Characterizing the specificity of activated Factor XIII for glutamine-containing substrate peptides

Activated Factor XIII (FXIIIa) is a transglutaminase that catalyzes the formation of gamma-glutamyl-varepsilon-lysine crosslinks in the fibrin network. To better understand the source of FXIIIa substrate specificity, Q-containing substrates based on beta-casein, K9-peptide, and alpha(2)-antiplasmin were characterized. alpha(2)AP (1-15, Q2, Q4) and alpha(2)AP (1-15, Q2, Q4N, K12R) are highly promising peptide models since they exhibited k(cat)/K(m) values comparable to intact beta-casein. In the absence of a lysine-like donor, FXIIIa could promote deamidation of a reactive Q to an E and solution NMR served as an effective strategy for monitoring this reaction. A tendency toward deamidation allowed greater investigations of the alpha(2)-antiplasmin based peptides. FXIIIa preferentially selects the Q2 residue for carrying out crosslinking processes. The E3 and Q4 provide supporting roles in binding. When a crosslinking reaction occurs at Q2, the Q4 position is sterically blocked from reactivity. By contrast, deamidation of Q2 to E2 allows, for the first time, observation of reactivity at Q4. The K12 position provides an additional favorable site of interaction with the FXIIIa surface. The sensitivity of alpha(2)AP (1-15, Q2, Q4) to amino acid changes at Q2, Q4, and K12 suggests the importance of individual FXIIIa subsites that are controlled by chemical environment and sterics.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides

Oxygen dependent degradation of hypoxia-inducible factor (HIF)-1α is triggered with hydroxylation by proline hydroxylase domain 2 (PHD2) under normoxic conditions. Some of previously developed PHD2 inhibitors show a considerable potency against factor inhibiting HIF (FIH), the HIF asparagine hydroxylase. For specific inhibition of PHD2, we have synthesized peptides containing 556-575 residues of HIF-1α with modifications at the Pro-564 and examined their inhibitory effect against PHD2. Adopting fluorescence polarization-based assays, we evaluated inhibitory potency of the peptides and selected potent inhibitors. These PHD2 inhibitor peptides showed no significant potency against FIH, demonstrating their specific inhibitory effect on PHD2.     

Structural features associated with the binding of glutamine-containing peptides to Factor XIII

Activated Factor XIII a2 catalyzes the formation of intermolecular gamma-glutamyl- epsilon -lysyl cross-links in the fibrin network. Solution NMR studies were carried out to characterize, the structural features associated with the binding of glutamine-containing peptides to Factor XIII. A coupled uv/vis kinetic assay demonstrated that K9 peptide (1-10), alpha2-antiplasmin (1-15), and alpha2-antiplasmin (1-15 Q4N) all function as glutamine-containing substrates for activated Factor XIII a2. 2D TOCSY spectra of the peptides exhibit upfield chemical shifts for the glutamine protons in the presence of Factor XIII. These results indicate that the reactive peptide glutamines are encountering a distinctive environment within the Factor XIII active site. 1D proton line-broadening and 2D transferred-NOESY studies reveal that the glutamines and residues located C-terminally come in direct contact with the enzyme and adopt an extended conformation. Substrates with sequences similar to alpha2-antiplasmin (1-15) are proposed to bind both at the catalytic site and at a neighboring apolar region.     

Inhibition of plant glutamine synthetases by substituted phosphinothricins

Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal α- and γ-substituted analogs of phosphinothricin (l-2-amino-4-(hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS₁ and chloroplastic GS₂ competitively versusl-glutamate, with K_i values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme-inhibitor dissociation. Herbicidal phosphinothricins exhibit low K_i values and slow enzyme turnover, as described by reactivation characteristics. Both the GS₁ and GS₂ isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series.     

Duplex structure formation between oligo(dA)''s and oligo(dT)''s generated by thymine-specific interaction with netropsin.

