Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, <i>Trypanosoma cruzi</i>, by proteinase inhibitors of the cystatin superfamily

Stoka, Veronika; Nycander, Maria; Lenarčič, Brigita; Labriola, Carlos; Cazzulo, Juán José; Björk, Ingemar; Türk, Vito

doi:10.1016/0014-5793(95)00798-e

Cited by 83 publications

(55 citation statements)

References 37 publications

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“…Q Sepharose-Fast Flow and Superdex 200 HR 10/30 columns were obtained from Pharmacia Biotech (Uppsala, Sweden). Chicken cystatin, bovine cathepsin S and cruzipain were purified as described previously [15][16][17]. Heparin, a gift from Dr. Steven T. Olson, University of Illinois at Chicago, had an average Mr of ~ 8000 and was similar to material used in a previous study [18].…”

Section: Methodsmentioning

confidence: 99%

High‐molecular‐weight kininogen binds two molecules of cysteine proteinases with different rate constants

et al. 1996

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Fluorescence titrations showed that high-molecularweight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped-flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (kas~,l = 23.0 X 106 M -1 s -1 and k~,2 = 3.4 X 106 M -1 s -1 ). As for low-molecular-weight kininogen, comparison of these kinetic constants with previous data for intact low-and high-molecular-weight kininogen and the separated domains indicated that the faster-binding site is also the tighter-binding site and is that of domain 3, whereas the slower-binding, lower-affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M~ of high-molecular-weight kininogen was determined to be 83 500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.Key words: Kininogen; Cathepsin; Cystatin; Cysteine proteinase; Kinetics disulfide bond, by limited proteolysis by kallikreins, thereby releasing the kinin segment [2]. The heavy chains of HK and LK are identical, whereas the light chain of HK is substantially larger than that of LK [8].About a decade ago, kininogens were shown to be potent inhibitors of papain-like cysteine proteinases [9,10]. Structural analyses demonstrated that the kininogen heavy chain consists of three domains, designated D1-D3, which share a high internal homology with low-molecular weight cystatins [2,11]. The kininogens therefore were classified as family 3 of the cystatin superfamily [12]. Isolated domains D2 and D3 were shown to inhibit papain-like cysteine proteinases (D2 and D3) and calpain (D2), whereas domain D1 was found to lack inhibitory activity [11,13].We have recently shown that intact human LK simultaneously can bind two molecules of cysteine proteinases with high affinity [14]. In this work we demonstrate that human HK can also bind two molecules of cathepsin S, cruzipain and papain independently. Moreover, kinetic studies of papain binding showed that the two sites bound the proteinase with different rate constants. Materials and methods

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Section: Methodsmentioning

confidence: 99%

High‐molecular‐weight kininogen binds two molecules of cysteine proteinases with different rate constants

et al. 1996

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“…1), following typical biphasic, slow-binding kinetics [18]. They were fitted by non-linear least-squares analysis to the following integrated rate equation [18]: [12]. The dissociation rate constant kn^ was calculated for each inhibitor concentration using the following equation [18]:…”

Section: Resultsmentioning

confidence: 99%

“…Cruzipain was purified as reported [12]. The Ii fragment, isolated as a complex with cathepsin L, was separated using reversed-phase HPLC [16], freeze-dried on a Speed-vac concentrator (model RC 100, Savant Instruments, Farmingdale, USA), and redissolved in distilled water.…”

Section: Methodsmentioning

confidence: 99%

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A fragment of the major histocompatibility complex class II – associated p41 invariant chain inhibits cruzipain, the major cysteine proteinase from Trypanosoma cruzi

et al. 1997

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A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits cruzipain, the cathepsin L-like enzyme from the protozoan parasite Trypanosoma cruzi. The binding of the Ii fragment to cruzipain is fast (k ass = 2.4 X10 7 M" 1 s" 1 ) and tight (Ki = 5.8 X10 n M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of cruzipain in vivo, i.e. as a potential drug to combat Chagas' disease.

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“…Because of their glandular localization they probably also act as inhibitors of exogenous peptidases. Accordingly, they were found to be effective inhibitors against cysteine peptidases from parasites, for example cruzipain from Trypanosoma cruzi (62). Furthermore, they might play a role in protection against viral infections, as they can suppress the infectivity of herpes simplex virus 1 (63) and were found up-regulated in children with seasonal influenza A infection (64).…”

Section: Cystatins and The Immune Responsementioning

confidence: 99%

Cystatins, cysteine peptidase inhibitors, as regulators of immune cell cytotoxicity

et al. 2017

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Cystatins comprise a superfamily of evolutionarily related proteins, present in all living organisms, from protozoa to mammals. They act as inhibitors of cysteine peptidases although they can also function independently of their inhibitory function. Cysteine cathepsins are implicated in various physiological and pathological processes. In the immune response they are involved in antigen processing and presentation, the cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), migration and adhesion of immune cells, cytokine and growth factor regulation and toll-like receptor signalling. Cystatins are probably involved in the regulation of all these processes; importantly, cystatin F has a crucial role in the regulation of immune cell cytotoxicity. NK cells and CTLs exploit the granzyme/perforinAmong those peptidases involved in immune processes, many studies have been focused on a group of endosomal/lysosomal cysteine peptidases, the cysteine cathepsins, whose involvement in antigen processing and presentation, cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), migration and adhesion of immune cells, cytokine and growth factor regulation and toll like receptor (TLR) signalling have been demonstrated (3,4). In addition to cathepsins, another lysosomal cysteine peptidase, legumain or asparaginyl endopeptidase, has also been associated with the immune response, most notably with antigen presentation and TLR signalling (4, 5). The activities of cysteine cathepsins and legumain are regulated on different levels. Their expression, for example, is regulated at transcriptional or translational levels. They are synthesised as inactive precursors, activated only at the site of action, and compartmentalised into lysosomes or other organelles. Their activity can be regulated by oxidation of the active site cysteine or by endogenous protein inhibitors (6), the latter being presented in more detail. CYSTEINE CATHEPSINS AND LEGUMAIN AS REGULATORS OF THE IMMUNE RESPONSEIn humans, cysteine cathepsins comprise a group of 11 lysosomal peptidases (cathepsins B, C, F, H, K, L, O, S, V, X and W). They are members of the papain family, classified as clan CA (7). The expression pattern, levels, localization and specificities of cysteine cathepsins differ, contributing to their various physiological roles. Cathepsins B, H, L and C are expressed ubiquitously in cells and tissues, while expression of others is restricted to specific cell types. They are endopeptidases, with the exception of cathepsins B, C, H and X. Cathepsins B and X are carboxypeptidases, cleaving substrates at the C-terminal end, while cathepsins B and H are aminopeptidases, cleaving substrates at the N-terminal end. Interestingly, in addition to exopeptidase activity, cathepsins B and H also exhibit endopeptidase activity (7,8). Cathepsin B can act as an endo-or exopeptidase due to the position of the structural element, termed the occluding loop, while in cathepsin H the type of activity is determined by a minicha...

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Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily

Cited by 83 publications

References 37 publications

High‐molecular‐weight kininogen binds two molecules of cysteine proteinases with different rate constants

High‐molecular‐weight kininogen binds two molecules of cysteine proteinases with different rate constants

A fragment of the major histocompatibility complex class II – associated p41 invariant chain inhibits cruzipain, the major cysteine proteinase from Trypanosoma cruzi

Cystatins, cysteine peptidase inhibitors, as regulators of immune cell cytotoxicity

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