Single Peptide Bond Research Articles

Development of a Functional cis‐Prolyl Bond Biomimetic and Mechanistic Implications for Nickel Superoxide Dismutase

During recent years several peptide-based Ni superoxide dismutase (NiSOD) models have been developed. These NiSOD models show an important structural difference compared to the native NiSOD enzyme, which could cause a completely different mechanism of superoxide dismutation. In the native enzyme the peptide bond between Leu4 and Pro5 is cis-configured, while the NiSOD models exhibit a trans-configured peptide bond between these two residues. To shed light on how the configuration of this single peptide bond influences the activity of the NiSOD model peptides, a new cis-prolyl bond surrogate was developed. As surrogate we chose a leucine/alanine-based disubstituted 1,2,3-triazole, which was incorporated into the NiSOD model peptide replacing residues Leu4 and Pro5. The yielded 1,5-disubstituted triazole nickel peptide exhibited high SOD activity, which was approximately the same activity as its parent trans-configured analogue. Hence, the conformation of the prolyl peptide bond apparently has of minor importance for the catalytic activity of the metallopeptides as postulated in literature. Furthermore, it is shown that the triazole metallopeptide is forming a stable cyanide adduct as a substrate analogue model complex.

The Proto-Ribosome: an ancient nano-machine for peptide bond formation.

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC) is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A to P-site passage of the tRNA 3' terminus during protein synthesis is performed by a rotary motion, synchronized with the overall tRNA/mRNA sideways movement and Guided by the PTC. This rotary motion leads to suitable stereochemistry for peptide bond formation as well as for substrate mediated catalysis. Analysis of the substrate binding modes to ribosomes led to the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, potentially in a similar manner to the modern PTC. Later in evolution, a mechanism, enabling some type of decoding genetic control triggered the emergence of the small ribosomal subunit or part of it. This seems to be the result of the appearance of reaction products that could have evolved after polypeptides capable of enzymatic function were generated sporadically, while an ancient stable RNA fold was converted into an old version of a tRNA molecule. As in the contemporary ribosome the symmetry relates only the backbone fold and nucleotides orientations but not nucleotide sequences, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC may have evolved by gene fusion or gene duplication.

Crystallization and preliminary X-ray crystallographic analysis of blood coagulation factor V-activating proteinase (RVV-V) from Russell's viper venom.

Russell's viper venom blood coagulation factor V activator (RVV-V) is a thrombin-like serine proteinase that specifically activates factor V by cleaving a single peptide bond between Arg1545 and Ser1546. Activated factor V combines with activated factor X produced by the enzyme RVV-X in the venom to form the prothombinase complex, which can induce disseminated intravascular coagulopathy in envenomated animals. In the current study, RVV-V was crystallized in order to attempt to understand its substrate specificity for factor V. Four distinct crystal forms of RVV-V were obtained using the sitting-drop vapour-diffusion method and diffraction data sets were collected on SPring-8 beamlines. The best crystal of RVV-V generated data sets to 1.9 A resolution.

Single-Molecule Measurements of Force-Induced Cleavage of VWF A1A2A3-Tridomain by ADAMTS13

