Cystatins – Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the elevated cellular cystatin levels seen in hereditary cystatin C amyloid angiopathy.     

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.     

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

The general potential of plant cystatins for the development of insect‐resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of digestive cysteine protease activities. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet assay with cystatin‐infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC‐I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z‐Phe‐Arg‐MCA‐hydrolyzing (cathepsin L–like) and Z‐Arg‐Arg‐MCA‐hydrolyzing (cathepsin B–like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the cysteine protease inhibitor E‐64 and to different plant cystatins including OsCYS1. In line with the broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatin‐infiltrated banana stem disks. These promising results, which illustrate the susceptibility of C. sordidus to plant cystatins, are discussed in the light of recent hypotheses suggesting a key role for cathepsin B–like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera. © 2009 Wiley Periodicals, Inc.     

Silencing barley cystatins HvCPI‐2 and HvCPI‐4 specifically modifies leaf responses to drought stress

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy‐2 and Icy‐4 cystatin genes. Artificial microRNA lines independently silencing the two drought‐induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay‐green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy‐2 and Icy‐4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap‐1, HvPap‐12, and HvPap‐16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.     

Modelling family 2 cystatins and their interaction with papain

Cystatins are extensively studied cysteine protease inhibitors, found in wide range of organisms with highly conserved structural folds. S-type of cystatins is well known for their abundance in saliva, high selectivity and poorer activity towards host cysteine proteases in comparison to their immediate ancestor cystatin C. Despite more than 90% sequence similarity, the members of this group show highly dissimilar binding affinity towards papain. Cystatin M/E is a potent inhibitor of legumain and papain like cysteine proteases and recognized for its involvement in skin barrier formation and potential role as a tumor suppressor gene. However, the structures of these proteins and their complexes with papain or legumain are still unknown. In the present study, we have employed computational methods to get insight into the interactions between papain and cystatins. Three-dimensional structures of the cystatins are generated by homology modelling, refined with molecular dynamics simulation, validated through numerous web servers and finally complexed with papain using ZDOCK algorithm in Discovery Studio. A high degree of shape complementarity is observed within the complexes, stabilized by numerous hydrogen bonds (HB) and hydrophobic interactions. Using interaction energy, HB and solvent accessible surface area analyses, we have identified a series of key residues that may be involved in papain–cystatin interaction. Differential approaches of cystatins towards papain are also noticed which are possibly responsible for diverse inhibitory activity within the group. These findings will improve our understanding of fundamental inhibitory mechanisms of cystatin and provide clues for further research.     

Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.     

Down-regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cysteine proteases, staphopain A and B

Of seven human cystatins investigated, none inhibited the cysteine proteases staphopain A and B secreted by the human pathogen Staphylococcus aureus. Rather, the extracellular cystatins C, D and E/M were hydrolyzed by both staphopains. Based on MALDI-TOF time-course experiments, staphopain A cleavage of cystatin C and D should be physiologically relevant and occur upon S. aureus infection. Staphopain A hydrolyzed the Gly11 bond of cystatin C and the Ala10 bond of cystatin D with similar Km values of approximately 33 and 32 microM, respectively. Such N-terminal truncation of cystatin C caused >300-fold lower inhibition of papain, cathepsin B, L and K, whereas the cathepsin H activity was compromised by a factor of ca. 10. Similarly, truncation of cystatin D caused alleviated inhibition of all endogenous target enzymes investigated. The normal activity of the cystatins is thus down-regulated, indicating that the bacterial enzymes can cause disturbance of the host protease-inhibitor balance. To illustrate the in vivo consequences, a mixed cystatin C assay showed release of cathepsin B activity in the presence of staphopain A. Results presented for the specificity of staphopains when interacting with cystatins as natural protein substrates could aid in the development of therapeutic agents directed toward these proteolytic virulence factors.     

Purification and Characterization of High Molecular Mass and Low Molecular Mass Cystatin from Goat Brain

Cystatin are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study two cystatins were isolated from goat brain using alkaline treatment, ammonium sulphate fractionation, gel filtration and ion exchange chromatography. The high molecular mass cystatin of 70.8 kDa was named as HM-GBC (high molecular mass goat brain cystatin) and the low molecular mass cystatin of 12.72 kDa was named as LM-GBC (low molecular mass goat brain cystatin). The molecular mass determined by SDS-PAGE was found to be 70.8 and 12.88 kDa for HM-GBC and LM-GBC, respectively, however with gel filtration the masses were found to be 70.8 and 12.58 kDa. Both the cystatins were found to be stable in broad range of pH and temperature. HM-GBC was found to have 2% carbohydrate content while LM-GBC lacks any carbohydrate content. Both cystatins were found to be devoid of any sulphydryl content. Stoke's radii of 36 and 16 A, and diffusion coefficient of 6.189 x 10(-15) and 1.392 x 10(-14) cm(2)/s were calculated for HM-GBC and LM-GBC. K (i) values with papain were found to be 1.875 x 10(-8) and 3.125 x 10(-8) M for HM-GBC and LM-GBC, respectively. K (+1), K (-1) and half-life calculated along with K (i) values obtained showed that HM-GBC inhibited papain more specifically as compared to LM-GBC. The IC(50) values obtained for HM-GBC and LM-GBC also showed that HM-GBC binds more effectively to papain than LM-GBC. Ultraviolet and fluorescence spectra indicated that upon formation of papain-HM-GBC/LM-GBC complex there is significant conformational change after interaction in one or both the proteins of the complex.     

