Inactivation Of Cysteine Proteases Research Articles

Thermal Inactivation of Cysteine Proteases: The Key Stages

Thermal Inactivation of Cysteine Proteases: The Key Stages

Evidence for inactivation of cysteine proteases by reactive carbonyls via glycation of active site thiols

Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O 2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H 2O 2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.

Inactivation of cysteine proteases by (acyloxy)methyl ketones using S'-P' interactions.

We have synthesized (acyloxy)methyl ketone inactivators of papain, cathepsin B, and interleukin-1beta conversion enzyme (ICE) that interact with both the S and S' subsites. The value of k(inact)/K(i) for these inactivators is strongly dependent on the leaving group. For example, Z-Phe-Gly-CH(2)-X is a poor inactivator of papain when X is OCOCH(3) (k(inact)/K(i) = 2.5 M(-)(1) s(-)(1)) but becomes a potent inactivator when X is OCO-L-Leu-Z (k(inact)/K(i) = 11 000 M(-)(1) s(-)(1)). Since these leaving groups have similar chemical reactivities, the difference in potency must be attributed to interactions with the S' sites. The potency of the leaving group correlates with the P' specificity of papain. Similar results are also observed for the inactivation of cathepsin B by these compounds. A series of inactivators with the general structure Fmoc-L-Asp-CH(2)-X were designed to inactivate ICE. No inhibition was observed when X was OCOCH(3). In contrast, ICE is inactivated when X is OCO-D-Pro-Z (k(inact)/K(i) = 131 M(-)(1) s(-)(1)). These results demonstrate that S'-P' interactions can be utilized to increase the efficacy and selectivity of (acyloxy)methyl ketone inactivators.

Inactivation of cysteine proteases by peptidyl epoxides: characterization of the alkylation sites on the enzyme and the inactivator

Inactivation of cysteine proteases by peptidyl epoxides: characterization of the alkylation sites on the enzyme and the inactivator

Inactivation of cysteine proteases by peptidyl epoxides: characterization of the alkylation sites on the enzyme and the inactivator

Erythro peptidyl epoxides are selective inactivators of cysteine proteases. The alkylation site, both on the enzyme papain and on the epoxide itself, was characterized. The inactivation of papain with the peptidyl epoxide erythro benzyloxycarbonyl-Phe-Ala-epoxide was followed by total hydrolysis by acid. Mass spectral analysis of the hydrolysate revealed, in addition to the expected amino acids, a unique signal of m/z 209 (MH(+)). Its high-resolution mass spectrum and daughter peak analysis correspond to the product of alkylation on cysteine and the expected fragmentation. A similar MS pattern was obtained for a synthetic model compound corresponding to the expected hydrolysis product. A (13)C NMR analysis of papain inactivated by a specifically (13)C-labelled peptidyl epoxide indicated that the alkylation of the enzyme's cysteine residue occurs on the primary carbon of the epoxide moiety.

Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism.

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.

Viral Inhibitors of Apoptosis

Publisher Summary This chapter reviews a number of regulators of apoptosis encoded by viruses from a variety of host organisms, detailing their discovery, their role in viral infection and replication, and in some cases the use of these genes as tools for the molecular dissection of apoptosis. Viruses manipulate the host cell cycle and macromolecular synthesis to facilitate their own replication. Multicellular organisms have evolved means of detecting these changes and responding by induction of apoptosis. It is clear that viral inhibition of this response improves viral fitness. Proteins such as BHRF1 and E1B 19kD appear to act as general suppressors of apoptosis. Other virus-encoded proteins, however, may act on more than one level. A variety of viral regulators of p53 are identified, but it is difficult to evaluate the relative importance of their effects on apoptosis versus their effects on the cell cycle. Cowpox response modifier A (CrmA) inhibits apoptosis through its inactivation of cysteine proteases, thus presumably allowing more time for viral replication. The ability of CrmA to prevent inflammation and pock formation, however, might be more dependent on its ability to inhibit production of mature IL-1β.

Inactivation of Cysteine Proteases

The cysteine proteases papain and cathepsin B are inactivated by a Michael acceptor, a peptidyl-β-chloro-α, β-unsaturated ester[formula]Inactivation occurred concomitant with chloride release which was stoichiometric with the amount of enzyme. This result is consistent with nucleophilic attack of the active site cysteine on the β-carbon of the inhibitor, followed by expulsion of chloride ion. Inactivation by this class of compounds requires the carbon skeleton about the double bond to be in thetransconfiguration. Thecisisomer was a competitive inhibitor. The difference in the mode of inhibition between the isomers is probably due to nonproductive binding of thecisisomer due to bulky chlorine substituent in the β-position.

Design of a new selective cysteine protease inactivator and its mechanistic implications

Cbz-Phe-epoxide was designed as a selective inactivator of cysteine proteases. It exhibits a time- and concentration-dependent inactivation of cysteine proteases, while showing no activity towards serine proteases. The inhibition is irreversible, correlated with loss of the free active-site thiol, and its rate is at least 10 4 faster than the rate of a model reaction in solution. These results support the proposed active-site directed, protonation-dependent, mechanism-based mode of inactivation of cysteine proteases by the new inhibitor.

Nature of papain products resulting from inactivation by a peptidyl O-acyl hydroxamate

Mass spectrometry has been used to provide insights into the mechanism of inhibition of cysteine proteases by a hydroxylamine derivative, CBZ-Phe-Gly-NH-O-CO-(2,4,6-Me 3)Ph. An oxidized form of papain resulting from the incubation of the enzyme with the peptidyl hydroxamate in the absence of a reducing agent has been identified as a sulfinic acid. The presence of a covalent enzyme-inhibitor complex of molecular mass consistent with a sulfenamide adduct of papain could also be detected by this method. Implications on the mechanism of inactivation of cysteine proteases by peptidyl hydroxamates are discussed.