Oligomerization of the human prion protein proceeds via a molten globule intermediate

The conformational transition of the human prion protein from an alpha-helical to a beta-sheet-rich structure is believed to be the critical event in prion pathogenesis. The molecular mechanism of misfolding and the role of intermediate states during this transition remain poorly understood. To overcome the obstacle of insolubility of amyloid fibrils, we have studied a beta-sheet-rich misfolded isoform of the prion protein, the beta-oligomer, which shares some structural properties with amyloid, including partial proteinase resistance. We demonstrate here that the beta-oligomer can be studied by solution-state NMR spectroscopy and obtain insights into the misfolding mechanism via its transient monomeric precursor. It is often assumed that misfolding into beta-sheet-rich isoforms proceeds via a compatible precursor with a beta-sheet subunit structure. We show here, on the contrary, evidence for an almost natively alpha-helix-rich monomeric precursor state with molten globule characteristics, converting in vitro into the beta-oligomer. We propose a possible mechanism for the formation of the beta-oligomer, triggered by intermolecular contacts between constantly rearranging structures. It is concluded that the beta-oligomer is not preceded by precursors with beta-sheet structure but by a partially unfolded clearly distinguishable alpha-helical state.     

Amyloid formation under physiological conditions proceeds via a native-like folding intermediate

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.     

Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.     

Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study

The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.     

Inhibition of thermolysin and human alpha-thrombin by cobalt(III) Schiff base complexes.

Cobalt(III) Schiff base complexes have been shown to inhibit the replication of the ocular herpes virus. It is well known that these complexes have a high affinity for nitrogenous donors such as histidine residues, and it is possible that they bind to (and inhibit) an enzyme that is crucial to viral replication. In model studies, we have found that [Co(acacen)(NH3)2]+ is an effective irreversible inhibitor of thermolysin at millimolar concentrations; it also inhibits human alpha-thrombin. Axial ligand exchange with an active-site histidine is the proposed mechanism of inhibition. The activity of thermolysin and thrombin can be protected by binding a reversible inhibitor to the active site before addition of the cobalt(III) complex.     

Evidence that catalysis by yeast inorganic pyrophosphatase proceeds by direct phosphoryl transfer to water and not via a phosphoryl enzyme intermediate

In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversion of configuration. Both the synthesis of chiral ATP gamma S and the determination of inorganic thiophosphate configuration were carried out as described by Webb [Webb, M. R. (1982) Methods Enzymol. 87, 301-316]. We also show in a single turnover experiment performed in H2(18)O that 1 mol each of 18O16O3P and 16O4P is produced per mol of inorganic pyrophosphate hydrolyzed, a strong indication that oxygen uptake to form inorganic phosphate on PPase catalysis of inorganic pyrophosphate hydrolysis comes directly from H2O. These two results provide strong evidence for the conclusion that PPase catalyzes inorganic pyrophosphate hydrolysis via a single-step direct phosphoryl transfer to water and does not involve formation of a phosphorylated enzyme intermediate.     

The interaction of heparin with human alpha-thrombin: effect on the hydrolysis of anilide tripeptide substrates.

The interaction of heparin with human α-thrombin was investigated in the present report. Hydrolysis of synthetic tripeptide anilide substrates by thrombin was enhanced in the presence of heparin. With both N-α-benzoyl-l-phenylalanyl-l-valyl-l-arginine-p-nitroanilide (BzPheValArgNaN) and N-α-p-tosyl-l-glycyl-l-prolyl-l-arginine-p-nitroanilide (TosGlyProArgNaN), saturating concentrations of heparin enhanced the binding of substrate two-to threefold as determined by a decrease in the apparent Michaelis constant value, while having a marginal inhibitory effect on V. Substrate inhibition was observed with BzPheValArgNaN, which was enhanced in the presence of heparin. The enhancing effect of heparin on the binding of TosGlyProArgNaN was used to determine a dissociation constant value of 1.7 × 10^?9m for the heparin · thrombin complex. This value is nearly two orders of magnitude lower than the dissociation constant value determined for the heparin · antithrombin III complex (B. Nordenman and I. Bjork, 1978, Biochemistry17, 3339–3344), suggesting strongly that heparin must bind to thrombin to account for the enhancing effect of heparin on the antithrombin III/thrombin reaction. Heparin also enhanced the rate of inactivation of thrombin by 1-chloro-3-tosylamido-7-amino-l-2-hepatonone, but had little effect on the inactivation rate with phenylmethanesulfonyl fluoride.     