Characteristics of C‐terminal, β‐amyloid peptide binding fragment of neuroprotective protease inhibitor,cystatin C

Cystatin C originally identified as a cysteine proteases inhibitor has a broad spectrum of biological roles ranging from inhibition of extracellular cysteine protease activities, bone resorption, and modulation of inflammatory responses to stimulation of fibroblasts proliferation. There is an increasing number of evidence to suggest that human cystatin C (hCC) might play a protective role in the pathophysiology of sporadic Alzheimer's disease. In vivo and in vitro results well documented the association of hCC with Aβ and the hCC‐induced inhibition of Aβ fibril formation. In our earlier work, using a combination of selective proteolytic methods and MS spectroscopy, C‐terminal fragment hCC(101‐117) was identified as the Aβ‐binding region. The fragment of Aβ peptide responsible for the complex formation with hCC was found in the middle, highly hydrophobic part, Aβ(17‐24). Structures and affinities of both Aβ and hCC binding sites were characterized by the enzyme‐linked immunosorbent assay‐like assay, by surface plasmon resonance, and by nano‐ESI‐FTICR MS of the hCC–Aβ‐ binding peptide complexes. In the in vitro inhibition studies, the binding cystatin sequence, hCC(101‐117), revealed the highest relative inhibitory effect toward Aβ‐fibril formation. Herein, we present further studies on molecular details of the hCC‐Aβ complex. With Ala substitution, affinity experiments, and enzyme‐linked immunosorbent assay‐like assays for the Aβ‐binding fragment, hCC(101‐117), and its variants, the importance of individual amino acid residues for the protein interaction was evaluated. The results were analyzed using hCC(101‐117) nuclear magnetic resonance structural data with molecular dynamics calculations and molecular modeling of the complexes. The results point to conformational requirements and special importance of some amino acid residues for the protein interaction. The obtained results might be helpful for the design of low molecular compounds modulating the biological role of both proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect

in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.     

Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.     

Increase of cyclin B by overexpression of cystatin α

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin α, was investigated. The cystatin α gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin α was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin α resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.     

Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site.

We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The Ki value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with Ki values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-Asn-NHPhNO2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low Mr kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (Ki 0.0016 nM) and relatively weak (Ki 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the alpha-helix and the first strand of the main beta-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (Ki 1.5 nM) but >5,000-fold lower affinity for legumain (Ki >1,000 nM) than wild-type cystatin C.     

Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin

When an excess of human cystatin C or chicken cystatin was mixed with papain, an enzyme-inhibitor complex was formed immediately. The residual free cystatin was then progressively converted to a form with different electrophoretic mobility and chromatographic properties. The modified cystatins were isolated and sequenced, showing that there had been cleavage of a single peptide bond in each molecule: Gly11-Gly12 in cystatin C, and Gly9-Ala10 in chicken cystatin. The residues Gly11 (cystatin C) and Gly9 (chicken cystatin) are among only three residues conserved in all known sequences of inhibitory cystatins. The modified cystatins were at least 1000-fold weaker inhibitors of papain than the native cystatins. An 18-residue synthetic peptide corresponding to residues 4-21 of cystatin C did not inhibit papain but was cleaved at the same Gly-Gly bond as cystatin C. When iodoacetate or L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane was added to the mixtures of either cystatin with papain, modification of the excess cystatin was blocked. Papain-cystatin complexes were stable to prolonged incubation, even in the presence of excess papain. We conclude that the peptidyl bond of the conserved glycine residue in human cystatin C and chicken cystatin probably is part of a substrate-like inhibitory reactive site of these cysteine proteinase inhibitors of the cystatin superfamily and that this may be true also for other inhibitors of this superfamily. We also propose that human cystatin C and chicken cystatin, and probably other cystatins as well, inhibit cysteine proteinases by the simultaneous interactions with such proteinases of the inhibitory reactive sites and other, so far not identified, areas of the cystatins. The cleavage of the inhibitory reactive site glycyl bond in mixtures of papain with excess quantities of cystatins is apparently due to the activity of a small percentage of atypical cysteine proteinase molecules in the papain preparation that form only very loose complexes with cystatins under the conditions employed and degrade the free cystatin molecules.     

Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica

The three-dimensional structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica, has been determined in solution at pH 6.8 and 25 degrees C by (1)H and (15)N NMR spectroscopy. The main body (Glu13-Asp97) of oryzacystatin-I is well-defined and consists of an alpha-helix and a five-stranded antiparallel beta-sheet, while the N- and C-terminal regions (Ser2-Val12 and Ala98-Ala102) are less defined. The helix-sheet architechture of oryzacystatin-I is stabilized by a hydrophobic cluster formed between the alpha-helix and the beta-sheet and is considerably similar to that of monellin, a sweet-tasting protein from an African berry, as well as those of the animal cystatins studied, e.g., chicken egg white cystatin and human stefins A and B (also referred to as human cystatins A and B). Detailed structural comparison indicates that oryzacystatin-I is more similar to chicken cystatin, which belongs to the type-2 animal cystatins, than to human stefins A and B, which belong to the type-1 animal cystatins, despite different loop length.     

