Inhibition of beta-amyloid(40) fibrillogenesis and disassembly of beta-amyloid(40) fibrils by short beta-amyloid congeners containing N-methyl amino acids at alternate residues.

A potential goal in the prevention or therapy of Alzheimer's disease is to decrease or eliminate neuritic plaques composed of fibrillar beta-amyloid (Abeta). In this paper we describe N-methyl amino acid containing congeners of the hydrophobic "core domain" of Abeta that inhibit the fibrillogenesis of full-length Abeta. These peptides also disassemble preformed fibrils of full-length Abeta. A key feature of the inhibitor peptides is that they contain N-methyl amino acids in alternating positions of the sequence. The most potent of these inhibitors, termed Abeta16-22m, has the sequence NH(2)-K(Me-L)V(Me-F)F(Me-A)E-CONH(2). In contrast, a peptide, NH(2)-KL(Me-V)(Me-F)(Me-F)(Me-A)-E-CONH(2), with N-methyl amino acids in consecutive order, is not a fibrillogenesis inhibitor. Another peptide containing alternating N-methyl amino acids but based on the sequence of a different fibril-forming protein, the human prion protein, is also not an inhibitor of Abeta40 fibrillogenesis. The nonmethylated version of the inhibitor peptide, NH(2)-KLVFFAE-CONH(2) (Abeta16-22), is a weak fibrillogenesis inhibitor. Perhaps contrary to expectations, the Abeta16-22m peptide is highly soluble in aqueous media, and concentrations in excess of 40 mg/mL can be obtained in buffers of physiological pH and ionic strength, compared to only 2 mg/mL for Abeta16-22. Analytical ultracentrifugation demonstrates that Abeta16-22m is monomeric in buffer solution. Whereas Abeta16-22 is susceptible to cleavage by chymotrypsin, the methylated inhibitor peptide Abeta16-22m is completely resistant to this protease. Circular dichroic spectroscopy of Abeta16-22m indicates that this peptide is a beta-strand, albeit with an unusual minimum at 226 nm. In summary, the inhibitor motif is that of alternating N-methyl and nonmethylated amino acids in a sequence critical for Abeta40 fibrillogenesis. These inhibitors appear to act by binding to growth sites of Abeta nuclei and/or fibrils and preventing the propagation of the network of hydrogen bonds that is essential for the formation of an extended beta-sheet fibril.     

Spatial separation of beta-sheet domains of beta-amyloid: disruption of each beta-sheet by N-methyl amino acids

In a recent model of beta-amyloid (Abeta) fibrils, based mainly on solid-state NMR data, a molecular layer consists of two beta-sheets (residues 12-23 and 31-40 of Abeta1-40), folded onto one another by a connecting "bend" structure (residues 25-29) in the side-chain dimension. In this paper, we use two N-methyl amino acids to disrupt each of the two beta-sheets individually (2NMe(NTerm), residues 17 and 19; and 2NMe(CTerm), residues 37 and 39), or both of them at the same time (4NMe, with the above four N-methylated residues). Our data indicate that incorporation of two N-methyl amino acids into one beta-sheet is sufficient to disrupt that sheet while leaving the other, unmodified beta-sheet intact and able to form fibrils. We show, however, that disruption of each of the two beta-sheets has strikingly different effects on fibrillogenesis kinetics and fibril morphology. Both 2NMe(NTerm) and 2NMe(CTerm) form fibrils at similar rates, but more slowly than that of unmodified Abeta1-40. Electron microscopy shows that 2NMe(NTerm) forms straight fibrils with fuzzy amorphous material coating the edges, while 2NMe(CTerm) forms very regular, highly twisted fibrils-in both cases, distinct from the morphology of Abeta1-40 fibrils. Both 2NMe peptides show a "CMC" approximately four times greater than that of Abeta1-40. CD spectra of these peptides also evolve differently in time: whereas the CD spectra of 2NMe(NTerm) evolve little over 10 days, those of 2NMe(CTerm) show a transition to high beta-sheet content at about day 4-5. We also show that disruption of both beta-sheet domains, as in 4NMe, prevents fibril formation altogether, and renders Abeta1-40 highly water soluble and monomeric, and with solvent-exposed side chains. In summary, our data show (1) that the two beta-sheet domains fold in a semiautonomous manner, since disrupting each one still allows the other to fold; (2) that disruption of the N-terminal beta-sheet has a more profound effect on fibrillogenesis than disruption of the C-terminal beta-sheet, suggesting that the former is the more critical for the overall structure of the fibril; and (3) that disruption of both beta-sheet domains renders the peptide monomeric and unable to form fibrils.     

