Dual concentration-dependent activity of thyroglobulin type-1 domain of testican: specific inhibitor and substrate of cathepsin L

The thyroglobulin type-1 (Tg-1) domain is a protein module that occurs in a variety of secreted and membrane proteins and is recognised as a potent inhibitor of cysteine peptidases. We present here some properties of the Tg-1 domain of human testican, a modularly organised proteoglycan secreted mainly by brain cells, the exact in vivo function of which is not yet clear. The domain was prepared as a recombinant protein in a Pichia pastoris expression system and its activity was demonstrated by specific and selective inhibition of cathepsin L (K i=0.14nM). Interaction at high enzyme and inhibitor concentrations resulted in degradation of the domain by cathepsin L, which was not observed under conditions used for the determination of kinetic parameters. No inhibitory activity could be detected for cathepsin K, but it exhibited a very similar degradation pattern. Homology modelling provided a good explanation for the different behaviour observed with the two enzymes. Firstly, the steric fit between the interfaces of testican domain and cathepsin L is stabilised by numerous favourable forces, while no such interactions are evident in the complex with cathepsin K, and repulsive interactions even prevent access of the domain to the active site of papain. Secondly, the prolonged first loop of the domain occupies a position near the catalytic cysteine residue in a more substrate-like manner, enabling cleavage of the Gly22-Ala23 bon

The catalytic domain of cathepsin C (dipeptidyl-peptidase I) alone is a fully functional endoprotease

Cathepsin C is a tetrameric lysosomal protease that acts as a dipeptidyl-peptidase due to the presence of the exclusion domain that is unique among papain-like cysteine proteases. Here we describe a recombinant form of cathepsin C lacking its exclusion domain (CatCΔEx) produced in a bacterial expression system (E. coli). CatCΔEx is a monomer with endoprotease activity and affinity for hydrophobic residues such as Phe, Leu or Pro, but not Val, in the P2 position. As opposed to cathepsin C, it does not require chloride ions for its activity. Despite lower turnover rates of hydrolysis of synthetic substrates, CatCΔEx has elastolytic and gelatinolytic activity comparable to other cysteine cathepsins

Current view on EpCAM structural biology

EpCAM, a carcinoma cell-surface marker protein and a therapeutic target, has been primarily addressed as a cell adhesion molecule. With regard to recent discoveries of its role in signaling with implications in cell proliferation and differentiation, and findings contradicting a direct role in mediating adhesion contacts, we provide a comprehensive and updated overview on the available structural data on EpCAM and interpret it in the light of recent reports on its function. First, we describe the structure of extracellular part of EpCAM, both as a subunit and part of a cis-dimer which, according to several experimental observations, represents a biologically relevant oligomeric state. Next, we provide a thorough evaluation of reports on EpCAM as a homophilic cell adhesion molecule with a structure-based explanation why direct EpCAM participation in cell–cell contacts is highly unlikely. Finally, we review the signaling aspect of EpCAM with focus on accessibility of signaling-associated cleavage sites

Probing the activity modification space of the cysteine peptidase cathepsin K with novel allosteric modifiers

Targeting allosteric sites is gaining increasing recognition as a strategy for modulating the activity of enzymes, especially in drug design. Here we investigate the mechanisms of allosteric regulation of cathepsin K as a representative of cysteine cathepsins and a promising drug target for the treatment of osteoporosis. Eight novel modifiers are identified by computational targeting of predicted allosteric sites on the surface of the enzyme. All act via hyperbolic kinetic mechanisms in presence of low molecular mass substrates, as expected for allosteric effectors. Two compounds have sizable effects on enzyme activity using interstitial collagen as a natural substrate of cathepsin K and four compounds show a significantly stabilizing effect on cathepsin K. The concept of activity modification space is introduced to obtain a global perspective of the effects elicited by the modifiers. Analysis of the activity modification space reveals that the activity of cathepsin K is regulated via multiple, different allosteric mechanisms

Cysteine Cathepsin Activity Regulation by Glycosaminoglycans

Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail

The central region of testican-2 forms a compact core and promotes cell migration

Testicans are modular proteoglycans of the extracellular matrix of various tissues where they contribute to matrix integrity and exert cellular effects like neurite outgrowth and cell migration. Using testican-2 as a representative member of the family, we tackle the complete lack of general structural information and structure–function relationship. First, we show using isothermal titration calorimetry and modeling that extracellular calcium-binding domain (EC) has only one active calcium-binding site, while the other potential site is inactive, and that testican-2 is within extracellular matrix always in the calcium-loaded form. Next, we demonstrate using various prediction methods that N- and C-terminal regions plus interdomain connections are flexible. We support this by small-angle X-ray-scattering analysis of C-terminally truncated testican-2, which indicates that the triplet follistatin-EC-thyroglobulin domain forms a moderately compact core while the unique N-terminal is disordered. Finally, using cell exclusion zone assay, we show that it is this domain triplet that is responsible for promoting cell migration and not the N- and C-terminal regions