The formation of oligomeric duplex molecules in the presence of the antibiotic netropsin in the series p(dA)n-p(dT)n is demonstrated using low-temperature CD measurements. Addition of Netropsin to mixtures of oligomers generates the same type of CD spectra as observed for poly(dA)-poly(dT) and maintains the duplex structure at temperatures at which base pairing of free oligomers is thermodynamically unstable. The shortest chain length forming a netropsin complex by thymine-specific interaction with the oligopeptide is represented by p(dA)4-p(dt)4. Studies with sequence isomers show that adjacent thymine residues strongly favour the complex formation with the oligopeptide.     

Structure of the hydration shells of oligo(dA-dT).oligo(dA-dT) and oligo(dA).oligo(dT) tracts in B-type conformation on the basis of Monte Carlo calculations

Monte Carlo simulations [(N, V, T)-ensemble] were performed for the hydration shell of poly(dA-dT).poly(dA-dT) in canonical B form and for the hydration shell of poly(dA).poly(dT) in canonical B conformation and in a conformation with narrow minor groove, highly inclined bases, but with a nearly zero-inclined base pair plane (B' conformation). We introduced helical periodic boundary conditions with a rather small unit cell and a limited number of water molecules to reduce the dimensionality of the configuration space. The coordinates of local maxima of water density and the properties of one- and two-membered water bridges between polar groups of the DNA were obtained. The AT-alternating duplex hydration mirrors the dyad symmetry of polar group distribution. At the dApdT step, a water bridge between the two carbonyl oxygens O2 of thymines is formed as in the central base-pair step of Dickerson's dodecamer. In the major groove, 5-membered water chains along the tetranucleotide pattern d(TATA).d(TATA) are observed. The hydration geometry of poly(dA).poly(dT) in canonical B conformation is distinguished by autonomous primary hydration of the base-pair edges in both grooves. When this polymer adopts a conformation with highly inclined bases and narrow minor groove, the water density distribution in the minor groove is in excellent agreement with Dickerson's spine model. One local maximum per base pair of the first layer is located near the dyad axis between adjacent base pairs, and one local maximum per base pair in the second shell lies near the dyad axis of the base pair itself. The water bridge between the two strands formed within the first layer was observed with high probability. But the water molecules of the second layer do not have a statistically favored orientation necessary for bridging first layer waters. In the major groove, the hydration geometry of the (A.T) base-pair edge resembles the main features of the AT-pair hydration derived from other sequences for the canonical B form. The preference of the B' conformation for oligo(dA).oligo(dT) tracts may express the tendency to common hydration of base-pair edges of successive base pairs in the grooves of B-type DNA. The mean potential energy of hydration of canonical B-DNA was estimated to be -60 to -80 kJ/mole nucleotides in dependence on the (G.C) contents. Because of the small system size, this estimation is preliminary.     

Potent inhibition of ribonuclease A by oligo(vinylsulfonic acid)

Ribonuclease A (RNase A) can make multiple contacts with an RNA substrate. In particular, the enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic interactions. Here, oligomers of vinylsulfonic acid (OVS) are shown to be potent inhibitors of RNase A that exploit these interactions. Inhibition is competitive with substrate and has Ki = 11 pm in assays at low salt concentration. The effect of salt concentration on inhibition indicates that nearly eight favorable Coulombic interactions occur in the RNase A.OVS complex. The phosphonic acid and sulfuric acid analogs of OVS are also potent inhibitors although slightly less effective. OVS is also shown to be a contaminant of MES and other buffers that contain sulfonylethyl groups. Oligomers greater than nine units in length can be isolated from commercial MES buffer. Inhibition by contaminating OVS is responsible for the apparent decrease in catalytic activity that has been observed in assays of RNase A at low salt concentration. Thus, OVS is both a useful inhibitor of RNase A and a potential bane to chemists and biochemists who use ethanesulfonic acid buffers.     

