Free Amino Terminus Research Articles

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

Identification of protease substrates by combinatorial profiling on TentaGel beads

Reacting a 65,536 member combinatorial library of octapeptides on TentaGel beads with various proteases followed by selective staining of the free amino termini at the reacted bead surface and sequence determination by amino acid analysis allowed the rapid identification of protease substrates.

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80–150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr 1, Pro 3 and Pro 5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [ 3H]Tyr by tyrosinase. Metabolic degradation of [ 3H]Tyr-5P was found to be considerably quicker than that of H-[ 3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.

Novel immobilizations of an adhesion peptide on the TiO2 surface: An XPS investigation

The covalent attachment of an adhesive peptide, reproducing the 351–359 sequence of human vitronectin, to oxidized titanium surfaces was investigated by XPS spectroscopy. The peptide enhances osteoblast adhesion to titanium, the most used biomaterial for implants and prostheses. Core level spectra of the TiO2 surface and of the biomimetic surface were investigated.Novel selective covalent immobilization of (351–359) HVP was carried out by treatment of the TiO2 surface with (3-aminopropyl) triethoxysilane, glutaric anhydride and a side chain protected peptide sequence presenting only a free terminal amino group, followed by side chain deprotection.An alternative strategy for covalent attachment consists in photoactivation of physisorbed (351–359) HVP directly on the TiO2 surface; samples were incubated with HVP solution and subsequently irradiated with UV light. A comparison with the results previously obtained for non-selective HVP immobilization will be discussed.

Nicastrin Functions as a γ-Secretase-Substrate Receptor

gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.

γ-Secretase Exists on the Plasma Membrane as an Intact Complex That Accepts Substrates and Effects Intramembrane Cleavage*

Research on Alzheimer's disease led to the identification of a novel proteolytic mechanism in all metazoans, the presenilin/gamma-secretase complex. This unique intramembrane-cleaving aspartyl protease is required for the normal processing of Notch, Jagged, beta-amyloid precursor protein (APP), E-cadherin, and many other receptor-like proteins. We recently provided indirect evidence of gamma-secretase activity at the cell surface in HeLa cells following inhibition of receptor-mediated endocytosis. Here, we directly identify and isolate gamma-secretase as an intact complex (Presenilin, Nicastrin, Aph-1, and Pen-2) from the plasma membrane, both in overexpressing cell lines and endogenously. Inhibition of its proteolytic activity allowed cell surface gamma-secretase to be captured in association with its plasma membrane-localized APP substrates (C83 and C99). Moreover, non-denaturing isolation of the intact enzyme complex revealed that cell surface gamma-secretase can specifically generate amyloid beta-protein from an APP substrate and similarly cleave a Notch substrate. These data directly establish the proteolytic function of gamma-secretase on the plasma membrane, independent of a hypothesized substrate trafficking role. We conclude that presenilin/gamma-secretase exists as a mature complex at the cell surface, where it interacts with and can cleave its substrates, consistent with an essential function in processing many adhesion molecules and receptors required for cell-cell interaction or intercellular signaling.

Aβ42 immunization in Alzheimer's disease generates Aβ N-terminal antibodies

Serum samples from Alzheimer's disease (AD) patients immunized with Abeta42 (AN1792) were analyzed to determine the induced antibody properties including precise amyloid-beta peptide (Abeta) epitopes and amyloid plaque-binding characteristics. The predominant response in these patients is independent of whether or not meningoencephalitis developed and is against the free amino terminus of Abeta. The immunostaining of amyloid plaques in brain tissue by patient sera is adsorbable by a linear Abeta1-8 peptide, demonstrating that the antibodies are directed predominantly to this epitope and not dependent on Abeta conformations or aggregates specific to plaques. Furthermore, the antibodies are not capable of binding amyloid precursor protein and would be predicted to be competent in facilitating clearance of amyloid plaques in AD brains.

Efficient and direct solid phase synthesis of ketomethylenimino and ketomethylenamino peptides

The aim of this study is the synthesis of pseudopeptides on solid supports, in order to quickly obtain modified peptides. We report a convenient step-by-step synthesis of ketomethylenimino ψ[CO–CH N] and ketomethylenamino ψ[CO–CH 2–NH] peptides. The key is the reaction between the free amino terminus of the supported peptide and a glyoxal-modified amino acid, leading to a ketomethylenimino bond, which can be reduced to a ketomethylenamino bond.

