Importance of the second binding loop and the C-terminal end of cystatin B (stefin B) for inhibition of cysteine proteinases.

The importance of residues in the second hairpin loop and the C-terminal end of mammalian cystatin B for binding of proteinases was elucidated by mutagenesis of the bovine inhibitor. Bovine cystatin B was modeled onto the crystal structure of the human inhibitor in complex with papain with minimal structural changes. Substitution of the two deduced contact residues in the second hairpin loop, Leu-73 and His-75, with Gly resulted in appreciably reduced affinities for papain and cathepsins H and B. These losses indicated that the two residues together contribute 20-30% of the free energy of binding of cystatin B to these enzymes and that Leu-73 is responsible for most of this contribution. In contrast, the small decrease in the affinity for cathepsin L suggested that the second hairpin loop is less important for inhibition of this proteinase. Replacement of the contact residue in the C-terminal end, Tyr-97, with Ala resulted in losses in affinity for papain and cathepsins L and H that were consistent with Tyr-97 contributing 6-12% of the energy of binding of cystatin B to these enzymes. However, this substitution minimally affected the affinity for cathepsin B, indicating that the C-terminal end is of limited importance for binding of this proteinase. All affinity decreases were due predominantly to increased dissociation rate constants. These results show that both the second hairpin loop and the C-terminal end of cystatin B contribute to anchoring the inhibitor to target proteinases, each of the two regions interacting with a different domain of the enzyme. However, the relative contributions of these two interactions vary with the proteinase.     

Putative alternative functions of human stefin B (cystatin B): binding to amyloid‐beta,membranes, and copper

We describe studies performed thus far on stefin B from the family of cystatins as a model protein for folding and amyloid fibril formation studies. We also briefly mention our studies on aggregation of some of the missense EPM1 mutants of stefin B in cells, which mimic additional pathological traits (gain in toxic function) in selected patients with EPM1 disease. We collected data on the reported interactors of stefin B and discuss several hypotheses of possible cytosolic alternative functions.     

Contributions of individual residues in the N-terminal region of cystatin B (stefin B) to inhibition of cysteine proteinases

The importance of individual residues in the N-terminal region of cystatin B for proteinase inhibition was elucidated by measurements of the affinity and kinetics of binding of N-terminally truncated, recombinant variants of the bovine inhibitor to cysteine proteinases. Removal of Met-1 caused an 8- to 10-fold lower affinity for papain and cathepsin B, decreased the affinity also for cathepsin L but only minimally affected cathepsin H affinity. Additional truncation of Met-2 further weakened the binding to papain and cathepsin B by 40-70-fold, whereas the affinity for cathepsins L and H was essentially unaffected. Removal of Cys-3 had the most drastic effects on the interactions, resulting in a further affinity decrease of approximately 1500-fold for papain, approximately 700-fold for cathepsin L and approximately 15-fold for cathepsin H; the binding to cathepsin B could not be assessed. The binding kinetics could only be evaluated for papain and cathepsin H and showed that the reduced affinities for these enzymes were predominantly due to increased dissociation rate constants. These results demonstrate that the N-terminal region of cystatin B contributes appreciably to proteinase inhibition, in contrast to previous proposals. It is responsible for 12-40% of the total binding energy of the inhibitor to the proteinases investigated, being of least importance for cathepsin H binding. Cys-3 is the most important residue of the N-terminal region for inhibition of papain, cathepsin L and cathepsin H, the role of the other residues of this region varying with the target proteinase.     

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.     

The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L.

The affinity and kinetics of binding of three N-terminally truncated variants of the cysteine proteinase inhibitor cystatin A to cysteine proteinases were characterized. Deletion of Met-1 only minimally altered the inhibitory properties of the protein. However, deletion also of Ile-2 resulted in reduced affinities of 900-, >/=3-, and 200-fold for papain and cathepsins L and B, respectively. Further truncation of Pro-3 substantially increased the inhibition constants to approximately 0.5 microM for papain and cathepsin L and to 60 microM for cathepsin B, reflecting additionally 2 x 10(3)-, 2 x 10(4)-, and 400-fold decreased affinities, respectively. The reductions in affinity shown by the latter mutant indicate that the N-terminal region contributes about 40% of the total free energy of binding of cystatin A to cysteine proteinases. Moreover, Pro-3 and to a lesser extent Ile-2 are the residues responsible for this binding energy. The reduced affinities for papain and cathepsin L were due only to higher dissociation rate constants, whereas both lower association and higher dissociation rate constants contributed to the decreased affinity for cathepsin B. These differential effects indicate that the N-terminal portion of cystatin A primarily functions by stabilizing the complexes with enzymes having easily accessible active-site clefts, e.g., papain and cathepsin L. In contrast, the N-terminal region is required also for an initial binding of cystatin A to cathepsin B, presumably by promoting the displacement of the occluding loop and allowing facile interaction of the rest of the inhibiting wedge with the active-site cleft of the enzyme.     

Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases

Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.     

In vitro study of stability and amyloid-fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-alpha-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.     

High affinity binding between copper and full-length prion protein identified by two different techniques

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.     

High affinity binding of the mastoparans by calmodulin

Calmodulin exhibits high affinity, calcium-dependent binding of the mastoparans — a group of cytoactive tetradecapeptides. The dissociation constants for the peptide-calmodulin complexes determined in 0.20 N KCl, 1.0 mM CaCl₂, pH 7.3 are ～0.3 nM for mastoparan, ～0.9 nM for mastoparan X, and ～3.5 nM for $\text{Polistes}$ mastoparan. The dissociation constant for the mastoparan-calmodulin complex is the smallest known for any calmodulin binding protein or peptide, suggesting that some type of peptide-calmodulin interaction could be physiologically significant.     

Essential role of proline isomerization in stefin B tetramer formation

Here we present the tetrameric structure of stefin B, which is the result of a process by which two domain-swapped dimers of stefin B are transformed into tetramers. The transformation involves a previously unidentified process of extensive intermolecular contacts, termed hand shaking, which occurs concurrently with trans to cis isomerization of proline 74. This proline residue is widely conserved throughout the cystatin superfamily, a member of which, human cystatin C, is the key protein in cerebral amyloid angiopathy. These results are consistent with the hypothesis that isomerization of proline residues can play a decisive role in amyloidogenesis.     

The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73.

The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by approximately 300-fold, >10-fold and approximately 4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by approximately 10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino-acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, approximately 45% of the total binding energy, for inhibition of cathepsin B.     

Cathepsins B and L and their inhibitors stefin B and cystatin C as markers for malignant progression of benign meningiomas

Meningiomas are, in general, slowly growing benign tumors attached to the dura mater and composed of neoplastic meningothelial (arachnoidal) cells. They have a wide range of histopathological appearances and are classified, according to the aggressiveness of their growth and the risk of recurrence, as WHO grade I (benign) meningiomas, WHO grade II (atypical) meningiomas and WHO grade III anaplastic (malignant) meningiomas. As invasion of normal tissue may occur in all grades, independent biological markers are needed to identify the more aggressive and recurrent meningiomas. The lysosomal cysteine proteinases, cathepsins B and L, have been associated with tumor invasiveness and the aim of this study was therefore to evaluate them, together with their endogenous inhibitors stefin B and cystatin C, as potential markers for the aggressiveness of meningiomas. The expression of cathepsins B and L and their inhibitors stefin B and cystatin C in 21 benign (grade I) and 9 atypical (grade II) meningiomas has been compared by immunohistochemical staining, QRT-PCR and Northern blot analysis. The protein levels of cathepsins B (p=0.050) and L (p=0.019) were found to be significantly higher in atypical than in benign meningiomas. In contrast, their mRNA levels did not differ, indicating that the synthesis of cathepsins was accelerated at the translational level. Protein and mRNA levels of stefin B (p= 0.007), but not cystatin C, were significantly lower in atypical compared with benign meningiomas. The expression of cathepsins and inhibitors was not different between central and peripheral meningioma tissue or between histological subtypes of meningiomas, with the exception of cathepsin L, the level of which was significantly lower in transitional meningiomas. We conclude that higher protein levels of cathepsins B and L and lower mRNA levels of stefin B are potential diagnostic markers for invasive and aggressive behavior of meningiomas. The diagnostic and prognostic value for relapse of meningioma needs to be confirmed in a larger population of patients.     

Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation

BackgroundCopper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson’s Disease. Hence, residue specific interaction of Cu²⁺ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail.MethodsWe investigated the residue specific mapping of Cu²⁺ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu²⁺ were studied using fluorescence, CD, and AFM respectively.ResultsCopper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu²⁺ showed an enhanced rate of fibril formation with a pronounced effect for G51D.ConclusionCu²⁺ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu²⁺ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q.General SignificanceThese findings will help in the better understanding of Cu²⁺ catalyzed aggregation of synucleins.     