Studies on C3 convertases. Inhibition of C5 convertase formation by peptides containing aromatic amino acids.

The influence of various peptides containing the aromatic amino acids phenylalanine and tyrosine on the formation of the enzyme EAC1423 of the complement system from component C3 and enzyme EAC142 was investigated. Kinetic analysis of enzyme EAC1423 formation and studies on the binding of the C3b fragment of 125I-labelled component C3 to enzyme EAC142 both showed that binding of the C3b fragment of component C3 was decreased by the peptides. Kinetic studies on component-C3 turnover in the fluid phase of enzyme EAC142 failed to reveal effects of the peptides. However, an initial lag in component-C3 turnover occurred that at constant component-C3 concentration was inversely proportional to enzyme EAC142 concentration. This lag in enzyme EAC142 activity is considered as an indication that the interaction of enzyme EAC142 with component C3 possibly does not follow simple Michaelis-Menten kinetics, as was previously assumed. It is shown that the stages after enzyme EAC1423 formation are not influenced by the peptides, suggesting a high degree of specificity of the peptides for the inhibition of enzyme EAC1423 formation.     

Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids

Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery.     

Aqueous solutions containing amino acids and peptides. Part 20. Volumetric behavior of some terminally substituted amino acids and peptides at 298.15 K

The densities at 298.15 K of aqueous solutions containing some terminally substituted amino acids and peptides containing the glycyl, L -and D -alanyl, L -leucyl, sarcosyl, and L -prolyl residues have been dertermined and standard state partial molar volumes and volumetric pairwise virial coefficients obtained. It is shown that the partial molar volumes can be represented using group volume contributions, but this approach is only approximate, and significant effects of N-terminal substitution and sequence dependence are observed. The volumetric virial coefficients for the amino acid amides have been expressed using a group-additivity approach, and the results obtained indicate that the dominant contributions come from peptide group interactions with other peptide groups and with hydrophobic groups. There is also some evidence of both sequence and chiral effects on the volumetric virial coefficients for proline-containing dipeptides.     

Automatic Edman microsequencing of peptides containing multiple unnatural amino acids

It is now routine using automatic Edman microsequencing to determine the primary structure of peptides or proteins containing natural amino acids; however, a deficiency in the ability to readily sequence peptides containing unnatural amino acids remains. With the advent of synthetic peptide chemistry, combinatorial chemistry, and the large number of commercially available unnatural amino acids, there is a need for efficient and accurate structure determination of short peptides containing many unnatural amino acids. In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer. Using a slightly modified gradient program, 19 of these 35 PTH amino acids can be readily resolved and distinguished from common PTH amino acids at low picomole levels. These unnatural amino acids in conjunction with the 20 natural amino acids can be used as building blocks to construct peptide libraries, and peptide beads isolated from these libraries can be readily microsequenced. To demonstrate this, we synthesized a simple tripeptide "one-bead one-compound" combinatorial library containing 14 unnatural and 19 natural amino acids and screened this library for streptavidin-binding ligands. Microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.     

Identification of metal ion binding peptides containing unnatural amino acids by phage display

The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni²⁺–nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pK_a’s of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni²⁺ with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities.     

Photolysis of N-methyl and N-acetyl substituted amino acids

Summary Experiments on the production of free radicals in aqueous solutions of N-methyl and N-acetyl substituted amino acids by UV light are reported. The ESR spectra observed at 77 K show that the bond of the CH_3–group of these substances is ruptured to a great deal compared with other bond ruptures producing paramagnetic centres. But in N-methyl substituted amino acids the methyl radical is less predominant than in N-acetyl substituted ones. At temperatures higher than 200 K the ESR spectra of all substances studied in our experiments are similar. It is supposed that a radical is formed with an unpaired spin near an oxygen atom.A part of this paper was reported at a Congress in Freiburg i. Br. (Germany) organized by the Deutsche Gesellschaft für Biophysik, October 2–4, 1974.     

Inhibition of chicken pyruvate kinases by amino acids.