Binding of glycoprotein Ib (GPIb) to von Willebrand factor (VWF) mediates platelet adhesion to injured vascular surface in hemostasis, but also contributes to thrombosis under pathological high shear stress. The adhesive activity of VWF is regulated by A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs 13 (ADAMTS13), which cleaves the ultra-large form of VWF newly secreted from activated endothelial cells to smaller and less adhesive forms. Cleavage is regulated mechanically by hemodynamic force as it occurs in the circulating blood. We used single-molecule experiments by atomic force microscopy (AFM) to test the hypothesis that mechanical stretch of VWF facilitates its proteolysis by ADAMTS13. Recombinant VWF A1A2A3 tridomain with a C-terminal histidine-tag either directly adsorbed to or captured by an anti-His monoclonal antibody (mAb) precoated on the Petri dish was stretched by either GPIb or an anti-A1 mAb (CR1) adsorbed on the AFM cantilever tip. Conformational changes, observed as abrupt length increases in the molecular complex, were induced by applied force. Times required for conformational change and for rupture of the molecular complex before or after conformational change were measured in a range of constant forces in the absence or presence of ADAMTS13. When stretched by GPIb, time-to-rupture before conformational change increased (catch) initially, reached a maximum, and then decreased (slip) with increasing force, confirming the catch-slip bond behavior we recently observed for GPIb interacting with VWF-A1 domain. Interestingly, time-to-conformational change also behaved as catch-slip bonds. Both sets of curves were independent of ADAMTS13. Time-to-rupture after conformational change exhibited the same force dependence as the time-to- rupture before conformational change. However, the time-to-rupture vs. force curve measured in the presence of ADAMTS13 was shifted downward towards shorter times relative to the curves measured in the absence of ADAMTS13. This effect was abolished by EDTA known to inhibit ADAMTS13 proteolysis. These results suggest that force-induced conformational change may have exposed the cryptic cleavage site to enable ADAMTS13 to cleave a single peptide bond in the A2 domain, thereby shortening the time-to-rupture. These data were corroborated by experiments using the CR1 mAb to stretch the A1A2A3 tridomain. The time-to-conformational change vs. force curves also exhibited catch-slip bonds, indicating that the mechanical characteristics of conformational change were indifferent to how the A1A2A3 tridomain was stretched. The time-to-rupture before conformational change vs. force curves behaved as slip bonds, consistent with the behavior of antibody-antigen interactions. Both sets of curves were independent of ADAMTS13, similar to the behavior observed when GPIb was used to stretch the A1A2A3 tridomain. Similar downward shifts were observed for the curve measured in the presence of ADAMTS13 relative to that measured in the absence of ADAMTS13. The rate of proteolytic cleavage, estimated from the downward shifts, decreased with increasing force for both cases when the A1A2A3 tridomain was stretched by GPIb and by CR1. These findings provide direct experimental evidence for mechanical force to play an important role in regulating VWF cleavage by ADAMTS13.

ADAMTS-13 Has a Disulfide Bond Reduction Activity on Von Willebrand Factor

Von Willebrand factor (VWF) multimers tether platelets to subendothelium exposed at the site of vessel injury to initiate the bleeding arrest. Upon synthesized, VWF multimers are either constitutively secreted or packed into the storage granules, where they are enriched in ultra-large (UL) multimers that are active in forming spontaneous high strength bonds with the GP Ib-IX-V complex on platelets. This hyper-reactivity of ULVWF multimers is in contrast to VWF multimers circulating in plasma (pVWF) that need to be activated by modulators or high fluid shear stress to aggregate platelets. The biochemical and structural bases for the functional difference between ULVWF and pVWF multimers are not known. We have recently shown that a portion of pVWF, but not ULVWF multimers contain surface exposed free thiols in the D3 and C domains. High fluid shear stress promotes the formation of new disulfide bonds utilizing the thiols to enhance VWF binding to platelets, suggesting that the shear-induced thiol-disulfide exchange may serve as a mechanism for the shear-induced activation of pVWF multimers. ULVWF freshly secreted from endothelial cells forms string-like structures that can be elongated by pVWF multimers through a covalent means. The different thiol distribution between ULVWF and its plasma counterpart may be caused by the former being cleaved by the zinc metalloprotease ADAMTS-13 at a single peptide bond of Y1065-M1606 in the A2 domain. Here, we provide several lines of evidence to demonstrate that ADAMTS-13 also contains a reductase-like activity that plays a role in cleaving ULVWF strings under flow conditions and maintaining circulating VWF multimers in an inactive (thiol) state. First, more than 90% of pVWF non-specifically adhered to the surface of a cone-plate viscometer when pVWF was exposed to a pathological high shear stress of 100 dyn/cm2 for 3 min at 37°C. The adhesion was prevented by recombinant (r) ADAMTS-13 or a truncation mutant that lacked the catalytic domain. Second, rADAMTS-13 prevented the shear-induced thiol-disulfide exchange so that free thiols remained in pVWF after shear exposure. This activity was not blocked by 5 mM of EDTA and was detectable with the N-terminal truncated mutant, suggesting that it is independent of the VWF-cleaving activity. We further found that rADAMTS-13 was able to reduce disulfide bonds, converting the disulfide forms of sheared pVWF to the thiol forms, suggesting that ADAMTS-13 prevents the thiol-disulfide exchange by disulfide bond reduction, not a steric hindered effect. Third, ADAMTS-13 contains the surface exposed thiol(s) that is necessary for the metalloprotease to attack and break a disulfide bond. Unlike VWF multimers, these thiols remained after the metalloprotease was exposed to a pathological high shear stress of 100 dyn/cm2. Fourth, using a series of N- and C-terminal truncation mutants, we located the thiol(s) potentially involved in VWF reduction to the 2nd to 8th TSP-1 motifs and CUB-1 domain of ADAMTS-13. Finally, ethylmaleimide (NEM), which blocks free thiols, did not inhibit rADAMTS-13 to cleave pVWF multimers under static conditions and in the presence of urea and barium. NEM-treated rADAMTS-13 retained only 27.5±4.9% activity in cleaving ULVWF strings under flow conditions as compared to untreated enzyme. These data characterizes a novel mechanism that plays a regulatory role in cleaving ULVWF strings and maintaining the circulating pVWF multimers in inactive forms.