Yeast mating for combinatorial Fab library generation and surface display

Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.

Syntheses of Heterobifunctional Candidate Ligands of P‐Selectin Containing Both Sulfated Lewis X Trisaccharide and Various Sulfated Peptides †

Various heterobifunctional glycopeptides containing both 3′‐sulfated Lewis x and peptides containing at least one sulfated tyrosine were constructed onto a pentaerythritol backbone following a common strategy that relies on the condensation, through reductive amination, of aldehydes 9 or 17 with various peptides having a free terminal amino group. The corresponding homodimer of 3′‐sulfated Lewis x linked to pentaerythritol was prepared for comparison of the inhibitory activities. †This paper is dedicated to Professor Gérard Descotes on the occasion of his 70th birthday.

Humoral immune response to fibrillar beta-amyloid peptide.

The beta-amyloid peptide (Abeta) is a normal product of the proteolytic processing of its precursor (beta-APP). Normally, it elicits a very low humoral immune response; however, the aggregation of monomeric Abeta to form fibrillar Abeta amyloid creates a neo-epitope, to which antibodies are generated. Rabbits were injected with fibrillar human Abeta(1-42), and the resultant antibodies were purified and their binding properties characterized. The antibodies bound to an epitope in the first eight residues of Abeta and required a free amino terminus. Additional residues did not affect the affinity of the epitope as long as the peptide was unaggregated; the antibody bound Abeta residues 1-8, 1-11, 1-16, 1-28, 1-40, and 1-42 with similar affinities. In contrast, the antibodies bound approximately 1000-fold more tightly to fibrillar Abeta(1-42). Their enhanced affinity did not result from their bivalent nature: monovalent Fab fragments exhibited a similar affinity for the fibrils. Nor did it result from the particulate nature of the epitope: monomeric Abeta(1-16) immobilized on agarose and soluble Abeta(1-16) exhibited similar affinities for the antifibrillar antibodies. In addition, antibodies raised to four nonfibrillar peptides corresponding to internal Abeta sequences did not exhibit enhanced affinity for fibrillar Abeta(1-42). Antibodies directed to the C-terminus of Abeta bound poorly to fibrillar Abeta(1-42), which is consistent with models where the carboxyl terminus is buried in the interior of the fibril and the amino terminus is on the surface. When used as an immunohistochemical probe, the antifibrillar Abeta(1-42) IgG exhibited enhanced affinity for amyloid deposits in the cerebrovasculature. We hypothesize either that the antibodies recognize a specific conformation of the eight amino-terminal residues of Abeta, which is at least 1000-fold more favored in the fibril than in monomeric peptides, or that affinity maturation of the antibodies produces an additional binding site for the amino-terminal residues of an adjacent Abeta monomer. In vivo this specificity would direct the antibody primarily to fibrillar vascular amyloid deposits even in the presence of a large excess of monomeric Abeta or its precursor. This observation may explain the vascular meningeal inflammation that developed in Alzheimer's disease patients immunized with fibrillar Abeta. Passive immunization with an antibody directed to an epitope hidden in fibrillar Abeta and in the transmembrane region of APP might be a better choice in the search for an intervention to remove Abeta monomers without provoking an inflammatory response.

The Cys-Xaa-His metal-binding motif: [N] versus [S] coordination and nickel-mediated formation of cysteinyl sulfinic acid.

A series of peptide ligands containing the sequence -Cys-Xaa-His- (CXH; Xaa=Gly or Lys) has been prepared and the coordination chemistry of these peptides with nickel(II) investigated. Selective protection of either the N-terminal cysteine thiol or amine group gave complexes with amino or thiolato coordination, respectively, to nickel(II). Insertion of CGH into a pentapeptide, N-acetyl-Ala-Cys-Gly-His-Ala-CONH(2), allowed the formation of a square-planar thiolato Cys-Gly-His complex with nickel(II) in an internal position of the peptide. Inclusion of an N-terminal cysteine residue with a free amino terminus gave rise to pH- and dioxygen-dependent coordination behavior. Solutions of CGH-CONH(2) with nickel(II) at neutral pH yielded a red nickel-thiolate complex, but at higher pH (8.5 or above) or with exposure to dioxygen, yellow nickel complexes with N-terminal amino coordination were observed. The disulfide-bridged dimers formed from Ni(CGH-CONH(2)) in the presence of air were characterized and found to have the typical coordination found in the amino-terminal binding motif of the serum albumins. Nickel(II) coordination and thiol reactivity were also studied by determination of rates of thiol alkylation and by monitoring air oxidation in the presence of various metals. Zinc(II) effectively inhibits thiol alkylation and oxidation (disulfide formation) in all the peptides studied. Nickel(II) inhibits aerobic oxidation and alkylation of N-terminal protected peptides such as N-acetyl-Cys-Gly-His, but does not inhibit air oxidation of free amino terminal peptides such as Cys-Gly-His. Instead, nickel(II) mediates the formation an additional product under aerobic conditions, a cysteinesulfinic acid.