Differences in specificity for the interactions of stefins A, B and D with cysteine proteinases

AbstractFour different stefin-type cysteine proteinase inhibitors have been isolated from porcine thymus and skin. Amino acid sequence determination revealed the presence of stefin A and stefin B type inhibitors and two new inhibitors, designated as porcine stefin D1 and stefin D2. Stefin D1 was identified as PLCPI, an inhibitor recently characterized from porcine polymorphonuclear leukocytes [Lenarčič et al. (1993) FEBS Lett. 336, 289–292]. Stefin A is composed of 101 amino acids and has an Mr of 11391 while stefin B contains 98 amino acids, has an Mr of 11174 and is N-terminally blocked. All inhibitors were found to be fast-acting inhibitors of papain, cathepsin L and cathepsin S (Ki = 0.009–0.161 nM). Stefins A and B also bind tightly and rapidly to cathepsin H (Ki = 0.027 and 0.069 nM, respectively), while stefins D1 and D2 have been shown to be very poor inhibitors of cathepsin H (Ki = 102–150 nM). he decreased affinity of these inhibitors toward cathepsin B (Ki = 2–1700 nM) was shown to be mainly due to the low second order association rate constants. The presence of a highly negatively charged N-terminus on stefin D1 constitutes a likely structural determinant of inhibitor specificity

The Central Region of Testican-2 Forms a Compact Core and Promotes Cell Migration

Testicans are modular proteoglycans of the extracellular matrix of various tissues where they contribute to matrix integrity and exert cellular effects like neurite outgrowth and cell migration. Using testican-2 as a representative member of the family, we tackle the complete lack of general structural information and structure–function relationship. First, we show using isothermal titration calorimetry and modeling that extracellular calcium-binding domain (EC) has only one active calcium-binding site, while the other potential site is inactive, and that testican-2 is within extracellular matrix always in the calcium-loaded form. Next, we demonstrate using various prediction methods that N- and C-terminal regions plus interdomain connections are flexible. We support this by small-angle X-ray-scattering analysis of C-terminally truncated testican-2, which indicates that the triplet follistatin-EC-thyroglobulin domain forms a moderately compact core while the unique N-terminal is disordered. Finally, using cell exclusion zone assay, we show that it is this domain triplet that is responsible for promoting cell migration and not the N- and C-terminal regions

Dual concentration-dependent activity of thyroglobulin type-1 domain of testican: specific inhibitor and substrate of cathepsin L

The thyroglobulin type-1 (Tg-1) domain is a protein module that occurs in a variety of secreted and membrane proteins and is recognised as a potent inhibitor of cysteine peptidases. We present here some properties of the Tg-1 domain of human testican, a modularly organised proteoglycan secreted mainly by brain cells, the exact in vivo function of which is not yet clear. The domain was prepared as a recombinant protein in a Pichia pastoris expression system and its activity was demonstrated by specific and selective inhibition of cathepsin L (K i=0.14nM). Interaction at high enzyme and inhibitor concentrations resulted in degradation of the domain by cathepsin L, which was not observed under conditions used for the determination of kinetic parameters. No inhibitory activity could be detected for cathepsin K, but it exhibited a very similar degradation pattern. Homology modelling provided a good explanation for the different behaviour observed with the two enzymes. Firstly, the steric fit between the interfaces of testican domain and cathepsin L is stabilised by numerous favourable forces, while no such interactions are evident in the complex with cathepsin K, and repulsive interactions even prevent access of the domain to the active site of papain. Secondly, the prolonged first loop of the domain occupies a position near the catalytic cysteine residue in a more substrate-like manner, enabling cleavage of the Gly22-Ala23 bon

Reaction scheme for the general modifier mechanism.

<p>The scheme describes the interaction of enzyme (E) with substrate (S) and modifier (A). <i>K</i>_S and <i>K</i>_A are equilibrium dissociation constants of the ES and EA complexes under rapid equilibrium conditions, <i>k</i>_cat is the catalytic constant and <i>α</i> and <i>β</i> are dimensionless coefficients that determine the effect of modifier on the substrate-binding affinity of the enzyme and the catalytic rate, respectively.</p