Substrate Recognition Mechanism of VAMP/Synaptobrevin-cleaving Clostridial Neurotoxins

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.

The Mechanism of Autocatalytic Activation of Plant-type L-Asparaginases

Plant l-asparaginases and their bacterial homologs, such as EcAIII found in Escherichia coli, form a subgroup of the N-terminal nucleophile (Ntn)-hydrolase family. In common with all Ntn-hydrolases, they are expressed as inactive precursors that undergo activation in an autocatalytic manner. The maturation process involves intramolecular hydrolysis of a single peptide bond, leading to the formation of two subunits (alpha and beta) folded as one structural domain, with the nucleophilic Thr residue located at the freed N terminus of subunit beta. The mechanism of the autocleavage reaction remains obscure. We have determined the crystal structure of an active site mutant of EcAIII, with the catalytic Thr residue substituted by Ala (T179A). The modification has led to a correctly folded but unprocessed molecule, revealing the geometry and molecular environment of the scissile peptide bond. The autocatalytic reaction is analyzed from the point of view of the Thr(179) side chain rotation, identification of a potential general base residue, and the architecture of the oxyanion hole.

Substitution of the Gla Domain in Factor X with That of Protein C Impairs Its Interaction with Factor VIIa/Tissue Factor

We previously reported that the first epidermal growth factor-like (EGF1) domain in factor X (FX) or factor IX (FIX) plays an important role in the factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. To assess the role of gamma-carboxyglutamic acid (Gla) domains of FX and FIX in FVIIa/TF induced coagulation, we studied four new and two previously described replacement mutants: FX(PCGla) and FIX(PCGla) (Gla domain replaced with that of protein C), FX(PCEGF1) and FIX(PCEGF1) (EGF1 domain replaced with that of protein C), as well as FX(PCGla/EGF1) and FIX(PCGla/EGF1) (both Gla and EGF1 domains replaced with those of protein C). FVIIa/TF activation of each FX mutant and the corresponding reciprocal activation of FVII/TF by each FXa mutant were impaired. In contrast, FVIIa/TF activation of FIX(PCGla) was minimally affected, and the reciprocal activation of FVII/TF by FIXa(PCGla) was normal; however, both reactions were impaired for the FIX(PCEGF1) and FIX(PCGla/EGF1) mutants. Predictably, FXIa activation of FIX(PCEGF1) was normal, whereas it was impaired for the FIX(PCGla) and FIX(PCGla/EGF1) mutants. Molecular models reveal that alternate interactions exist for the Gla domain of protein C such that it is comparable with FIX but not FX in its binding to FVIIa/TF. Further, additional interactions exist for the EGF1 domain of FX, which are not possible for FIX. Importantly, a seven-residue insertion in the EGF1 domain of protein C prevents its interaction with FVIIa/TF. Cumulatively, our data provide a molecular framework demonstrating that the Gla and EGF1 domains of FX interact more strongly with FVIIa/TF than the corresponding domains in FIX.

On Ribosome Conservation and Evolution

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A-> P-site passage of the tRNA terminus in the peptidyl transferase center is performed by a rotatory motion, synchronized with the overall tRNA/mRNA sideways movement. Guided by the PTC, the rotatory motion leads to stereochemistry suitable for peptide bond formation, as well as for substrate-mediated catalysis, consistent with quantum mechanical calculations elucidating the transition state mechanism for peptide bond formation and indicating that the peptide bond is being formed during the rotatory motion. Analysis of substrate binding modes to inactive and active ribosomes illuminated the significant PTC mobility and supported the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, utilizing free amino acids. Genetic control of the reaction evolved after poly-peptides capable of enzymatic function were created, and an ancient stable RNA fold was converted into tRNA molecules. As the symmetry relates only the backbone fold and nucleotide orientations, but not nucleotide sequence, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC has evolved by gene fusion, presumably by taking advantage of similar RNA fold structures.