Alanine, serine, and phenylalanine behave as inhibitors competitive with phosphoenolpyruvate for the activated forms of the chicken pyruvate kinases. On the other hand, phenylalanine and alanine behave as K-type inhibitors and serine behaves as a heterotropic activator of pyruvate kinase variants which undergo homotropic activation. Tryptophan lowers the Vm and tends to yield complex plots with all variants studied. Kinetic patterns obtained in the presence of phenylalanine also show some characteristics not generally associated with a competitive mechanism. These observations are related to data previously obtained using the rat isozymes and are used to formulate a mechanism which explains the effects of the amino acids. This mechanism hypothesizes that all the effector amino acids bind to the phosphoenolpyruvate site; however, amino acids with nonpolar side chains also interact with a nonpolar region of the T conformer and thereby stabilize it. It is further proposed that there are two such nonpolar regions on the various pyruvate kinases--the one which reacts with the nonbulky side chains, and another which reacts only with relatively bulky side chains. The stabilizing effect of this second nonpolar interaction imparts inhibitory characteristics which are not competitive in nature. Serine and perhaps other polar compounds may also bind at the phosphoenolpyruvate site, but because of their polarity exert a repulsive force at the same nonpolar site with which the nonbulky nonpolar amino acids interact. This repulsion stabilizes the R conformation. Presumably the homotropic activating effects of phosphoenolpyruvate operate via this same mechanism. The data are also used to support a specific sequential-concerted mechanism for the homotropic activating effect of phosphoenolpyruvate. According to this mechanism, phosphoenolpyruvate adds sequentially to the first two subunits. This interaction causes the respective subunits to convert to the R conformation but, once two subunits are in R conformation, the remaining two subunits convert in concert.     

Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

Partial intercalation with DNA of peptides containing two aromatic amino acids

The interactions with DNA of tetrapeptide amides containing lysine at the N-terminal position and aromatic amino acids at the second and fourth positions (Ala at position three), 1-6, have been investigated by nmr, CD, and viscometric methods. Tetrapeptides with N-terminal lysine and a single aromatic amino acid, 7-10, were investigated as controls. Significant decreases in DNA viscosity occurred on addition of 7, with the aromatic group at the second position, but not with any of the other single aromatic amino acid peptides. All of the tetrapeptides with two aromatic groups caused DNA viscosity decreases which were two to three times larger than with 7. Peptides with p-nitrophenylalanine (p-NO2Phe) as the aromatic group were synthesized for nmr studies because of its simpler aromatic nmr spectrum relative to Phe. Large upfield shifts of the aromatic proton signals were obtained when the amino acid in the second position was L-p-NO2Phe, and the fourth position contained either p-NO2Phe or Phe. Such peptides also caused the largest DNA viscosity decreases on complex formation. Smaller upfield shifts of the aromatic signals were obtained when the amino acid in the second position was L-Phe or a D isomer of Phe or p-NO2Phe. With all peptides, larger upfield nmr shifts were obtained with heat-denatured, recooled DNA than with native DNA under the same conditions. As with nmr, CD results are quite different for the peptides with L and D amino acids at the second position. All of the results can be interpreted in terms of a model in which lysine interacts stereospecifically with the backbone in a DNA double helix and the aromatic group at the second position stacks strongly with the base pairs when the amino acid is an L isomer. The aromatic group at the fourth position can also interact with the base pairs, but primarily through a sideways stacking of the aromatic group with base pairs for either L or D isomers. Because of covalent constraints on the separation distance for the two aromatic groups in the tetrapeptides, they must stack on opposite sides of the same base pair in violation of the neighbor exclusion principle observed with classical intercalators. This stacking at the same base pair no doubt accounts for the larger viscosity decreases in DNA with the peptides containing two aromatic groups relative to those with a single aromatic group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human liver alanine aminopeptidase. Inhibition by amino acids.

Human liver alanine aminopeptidase is inhibited by L-amino acids having hydrophobic side chains such as Phe, Tyr, Trp, Met, and Leu. Blocking of the amino group or the carboxyl group greatly reduces the inhibitory capacity of the amino acid. Kinetic studies demonstrate that inhibition of hydrolysis of the substrate L-Ala-beta-naphthylamide is of the noncompetitive type. Inhibition of the substrate L-Leu-L-Leu is of the mixed type. Inhibition of the substrate L-Ala-L-Ala-L-Ala is of the competitive type. These changes in the mechanism of inhibition are thought to be the result of the binding of the amino acid to the third residue binding site on the enzyme. This is the part of the active center to which the third residue from the amino end of a peptide substrate is normally bound. The inhibitor constants of several alanine oligopeptides are shown to decrease with increasing length through L-Ala-L-Ala-L-Ala-L-Ala, demonstrating that alanine aminopeptidase is a multisited enzyme with three and possibly four residue sites per active center. The inhibitor constant for Gly-Gly--Phe suggesting that indeed the third residue site preferentially binds large hydrophobic residues.     