Inhibition of thrombin by synthetic hirudin peptides

To investigate the role of different regions of hirudin in the interaction with the proteinase thrombin, segments of hirudin containing 15-51 residues were synthesized. The C-terminal segment 40-65 inhibited the fibrinogen clotting activity of thrombin but not amidolysis of tosyl-Gly-Pro-Arg-p-nitroanilide. Central peptide 15–42 was insoluble at pH 7, but peptide 15-65 inhibited fibrinogen clotting and amidolysis to an equal extent. The N-terminal loop peptide 1-15 had no inhibitory activity and did not affect the potency of peptide 15-65. These data suggest that the central region inhibits catalysis.     

Inhibition of mammalian collagenases by thiol-containing peptides

The following thiol-containing peptide analogues of the carboxyl side of the collagenase-sensitive bond of collagen were synthesized and tested as inhibitors of collagenases partially purified from homogenates of rabbit V-2 tumor and culture medium of pig synovium: HSCH2CH(CH3)CO-Ala-OEt (I), HSCH2CH(CH2Ph)CO-Ala-OEt (II), HSCH2CH[CH2CH(CH3)2]CO-Ala-OEt (III); HSCH2 CH-[CH2CH(CH3)2]CO-Ala-Gly-OEt (IV); HSCH2CH[CH2CH(CH3)2]CO-Ala-Gly-Gln (V). The compounds are listed in order of their inhibitory potency when assayed with nonfibrillar-acid-soluble calf skin collagen at pH 7.6, 35 degrees C. The best inhibitor (III) gave 50% inhibition between 1 and 4 microM. II was a competitive inhibitor with a Ki value of 75 microM. The enzymes preferred an isobutyl side chain at the 2-carbon position, and, where tested (III, IV), did not discriminate strongly between stereoisomers at the chiral 2-carbon. Increasing the length of the inhibitor did not markedly increase potency.     

Inhibition of virus multiplication by immunoactive peptides

In the present study we show that peritoneal macrophages obtained from the mice treated with the immunoactive peptides inhibit the multiplication of Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), but not that of vesicular stomatitis virus (VSV), and that the intraperitoneal administration of the peptides suppresses the infection with HSV-1 in mice.     

NMR studies of a deoxyribodecanucleotide containing an extrahelical thymidine surrounded by an oligo(dA).oligo(dT) tract

One- and two-dimensional NMR experiments were carried out on a decamer, d-(CGCTTTTCGC).d(GCGAAAAGCG), and on the same sequence with the addition of an unpaired thymidine, d(CGCTTTTCGC).d(GCGAATAAGCG), which will be referred to as the T-bulge decamer. Evidence from one-dimensional NOE experiments on the exchangeable protons indicates that the unpaired thymidine is extrahelical. This conclusion is also supported by numerous cross-peaks in the two-dimensional NOESY spectrum of the nonexchangeable protons. Assignments for all of the resonances, with the exception of the H5' and H5" resonances, have been made for both oligonucleotide duplexes through the use of 2D NOESY, COSY, and relayed COSY experiments. Temperature dependence of the methyl resonance chemical shifts indicates that the unpaired thymidine shows unusual behavior compared to other thymidines in the duplex. Two-dimensional NOESY experiments carried out from 5 to 35 degrees C indicate the unpaired thymidine remains extrahelical throughout this temperature range. A similar temperature dependence for the methyl chemical shift is found in the corresponding single-strand d(GCGAATAAGCG). The oligo-(dA).oligo(dT) tracts in both the decamer and the T-bulge decamer have structures different from B-form DNA and exhibit NOEs similar to those observed in other oligonucleotides containing A.T tracts. The formation of this unusual A.T tract structure may induce the extrahelical conformation of the unpaired thymidine.     

Inhibition of NF-kappa B activation by peptides targeting NF-kappa B essential modulator (nemo) oligomerization

NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.     

An achiral (oligo)nucleotide analog

An achiral nucleotide analog based on barbituric acid has been synthesized. The analog, which is 5,5-di(2-phosphoethyl)barbituric acid, undergoes extensive oligomerization in aqueous solution, when activated, to produce pyrophosphate-linked chains. In contrast to a number of other bisphosphorylated nucleoside analogs which have been studied, the compound has little tendency to cyclize. The possible prebiotic implications are discussed. Correspondence to: AM. Schwartz     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.