A quenched fluorescent dipeptide for assaying dispase- and thermolysin-like proteases

Metalloproteases such as dispase and thermolysin play a crucial role in the life cycle of bacteria. Commonly, they prefer hydrophobic amino acids at P1′ of substrate proteins, thereby cleaving the peptide bond at the alpha amino group. Activity of such proteases has been measured by the use of tailor-made oligopeptides provided with fluorescence resonance energy transfer dyes. We can now show that the short dipeptide Dabcyl-Ser-Phe-EDANS is an appropriate substrate of dispase and thermolysin. It was cleaved by both enzymes at the single peptide bond accompanied by a steep increase in fluorescence. Substantial quenching effects of the formed products were observed only when more than 80 μM substrate was hydrolyzed. High affinity of the proteases for the dipeptide resulted in low K m values of 91 ± 9 and 104 ± 18 μM, which are comparable to those measured for longer peptides. Dabcyl-Ser-Phe-EDANS was also used to determine the pH and optimal temperature of dispase, which were found at pH 7.0 and 50 °C. Buffer substances such as acetate, citrate, and tris(hydroxymethyl)aminomethane had no significant effect on enzyme activity. Measurements up to 100 °C revealed that hydrolysis of the quenched fluorescent dipeptide took place only in the presence of active dispase.

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization, Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.

Botulinum Neurotoxin Types A, B, and E: Fragmentations by Autoproteolysis and Other Mechanisms Including by O-Phenanthroline–Dithiothreitol, and Association of the Dinucleotides NAD+/NADH with the Heavy Chain of the Three Neurotoxins

The first evidence of autoproteolytic activity of the approximately 50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their approximately 100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183-1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249-Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221-231; Ahmed et al. (2003). Biochemistry 42:12539-12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (-P1-P1' -) in one serotype (except Asp-Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249-Tyr250, Gln258-Ser259 and Ile243-Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT's structure-function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418-Thr419 or Phe422-Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT's entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs-the 9-residuelong motifs present on the NT's natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD(+)/NADH associated with the NTs type A, B and E (2-3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase-a representative NAD(+)/NADH binding protein.

Single Peptide Bonds Exhibit Poly(Pro)II (“Random Coil”) Circular Dichroism Spectra

The far-UV circular dichroism spectra of a series of amino acid derivatives containing single peptide bonds have been measured. The N-acetyl-alanine displays a polyproline (PP) II-like spectrum, but alaninamide shows a very weak positive signal. Similarly Gly-Ala shows a PPII spectrum, but Ala-Gly does not. On heating, the spectrum shows a two-state transition also shown by long PPII polypeptides. Thus the characteristic PPII negative maximum at <200 nm results from the coupling of a peptide bond N-terminal to the chiral alpha-carbon, and therefore the simplest peptide bonds have a preferred conformation that defines the spectrum of disordered proteins of any size.

Protein dissection enhances the amyloidogenic properties of α‐lactalbumin

Alpha-lactalbumin (LA) in its molten globule (MG) state at low pH forms amyloid fibrils. Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1-40/41-123, named Th1-LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the beta-subdomain of the native protein (1-40/53-123, named desbeta-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. Th1-LA and desbeta-LA aggregate at pH 2.0 much faster than the intact protein and form long and well-ordered fibrils. Furthermore, in contrast to intact LA, the LA derivatives form regular fibrils also at neutral pH, even if at much reduced rate. In acidic solution, Th1-LA and desbeta-LA adopt a MG state which appears to be similar to that of intact LA, as given by spectroscopic criteria. At neutral pH, both Th1-LA and desbeta-LA are able to bind the hydrophobic dye 1-anilinonaphtalene-8-sulfonate, thus indicating the presence of exposed hydrophobic patches. It is concluded that nicked Th1-LA and gapped desbeta-LA are more relaxed and expanded than intact LA and, consequently, that they are more suitable protein species to allow the large conformational transitions required for the polypeptide chain to form the amyloid cross-beta structure. As a matter of fact, the MG of LA attains an even more flexible conformational state during the early phases of the aggregation process at acidic pH, as deduced from the enhancement of its susceptibility to proteolysis by pepsin. Our data indicate that deletion of the beta-subdomain in LA does not alter the ability of the protein to assemble into well-ordered fibrils, implying that this chain region is not essential for the amyloid formation. It is proposed that a proteolytic hydrolysis of a protein molecule at the cellular level can trigger an easier formation of amyloid precipitates and therefore that limited proteolysis of proteins can be a causative mechanism of protein aggregation and fibrillogenesis. Indeed, a vast majority of protein deposits in amyloid diseases are given by protein fragments derived from larger protein precursors.