Cathepsin B but not cathepsins L or S contributes to the pathogenesis of Unverricht‐Lundborg progressive myoclonus epilepsy (EPM1)

The inherited epilepsy Unverricht‐Lundborg disease (EPM1) is caused by loss‐of‐function mutations in the cysteine protease inhibitor, cystatin B. Because cystatin B inhibits a class of lysosomal cysteine proteases called cathepsins, we hypothesized that increased proteolysis by one or more of these cathepsins is likely to be responsible for the seizure, ataxia, and neuronal apoptosis phenotypes characteristic of EPM1. To test this hypothesis and to identify which cysteine cathepsins contribute to EPM1, we have genetically removed three candidate cathepsins from cystatin B‐deficient mice and tested for rescue of their EPM1 phenotypes. Whereas removal of cathepsins L or S from cystatin B‐deficient mice did not ameliorate any aspect of the EPM1 phenotype, removal of cathepsin B resulted in a 36–89% reduction in the amount of cerebellar granule cell apoptosis depending on mouse age. The incidence of an incompletely penetrant eye phenotype was also reduced upon removal of cathepsin B. Because the apoptosis and eye phenotypes were not abolished completely and the ataxia and seizure phenotypes experienced by cystatin B‐deficient animals were not diminished, this suggests that another molecule besides cathepsin B is also responsible for the pathogenesis, or that another molecule can partially compensate for cathepsin B function. These findings establish cathepsin B as a contributor to the apoptotic phenotype of cystatin B‐deficient mice and humans with EPM1. They also suggest that the identification of cathepsin B substrates may further reveal the molecular basis for EPM1. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 315–327, 2003     

Interactions of papaya proteinase IV with inhibitors.

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.     

Mammalian cystatin and protagonists in brain diseases

Abstract

Cystatins are the thiol Proteinase inhibitors, present ubiquitously in mammalian body. They prevent unwanted proteolysis and play important role in several diseases. Regulation of cysteine Proteinase and their inhibitors is of utmost importance in neurodegenerative diseases like Alzheimer, amyloid angiopathy and in many other diseases. The action of these cysteine proteases is biologically controlled by proteinase inhibitors namely cystatins(cys) they constitute a superfamily of homologous proteins. The major role of cystatins is to protect the organism against endogenous proteases released from lysosomes, invading microorganisms and parasites that use cysteine proteases to enter the body. An enormous progress has been made in understanding of protein degradation process under normal and pathological conditions; in fact proteases are now clearly viewed as important drug targets. Some studies have suggested that cystatin C is a target for intervention in neurological disorders because its expression increases in response to human neurological disorders and in animal models of neurodegenerative states. Although, these studies did not clarify whether CysC up-regulation is a pathogenic factor in neurodegenerative disorders or whether it represents a neuroprotective compensatory response of the organisms aimed to prevent progression of the disease. However, for other diseases in some cases cystatins other than cys C are up regulated and in some it is down regulated.

Cystatins have been implicated in the processes of neuronal degeneration and repair of the nervous system. Both CysC and CysB are potent, reversible inhibitors of most of the currently known cathepsins. The extent of proteolytic activity at any given time and location is the result of a balance between active proteases and physiological inhibitors. Uncontrolled proteolysis as a result of imbalance between active proteases and their endogenous inhibitors has been associated with neuronal cell death in different neuronal diseases, including brain tumors, stroke, some forms of epilepsy, Alzheimer’s disease, and neurological autoimmune diseases.

An antidepressant is a psychiatric medication used to alleviate mood disorders, major depression and other brain diseases. Drugs including the monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), and serotonin-norepinephrine reuptake inhibitors (SNRIs) are most commonly used antidepressant. They are also used to treat other conditions, such as anxiety disorders, obsessive compulsive disorder, eating disorders, and chronic pain. Although the mechanisms of the action of these antidepressants are not precisely understood, their principal target of action is at the monoamine transporter proteins located at nerve endings. Monoamine neurotransmitter transporters act to terminate synaptic neurotransmission. Selective serotonin reuptake inhibitors or SSRIs are also most widely used class of antidepressants. They work by increasing the level of serotonin in the brain. SSRIs have fewer and milder side effects, fewer drug interactions, and are much less likely to be associated with suicide than TCAs.

These antidepressants shows binding when incubated with cystatin, presenting the involvement of these antidepressant in cascade of disease, as leaving no cystatin to inhibit the cathepsin showing the myriad side effect after the administration of antidepressant. This might be one of the reason in the mechanism of action of antidepressant.

So this review expound about the role of cystatins in neurological diseases which is considered to be highly significant as it pave the way for commanding tool in the drug design.

Communicated by Ramaswamy H. Sarma