High affinity (3H)tryptamine binding sites in various organs of the rat

(3H)Tryptamine binds with high affinity (KD values around 4 nmol/l) to membranes from different organs of the rat. The number of binding sites was high in the liver, intermediate in the heart and cerebral cortex and low in other organs including ileum, kidney, spleen, stomach and lung. Binding to kidney membranes was investigated in more detail. It was rapid and reversible (t1/2 7 min.). The structure-activity profile was characterized by high affinity of some beta-carbolines (IC50 = 20 - 200 nmol/l), medium affinity of serotonin (IC50 = 858 nmol/l) and low of methysergide (IC50 greater than 20 mumol/l). The findings correlate very well with binding characteristics to cerebral cortex membranes. Therefore, rat brain cortex and other organs contain the same type of (3H)tryptamine binding site and may be equally appropriate for further analysis.     

Characterization of the high affinity heparin binding site of the Alzheimer's disease βA4 amyloid precursor protein (APP) and its enhancement by zinc(II)

The Alzheimer's disease βA4 amyloid precursor protein (APP) has been shown to be involved in a diverse set of biological protein precursor-like proteins (APLP1 and APLP2) belong to a superfamily of proteins that are probably functionally related. In order to characterize the cell adhesion properties of APP the brain specific isoform APP₆₉₅ was purified and used to assess the binding to herparin, a structural and functional analogue of the glycosaminoglycan heparan sulfate. We show that APP binds in a time dependent and saturable manner to heparin. The salt concentration of 620 mM at which APP elutes from heparin Sepharose is greater than physiological. Tha apparent equilibrium constant for dissociation was determined to be 300 pM for APP binding to heparin Sepharose. A high affinity heparin binding site was identified within a region conversed in rodent and human APP, APLP1 and APLP2. This binding site was located between residues 316-337 of APP₆₉₅ which is within the carbohydrate domain of APP. We also demonstrate an interaction between this heparin binding site and the zinc(II) binding site which is conserved in all members of the APP superfamily. We show by using an automated surface plasmon resonance biosensor (BIAcore, Pharmacia) that the affinity for heparin is increased two- to four-fold in the presence of micromolar zinc(II). The identification of zinc-enhanced binding of APP to heparin sulfate side chains of proteoglycans offers a molecular link between zinc(II), as a putative environmental toxin for Alzheimer's disease, and aggregation of amyloid βA4 protein.     

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.     

Fluorescence microscopy studies on islet amyloid polypeptide fibrillation at heterogeneous and cellular membrane interfaces and its inhibition by resveratrol

Type II diabetes mellitus (T2DM) is a disease characterized by progressive deposition of amyloid in the extracellular matrix of β-cells. We investigated the interaction of the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS-1E cells using fluorescence microscopy techniques. Following preferential partitioning of IAPP into the fluid lipid phase, the membrane suffers irreversible damage and predominantly circularly-shaped lipid-containing IAPP amyloid is formed. Interaction studies with the pancreatic β-cell line INS-1E revealed that growing IAPP fibrils also incorporate substantial amounts of cellular membranes in vivo. Additionally, the inhibitory effect of the red wine compound resveratrol on IAPP fibril formation has been studied, alluding to its potential use in developing therapeutic strategies against T2DM.     

Amyloid fibril formation by human stefin B in vitro: immunogold labelling and comparison to stefin A

The mechanism by which proteins form amyloid fibrils is of high interest to the scientific community as its understanding could resolve questions relevant to conformational diseases. The structural and energetic basis of the process is still largely unknown. The main controversial issue is the co-existence of several protein conformations. Three models for the mechanism of protein fibrillogenesis have been proposed which need to be tested by experiments. In this report, amyloid fibrils grown from human stefin B (type I cystatin) are described. This physiologically relevant protein readily forms fibrils in vitro, in contrast to the homologue--human stefin A--which forms fibrils under extreme conditions only. In order to specifically label stefin B fibrils in vitro, rabbit polyclonal antibody and mouse monoclonal antibody A6/2 against human stefin B were used for immunogold labelling. Samples were examined by transmission electron microscopy. Fibrils of stefin B were strongly labelled using polyclonal antibody and Protein A gold, whereas no positive reaction was observed with monoclonal antibody A6/2.     

-NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings

Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.