Resolution of parvovirus dimer junctions proceeds through a novel heterocruciform intermediate

The minute virus of mice initiator protein, NS1, excises new copies of the left viral telomere in a single sequence orientation, dubbed flip, during resolution of the junction between monomer genomes in palindromic dimer intermediate duplexes. We examined this reaction in vitro using both (32)P-end-labeled linear substrates and similar unlabeled templates labeled by incorporation of [alpha-(32)P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriL(TC), the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3' nucleotide to prime DNA synthesis and, in a reaction we call "read-through synthesis," forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5' end of the DNA, where it can serve as a 3'-to-5' helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriL(GAA).     

Inhibition of chymotrypsin by phosphonate and phosphonamidate peptide analogs

A series of mono-, di-, and tetrapeptide analogs incorporating an activated, tetrahedral phosphorus moiety in place of the scissile carbonyl group have been synthesized and evaluated as inactivators of porcine pancreatic elastase (EC 3.4.21.11) and bovine pancreatic chymotrypsin (EC 3.4.21.1). As previously reported [8.]Biochem. Biophys. Res. Commun.112, 1085–1090), the simple phosphonofluoridate analogs of alanine (6a-F) and phenylalanine (6b-F) are the most rapid inactivators yet reported for these enzymes. More hydrolytically stable derivatives of 5b and 6b were explored for chymotrypsin, and the thiophenyl, thiomethyl, and phenyl esters were shown to be effective inactivators, with the latter being the most stable. This strategy was extended to the tetrapeptide level by the synthesis of the four diastereomers of analog 8. However, each isomer proved to be comparable in its behavior toward chymotrypsin. Potential reasons for this unexpected lack of stereoselectivity were investigated, and it was shown that the phosphorus amide substituent is capable of functioning as a leaving group. In a related dipeptide diester analog, -15, a threefold difference in reactivity between phosphorus diastereomers was seen.     

Inhibition of adenosine kinase by phosphonate and bisphosphonate derivatives

The enzyme adenosine kinase (AK) plays a central role in regulating the intracellular and interstitial concentration of the purine nucleoside adenosine (Ado). In view of the beneficial effects of Ado in protecting tissues from ischemia and other stresses, there is much interest in developing AK inhibitors, which can regulate Ado concentration in a site- and event-specific manner. The catalytic activity of AK from different sources is dependent upon the presence of activators such as phosphate (Pi). In this work we describe several new phosphorylated compounds which either activate or inhibit AK. The compounds acetyl phosphate, carbamoyl phosphate, dihydroxyacetone phosphate and imidodiphosphate were found to stimulate AK activity in a dose-dependent manner comparable to that seen with Pi. In contrast, a number of phosphonate and bisphosphonate derivatives, which included clodronate and etidronate, were found to inhibit the activity of purified AK in the presence of Pi. These AK inhibitors (viz. clodronate, etidronate, phosphonoacetic acid, 2-carboxyethylphosphonic acid, N-(phosphonomethyl)-glycine and N-(phosphonomethyl)iminodiacetic acid), at concentrations at which they inhibited AK, were also shown to inhibit the uptake of ³H-adenosine and its incorporation into macromolecules in cultured mammalian cells, indicating that they were also inhibiting AK in intact cells. The drug concentrations at which these effects were observed showed limited toxicity to the cultured cells, indicating that these effects are not caused by cellular toxicity. These results indicate that the enzyme AK provides an additional cellular target for the clinically widely used bisphosphonates and related compounds, which could possibly be exploited for a new therapeutic application. Our structure–activity studies on different AK activators and inhibitors also indicate that all of the AK activating compounds have a higher partial positive charge (δ⁺) on the central phosphorous atom in comparison to the inhibitors. This information should prove helpful in the design and synthesis of more potent inhibitors of AK.     

Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.     

Inhibition of DNA polymerases by tripeptide derivative protease inhibitors.

Benzyloxycarbonyl(Z)-Leu-Leu-Leu-al and dansyl(Dns)-Leu-Leu-Leu-CH2Cl, well known as protease inhibitors, effectively inhibit the activities of DNA polymerases alpha, beta and gamma from rat liver and pol I from Escherichia coli, but the ability of these inhibitors to inhibit terminal deoxynucleotidyl transferase (TdT) is weak. The mode of inhibition by these tripeptide analogues is non-competitive with dNTP. The Ki values for Z-Leu-Leu-Leu-al and Dns-Leu-Leu-Leu-CH2Cl are 6.25 x 10(-5) M and 6.56 x 10(-5) M, respectively.     