The interaction of cationic dendrons with albumin and their diffusion through cellulose membranes

Amphipathic dendrons (partial dendrimers) having three lipidic (C 14) chains coupled to dendritic lysine head groups with 8, 16 or 32 free terminal amino groups have been synthesised by solid phase peptide synthesis. Their interaction with albumin was studied in the presence of NaCl using dynamic dialysis, the diffusion of the dendrons through regenerated cellulose membranes also being studied. The stoichiometry of dendron: albumin interactions was found to be 1:1.5, 1:4 and 1:5 for the dendrons with 8, 16 and 32 amino groups, respectively. Membrane permeability P, membrane diffusion coefficient D and the membrane partition coefficient K values were calculated for each dendron. P and D values were low but highest for the 8 amino group dendron. The membrane partition coefficient K was greatest for the 8 amino group dendron. This was also the case with octanol/water partition coefficient studies. Considerable adsorption of the dendrons to the cellulose membrane occurred but NaCl decreased adsorption and improved diffusion of the dendrons through the cellulose membrane.

Adsorption of amphipathic dendrons on polystyrene nanoparticles

Adsorption of dendrons onto nanoparticles may provide new model structures which may be useful in drug and gene delivery. Tritiated amphipathic dendrons having three lipidic (C 14) chains coupled to branched (dendritic) lysine head groups with 8, 16 or 32 free terminal amino groups have been synthesised by solid phase peptide techniques. The interaction between these tritiated dendrons and 200 nm polystyrene latex nanoparticles was investigated in phosphate buffered saline. The amount of dendron adsorbed increased with increasing concentration of dendrons and then decreased. Maximum adsorption of dendrons per gram of nanoparticles was found to be between 8.2 and 84×10 –6 M, the amounts adsorbed being inversely proportional to the number of amino groups present in the molecule. The number of dendron molecules adsorbed per nanoparticle was found to be between 430 and 4421. The degree of adsorption was found to be slightly altered by the temperature.

Structural changes in urea-formaldehyde resins during storage

C NMR spectroscopy. The three-step synthesis produces the resin consisting of two parts: the polymeric part from condensation of formaldehyde with first amount of urea, and monomeric part formed after addition of second part of urea. Some differences in structure depending on details of synthesis are of temporary character and level mainly in transhydroxymethylation from polymeric to monomeric part of resin. The reactions occuring after addition of second urea and following during storage are considered as one continuous process. Methylenes adjacent to secondary and tertiary amino groups form only during acid condensation. The main reaction during storage is the formation of methylenes adjacent to secondary amino groups. This reaction occurs between free terminal hydroxymethyl and amino groups of both resin parts. The physical and mechanical properties of particleboards confirm the close performance characteristics of studied resins. Particleboards satisfy requirements for general purpose boards P2 and emission standard E1.

Absorption of biologically active peptide hormones from the small intestine of rat.

Absorption of the 4, 10 and 34 amino acid forms of gastrin from the small intestine has been investigated in anaesthetized rats. The method of assessment of successful absorption of the hormone into the systemic circulation was when the amount of acid secreted by the stomach over consecutive 15-min periods was increased. When the natural hormones were infused into the ileum in a relatively high dose, there was no increase in gastric acid secretion, indicating that they had not been absorbed. Each of the forms of gastrin was conjugated at the free amino terminus to the carboxyl group of cholic acid. Subsequent infusion of the conjugated form of gastrin into the ileum, this time in relatively low doses, resulted in substantial and prolonged increases in gastric acid secretion, indicating that these hormones had been successfully absorbed. In addition, conjugation of the 10 and 34 amino acid forms of gastrin with cholic acid was shown to increase markedly the potency in evoking an increase in gastric acid secretion in response to intravenous injection of the hormone. Absorption of the gastrin conjugates was specific to the ileum thus indicating that they had been absorbed through the bile salt transporters.