Enzymatic synthesis of acyl peptides containing p-nitroanilides of basic amino acids]

A method is suggested for synthesis of acylpeptides, containing arginine or lysine p-nitroanilides at the C-terminus, via the acyl transfer reaction catalyzed by the Bacillus subtilis serine proteinase. Acyl-di- and acyltripeptide ethers with L- and D-amino acids were used as the carboxyl component taken in a twofold excess. When the concentration of dimethylformamide increases, the hydrolysis of the initial ether and the reaction product diminishes. Because of the enzyme inactivation by dimethylformamide the latter's optimal concentration is 70-80%.     

Solid phase synthesis of peptides containing novel amino acids, substituted 3-benzimidazolealanines

A direct solid-phase synthesis of a series of substituted benzimidazole-containing peptides is described. The method involves on-resin formation of new amino acids containing benzimidazole derivatives in the side chain. The heterocycle conjugates were obtained by reaction between aldehydes and peptides containing β-(3,4-diaminophenyl)alanine residue, immobilized on a polymeric solid support.     

Fermentation of peptides and amino acids by a monensin-sensitive ruminal Peptostreptococcus.

A monensin-sensitive ruminal peptostreptococcus was able to grow rapidly (growth rate of 0.5/h) on an enzymatic hydrolysate of casein, but less than 23% of the amino acid nitrogen was ever utilized. When an acid hydrolysate was substituted for the enzymatic digest, more than 31% of the nitrogen was converted to ammonia and cell protein. Coculture experiments and synergisms with peptide-degrading strains of Bacteroides ruminicola and Streptococcus bovis indicated that the peptostreptococcus was unable to transport certain peptides or hydrolyze them extracellularly. Leucine, serine, phenylalanine, threonine, and glutamine were deaminated at rates of 349, 258, 102, 95, and 91 nmol/mg of protein per min, respectively. Deamination rates for some other amino acids were increased when the amino acids were provided as pairs of oxidized and reduced amino acids (Stickland reactions), but these rates were still less than 80 nmol/mg of protein per min. In continuous culture (dilution rate of 0.1/h), bacterial dry matter and ammonia production decreased dramatically at a pH of less than 6.0. When dilution rates were increased from 0.08 to 0.32/h (pH 7.0), ammonia production increased while production of bacterial dry matter and protein decreased. These rather peculiar kinetics resulted in a slightly negative estimate of maintenance energy and could not be explained by a change in fermentation products. Approximately 80% of the cell dry matter was protein. When corrections were made for cell composition, the yield of ATP was higher than the theoretical maximum value. It is possible that mechanisms other than substrate-level phosphorylation contributed to the energetics of growth.     

Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.     

Structure-function relationships for inhibitors of beta-amyloid toxicity containing the recognition sequence KLVFF.

Beta-amyloid (Abeta), the primary protein component of Alzheimer's plaques, is neurotoxic when aggregated into fibrils. We have devised a modular strategy for generating compounds that inhibit Abeta toxicity. These compounds contain a recognition element, designed to bind to Abeta, linked to a disrupting element, designed to interfere with Abeta aggregation. On the basis of this strategy, a hybrid peptide was synthesized with the sequence KLVFF (residues 16-20 of Abeta) as the recognition element and a lysine hexamer as the disrupting element; this compound protects cells in vitro from Abeta toxicity [Pallitto, M. M., et al. (1999) Biochemistry 38, 3570]. To determine if the length of the disrupting element could be reduced, peptides were synthesized that contained the KLVFF recognition element and a sequence of one to six lysines as disrupting elements. All compounds enhanced the rate of aggregation of Abeta, with the magnitude of the effect increasing as the number of lysines in the disrupting element increased. The greatest level of protection against Abeta toxicity was achieved with compounds containing disrupting elements of three or more lysines in sequence. A peptide with an anionic disrupting element, KLVFFEEEE, had activity similar to that of KLVFFKKKK, in both cellular toxicity and biophysical assays, whereas a peptide with a neutral polar disrupting element, KLVFFSSSS, was ineffective. Protective compounds retained activity even at an inhibitor:Abeta molar ratio of 1:100, making these some of the most effective inhibitors of Abeta toxicity reported to date. These results provide critical insight needed to design more potent inhibitors of Abeta toxicity and to elucidate their mechanism of action.