ADAMTS-13 Deficiency in Children with Septic Shock.

Von Willebrand factor (VWF) is synthesized and released from endothelial cells and megakaryocytes/platelets in a form that is ultra-large (UL) in size and hyper-reactive in functions, capable of spontaneously activating and aggregating platelets. Upon its release, ULVWF is cleaved by the metalloprotease ADAMTS-13 at a single peptide bond of Tyr842 and Met843 in the A2 domain of VWF, converting this hyper-reactive ULVWF to smaller and less active form. If it is left uncleaved, ULVWF could activate and aggregate platelets, leading to systemic thrombosis, as often found in patients with thrombotic thrombocytopenic purpura. However, defects in ULVWF proteolysis may also occur in other conditions where ADAMTS-13 activity may be suppressed and ULVWF release from endothelial cells is systemic and persistent. To test this hypothesis, we have measured changes in ADAMTS-13 activity and ULVWF proteolysis in pediatric patients with severe sepsis with one or more organ failure, an extremely severe form of systemic inflammation that is likely to trigger the release of a large amount of ULVWF. Ten patients (and 6 matching controls) were recruited and followed for a period of 7 to 28 days in a pediatric intensive care unit. We found that the mean ADAMTS-13 activity in patients on admission as determined by a flow assay was 60.1±30.4%, significantly lower than 97.7±1.5% found control subjects. Among these patients, 70% presented severe to moderate thrombocytopenia (22,000–150,000/ml) on admission. Three patients (30%) had ADAMTS-13 activity less than 30%, below the threshold level of deficiency predetermined for this assay. Two of these three patients also presented severe thrombocytopenia (platelet counts of 22,000 and 66,000/ml, respectively). During the follow-up period, changes in ADAMTS-13 activity correlated approximately with changes in platelet counts. Patients with little or no improvement in ADAMTS-13 activity often showed persistent thrombocytopenia, whereas improvement in the enzyme activity associated with increase in platelet counts. We have also measured plasma levels of four inflammatory cytokines and found that IL-6 and TNF-α were 98.7±10.8 and 516.2±66.0 pM in patients with severe ADAMTS-13 deficiency (< 30% in activity), significantly higher than those with the enzyme activity greater than 30% (49.2±21.3 and 46.3±19.1 pM for IL-6 and TNF-a, respectively, p < 0.001). In comparison, plasma levels of IL-1β and IL-8 were elevated, but showed no difference between the two groups. The reduction in plasma IL-6 and TNF-α levels in some patients correlated with increase in ADAMTS-13 activity and platelet counts. These results suggest that severe sepsis may result in a transient deficiency in ULVWF proteolysis, likely due to deficiency in ADAMTS-13 activity (consumptive or direct inhibition). The suppression of ULVWF proteolysis may contribute to thrombocytopenia often seen in these patients. The deficiency in ULVWF proteolysis may therefore play a bridge role between inflammation and thrombosis.

Fluorescence anisotropy assay for proteolysis of specifically labeled fusion proteins

A cloning method and plasmid vectors that permit fluorescence-anisotropy-based measurement of proteolysis are reported. The recombinant protein substrates produced by this method contain a tetracysteine motif that can be site-specifically labeled with bis-arsenical fluorophore [Science 281 (1998) 269]. Six protein substrates with an N-terminal fusion of the tetracysteine motif and different protease recognition sites were created and tested for reaction with commercial proteases commonly used to process recombinant fusion proteins. In each case, proteolysis of a single susceptible peptide bond could be monitored in real time and with sufficient data quality to allow numerical analysis of proteolysis reaction kinetics. Measurement of proteolysis extent using fluorescence anisotropy is shown to be comparable to densitometry measurements made on denaturing polyacrylamide gels but with the added advantages implicit in a time-resolved measurement, quantification by a spectroscopic measurement, and facile extensibility to high-throughput formats. The assay was also demonstrated as a general tool for monitoring proteolysis of multidomain fusion proteins containing an internal protease site such as are being created in structural genomics studies worldwide.