Neuronal ceroid lipofuscinoses caused by defects in soluble lysosomal enzymes (CLN1 and CLN2).

Infantile and classical late infantile neuronal ceroid lipofuscinoses (NCL) are two recent additions to the expanding spectrum of lysosomal storage disorders caused by deficiencies in lysosomal hydrolases. They are latecomers to the lysosomal storage disorders, probably because of the heterogeneous nature of the storage material, which precluded meaningful biochemical analysis. Infantile NCL is caused by deficiency in palmitoyl-protein thioesterase, an enzyme that hydrolyzes fatty acids from cysteine residues in lipid-modified proteins. Classical late-infantile NCL is caused by a deficiency in tripeptidyl amino peptidase-I, a lysosomal peptidase that removes three amino acids from the free amino terminus of peptides or small proteins. Late-onset forms of these disorders have been described. The clinical, biochemical, and molecular genetic aspects of these two latest lysosomal storage disorders are discussed in this review. In addition, approaches to treatment and future directions for research are examined.

Mass spectrometric noncovalent probing of amino acids in peptides and proteins

Mass spectrometric methods are becoming available that probe structural or topological aspects of peptides and proteins. However, most of them require extensive chemical modification or covalent crosslinking of the biomolecules. We present a novel strategy based on the formation of noncovalent complexes between basic acidic amino acid residues and selective ligands. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) are employed to analyze the noncovalent assemblies. Different basic sites are probed with sulfonic acid derivatives. Naphthalene-disulfonic acid selectively binds to arginine residues and free amino termini; here we provide a chemical explanation for this behavior. Acidic sites are probed with molecules carrying guanidinium groups. However, only cysteic acid residues can so far be detected in this fashion; identification of glutamic and aspartic acid still awaits further investigation.

Synthesis and characterization of immobilized dendritic silica surfaces

A dendritic family of isocyanate monomers containing t-butyl esters has been synthesized and characterized. Treatment of free terminal amino groups immobilized on an activated glass with these reagents afforded a surface coating that instilled the tree-like properties of these dendrons to the surface. The amount of branched reagent bound to the silica was determined to be between 0.07 and 0.68 mmol/g.

Streptokinase Triggers Conformational Activation of Plasminogen through Specific Interactions of the Amino-terminal Sequence and Stabilizes the Active Zymogen Conformation

Cleavage of Arg(561)-Val(562) in plasminogen (Pg) generates plasmin (Pm) through a classical activation mechanism triggered by an insertion of the new amino terminus into a binding pocket in the Pg catalytic domain. Streptokinase (SK) circumvents this process and activates Pg through a unique nonproteolytic mechanism postulated to be initiated by the intrusion of Ile(1) of SK in place of Val(562). This hypothesis was evaluated in equilibrium binding and kinetic studies of Pg activation with an SK mutant lacking Ile(1) (SK(2--414)). SK(2--414) retained the affinity of native SK for fluorescein-labeled [Lys]Pg and [Lys]Pm but induced no detectable conformational activation of Pg. The activity of SK(2--414) was partially restored by the peptides SK(1--2), SK(1--5), SK(1--10), and SK(1--15), whereas Pg(562--569) peptides were much less effective. Active site-specific fluorescence labeling demonstrated directly that the active catalytic site was formed on the Pg zymogen by the combination of SK(1--10) and SK(2--414), whereas sequence-scrambled SK(1-10) was inactive. The characterization of SK(1--10) containing single Ala substitutions demonstrated the sequence specificity of the interaction. SK(1--10) did not restore activity to the further truncated mutant SK(55-414), which was correlated with the loss of binding affinity of SK(55--414) for labeled [Lys]Pm but not for [Lys]Pg. The studies support a mechanism for conformational activation in which the insertion of Ile(1) of SK into the Pg amino-terminal binding cleft occurs through sequence-specific interactions of the first 10 SK residues. This event and the preferentially higher affinity of SK(2--414) for the activated proteinase domain of Pm are thought to function cooperatively to trigger the conformational change and stabilize the active zymogen conformation.