Inhibition of TRAP-induced angiogenesis by the tripeptide Phe-Pro-Arg, a thrombin-receptor-derived peptide analogue

Summary We have recently shown that thrombin promotes angiogenesis by a mechanism independent of fibrin formation. In the present paper, we investigated the effect of the thrombin-receptor-activating tetradecapeptide (TRAP_1–14, S₄₂FLLRNPNDKYEPF₅₅) for its effects on angiogenesis in the chick chorioallantoic membrane (CAM) system of angiogenesis. A dose-dependent promotion of angiogenesis is evident with TRAP. In contrast, a thrombin-receptor-derived tripeptide analogue H-Phe-Pro-Arg-OH (FPR), which was designed based on the S₄₂FLLR₄₆ sequence, caused an inhibition of angiogenesis in the CAM, and when it was combined with TRAP it caused a complete reversal of the angiogenesis-promoting effect of TRAP. These results indicate that the proteolytic exposure of the receptor N-terminal tetradecapeptide by thrombin can activate the post-thrombotic events related to angiogenesis. These events can be modulated by constrained peptide analogues such as FPR.     

Fractionation of heparin by affinity chromatography on covalently-bound human alpha-thrombin

Commerical heparin, 135 USP units/mg, was fractionated by human α-thrombin-agarose affinity chromatography. Heparin was applied to an α-thrombin-agarose column equilibrated with 0.01 M Tris HCl (pH 7.4). Unbound heparin was washed from the column with the equilibration buffer. Bound heparin could be eluted with buffer containing 0.025 M NaCl. The specific activity of bound heparin was as great as 500 USP units/mg. Gel filtration was used to fractionate the heparin into molecular size classes. Low molecular weight heparin, with an average specific activity of 100 USP units/mg, was applied to the α-thrombin-agarose column. Gel filtration of the unbound heparin indicated that larger heparin molecules been selectively removed by the α-thrombin-agarose column. Bound heparin had a specific activity of 270 units/mg. Kinetic results of $\text{N-α-tosyl-L-glycyl-L-prolyl-L-arginine-}\text{p}\text{-nitroanilide}$ hydrolysis by α-thrombin in the presence of heparin correlated with the anticoagulant activity.     

Inhibition of enzymatic activity of alpha-thrombin by low molecular weight synthetic inhibitor]

The effect of the organophosphoric inhibitor, SA-152, on the fibrinogen-coagulating and TAME-esterase activity of bovine alpha-thrombin was studied. The irreversible inhibition constants (k11 = 1.1 x 10(4) M-1.min-1,Ki = 0.7 x 10(-4) M, k2 = 0.8 min-1 towards the coagulating activity and kII = 0.7 x 10(4) M-1.min-1, Ki = 0.3 x 10(-4) M, k2 = 0.2 min-1 towards the esterase activity) were determined. The SA-152 inactivated alpha-thrombin was dialyzed and incubated with 0.5 M and 2.5 M NaCl and 10 mM TAME. There was no reconstitution of activity of the SA-152 modified alpha-thrombin after dialysis and treatment with high concentrations of NaCl and TAME. Heparin interactions with the anion-binding site of the high molecular weight recognition center in the alpha-thrombin molecule did not significantly influence the values of the kinetic constants for the enzyme inhibition by SA-152. This finding is consistent with the hypothesis on the irreversible binding of SA-152 in the active center of the enzyme.     

Analysis of human alpha-thrombin by hydrophobic interaction high-performance liquid chromatography

The major active form of human thrombin, alpha-thrombin, was analyzed by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). The chromatographic system included a polymeric phenyl column and elution was performed by a gradient, 2-0M sodium chloride (5-20 min). Total analysis time was 30 min per injection. By this method, a good resolution between alpha-thrombin and the proteolytically modified thrombin forms, beta- and gamma-thrombin, was obtained. In addition, the thrombin preforms, prothrombin, prethrombin 1, and prethrombin 2, were also resolved from alpha-thrombin in the system. The results from the HIC method were compared to those obtained from non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By this high-resolution chromatographic method, the rapid analysis of purified alpha-thrombin is possible.     

Reduction of cysteine sulfinic acid in peroxiredoxin by sulfiredoxin proceeds directly through a sulfinic phosphoryl ester intermediate

Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, Cys-SO(2)(-). This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the gamma-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8A resolution crystal structure of Srx in complex with ATP and Mg(2+). This structure reveals the role of the Mg(2+) ion to position the gamma-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the Prx-SO(2)(-) species, support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.     

Inhibition of Yersinia protein tyrosine phosphatase by phosphonate derivatives of calixarenes

Inhibition of Yersinia protein tyrosine phosphatase by calix[4]arene mono-, bis-, and tetrakis(methylenebisphosphonic) acids as well as calix[4]arene and thiacalix[4]arene tetrakis(methylphosphonic) acids have been investigated. The kinetic studies revealed that some compounds in this class are potent competitive inhibitors of Yersinia PTP with inhibition constants in the low micromolar range. The binding modes of macrocyclic phosphonate derivatives in the enzyme active center have been explained using computational docking approach. The results obtained indicate that calix[4]arenes are promising scaffolds for the development of inhibitors of Yersinia PTP.