue 4), SLAH1 (SLAC1 Homologue 1), zinc-finger proteins, AN1-type proteins, auxin-repressed proteins, thioredoxin and nuclear transport factor 2 (NTF-2). Other genes were also found exclusively in nematode stressed soybean roots, such as NAC domain-containing proteins, MADS-box proteins, SOC1 (suppressor of overexpression of constans 1) proteins, thioredoxin-like protein 4-Coumarate-CoA ligase and the transcription factor (TF) MYBZ2. Among the genes identified in non-stressed roots only were Ser/Thr protein kinases, wound-induced basic protein, ethylene-responsive family protein, metallothionein-like protein cysteine proteinase inhibitor (cystatin) and Putative Kunitz trypsin protease inhibitor. An understanding of the roles of these differentially expressed genes will provide insights into the resistance mechanisms and candidate genes involved in soybean-M. javanica interaction and contribute to more effective control of this pathogen.

Cysteinyl Cathepsins: Multifunctional Enzymes in Cardiovascular Disease

Until recently, the role of lysosomal cysteine protease cathepsins in intracellular protein degradation was believed to be mainly restricted to scavenging. However, recent studies have revealed nontraditional roles for cysteine protease cathepsins in the extracellular space during the development and progression of cardiovascular disease. Although the precise mechanisms are unknown, data from animal studies suggest that members of the cathepsin family, like other extracellular proteases, contribute to extracellular matrix protein remodeling and interstitial matrix degradation, as well as to cell signaling and cell apoptosis in heart disease. Inflammatory cytokines and hormones regulate the expression and secretion of cathepsins in cultured cardiovascular cells and macrophages. Serum levels of cathepsins L, S, and K and their endogenous inhibitor cystatin C may be useful predictive biomarkers in patients with coronary artery disease and cardiac disease. Furthermore, in vivo pharmacological intervention with a synthetic cathepsin inhibitor and cardiovascular drugs (including statins and angiotensin II type 1 receptor antagonists) has the potential for pharmacologic targeting of cathepsins in cardiovascular disease. This review focuses on cathepsin biology (structure, synthesis, processing, activation, secretion, activity regulation, and function) and the involvement of cysteinyl cathepsins in the pathogenesis of several heart and vessel diseases, especially with respect to their potential application as diagnostic and prognostic markers and drug targets to prevent inappropriate proteolysis in cardiovascular disease.