Pharmacokinetics (PK) of the angiogenesis inhibitor ABT-510 in healthy subjects

3080 Background: ABT-510 is a subcutaneously (SC) administered nonapeptide thrombospondin analogue in phase 2 clinical development for treatment of advanced malignancies. In mouse, rat, dog and monkey, t1/2 ranged from 0.2 h (mouse) to 1.3 h (monkey); metabolism was dominated by a single peptide bond cleavage yielding two inactive peptides (including M-1) which were rapidly excreted in bile and urine. Methods: To assess the safety, tolerability and PK of ABT-510, a first-time-in-man, single-escalating-dose, double-blind study was conducted in 43 healthy male subjects (mean±SD 34±12 yrs, 76±8 kg), randomly assigned to receive ABT-510 by SC bolus (10, 50, 75, 100 or 130 mg), SC infusion of 100 mg over 24 h, or 30-minute IV infusion (10, 50 or 100 mg); one subject per group received placebo. Samples for ABT-510 and M-1 assay were collected predose and over 24 h (blood) or 48 h (urine); concentrations were determined using a validated LC/MS/MS method. Results: PK results for SC bolus groups are shown below: ABT-510 PK were dose-proportional both for IV and SC regimens. Following IV dosing, CL, Vd and t1/2 were 35±8 L/h, 44±9 L and 0.9±0.1 h. SC doses were completely bioavailable relative to IV. The SC infusion produced a mean steady-state concentration of 150 ng/mL. M-1 plasma exposure averaged 50% greater than parent drug. About 78% of the dose was recovered as M-1 in urine. ABT-510 was generally well-tolerated, most AEs were mild in severity and resolved quickly. Conclusions: ABT-510 PK were linear in the range studied, interindividual variability was low. SC doses were rapidly absorbed and completely bioavailable relative to IV. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Abbott Laboratories Abbott Laboratories

Pharmacokinetics (PK) of the angiogenesis inhibitor ABT-510 in healthy subjects

3080 Background: ABT-510 is a subcutaneously (SC) administered nonapeptide thrombospondin analogue in phase 2 clinical development for treatment of advanced malignancies. In mouse, rat, dog and monkey, t1/2 ranged from 0.2 h (mouse) to 1.3 h (monkey); metabolism was dominated by a single peptide bond cleavage yielding two inactive peptides (including M-1) which were rapidly excreted in bile and urine. Methods: To assess the safety, tolerability and PK of ABT-510, a first-time-in-man, single-escalating-dose, double-blind study was conducted in 43 healthy male subjects (mean±SD 34±12 yrs, 76±8 kg), randomly assigned to receive ABT-510 by SC bolus (10, 50, 75, 100 or 130 mg), SC infusion of 100 mg over 24 h, or 30-minute IV infusion (10, 50 or 100 mg); one subject per group received placebo. Samples for ABT-510 and M-1 assay were collected predose and over 24 h (blood) or 48 h (urine); concentrations were determined using a validated LC/MS/MS method. Results: PK results for SC bolus groups are shown below: ABT-510 PK were dose-proportional both for IV and SC regimens. Following IV dosing, CL, Vd and t1/2 were 35±8 L/h, 44±9 L and 0.9±0.1 h. SC doses were completely bioavailable relative to IV. The SC infusion produced a mean steady-state concentration of 150 ng/mL. M-1 plasma exposure averaged 50% greater than parent drug. About 78% of the dose was recovered as M-1 in urine. ABT-510 was generally well-tolerated, most AEs were mild in severity and resolved quickly. Conclusions: ABT-510 PK were linear in the range studied, interindividual variability was low. SC doses were rapidly absorbed and completely bioavailable relative to IV. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Abbott Laboratories Abbott Laboratories

Enzymatic and Molecular Characteristics of the Efficiency and Specificity of Exfoliative Toxin Cleavage of Desmoglein 1

Exfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu381-Gly382, located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilized structure of Dsg1. Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage of Dsg1 by ETA, ETB, and ETD demonstrated kcat/Km values of 2-6 x 10(4) m(-1) s(-1), suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine protease inhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate. Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we show that for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond. If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisite specificity and efficiency of ETA may depend on the enzyme's binding upstream of the cleavage site with a very specific fit, like a key in a lock.

Structure-based development of a novel collagen inhibitor for MMP-1: re-designing the functions of a matrix protein.

Collagenases are a highly specific class of enzymes. In their native states, collagenases cleave only native triple helical collagen molecules at a single peptide bond between Gly775-Leu776 for Type I collagen and Gly775-Ile776 for Type II collagen. The linear sequence of collagen is about 1050 amino acids in length, where three linear peptide sequences are required to form a triple helical collagen molecule. At present, there exist no crystallographic structures of collagenase bound to native triple helical collagen; nor has it been shown that collagenase recognizes the triple helical conformation of collagen. In our study, we have used an inhibitor design structure-activity based approach to show that collagenase recognizes and cleaves triple helical collagen conformations in preference to non-triple helical collagen conformations.