Chaperone‐like activity of a synthetic peptide toward oxidized γ‐crystallin

αA‐Crystallin can function like a molecular chaperone. We recently reported that the αA‐crystallin sequence, KFVIFLDVKHFSPEDLTVK (peptide‐1, residues 70–88) by itself possesses chaperone‐like (anti‐aggregating) activity during a thermal denaturation assay. Based on the above data we proposed that the peptide‐1 sequence was the functional site in αA‐crystallin. In this study we investigated the specificity of peptide‐1 against γ‐crystallin aggregation in the presence of H₂O₂ and CuSO_4. Peptide‐1 was able to completely protect against the oxidation‐induced aggregation of γ‐crystallin. Removal of N‐terminal Lys or the replacement of Lys with Asp ( D FVIFLDVKHFSPEDLTVK, peptide‐2) did not alter the anti‐aggregation property of peptide‐1. However, deletion of KF residues from the N‐terminus of peptide‐1 resulted in a significant loss of its anti‐aggregation property. Bio‐gel P‐30 size‐exclusion chromatography of γ‐crystallin incubated with peptide‐2 under oxidative conditions revealed that a major portion of the peptide elutes in the void volume region along with γ‐crystallin, suggesting the binding of the peptide to the protein. Peptide‐1 and ‐2 were also able to prevent the UV‐induced aggregation of γ‐crystallin. These data indicate that the same amino acid sequence in αA‐crystallin is likely to be responsible for suppressing the heat‐denatured, oxidatively modified and UV‐induced aggregation of proteins.     

Insights into the determinants of β‐sheet stability: 1H and 13C NMR conformational investigation of three‐stranded antiparallel β‐sheet‐forming peptides

Abstract: In a previous study we designed a 20‐residue peptide able to adopt a significant population of a three‐stranded antiparallel β‐sheet in aqueous solution (de Alba et al. [1999]Protein Sci. 8, 854–865). In order to better understand the factors contributing to β‐sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and ¹³C_α conformational shifts. The populations of the target β‐sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic β‐sheet propensities greatly disfavor β‐sheet folding and that, as already found in other β‐sheet models, specific cross‐strand side chain–side chain interactions contribute to β‐sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross‐strand side chain–side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor β‐sheet‐forming residue, such as Gly.     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Influence of EGCG on α‐synuclein (αS) aggregation and identification of their possible binding mode: A computational study using molecular dynamics simulation

The accumulation of intrinsically disordered α‐synuclein (αS) protein that can form β‐sheet‐rich fibrils is linked to Parkinson's disease. (−)‐Epigallocatechin‐3‐gallate (EGCG) is the most abundant active component in green tea and can inhibit the fibrillation of αS. The elucidation of this molecular mechanism will be helpful to understand the inhibition mechanism of EGCG to the fibrillation of αS and also to find more potential small molecules that can inhibit the aggregation of αS. In this work, to study the influence of EGCG on the structure of β‐sheet‐rich fibrils of αS and identification of their possible binding mode, molecular dynamics simulations of pentamer and decamer aggregates of αS in complex with EGCG were performed. The obtained results indicate that EGCG can remodel the αS fibrils and break the initial ordered pattern by reducing the β‐sheet content. EGCG can also break the Greek conformation of αS by the disappeared H‐bond in the secondary structure of turn. The results from our study can not only reveal the specific interaction between EGCG and β‐sheet‐rich fibrils of αS, but also provide the useful guidance for the discovery of other potential inhibitors.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Effects of single d‐amino acid substitutions on disruption of β‐sheet structure and hydrophobicity in cyclic 14‐residue antimicrobial peptide analogs related to gramicidin S

Abstract: Gramicidin S (GS) is a 10‐residue cyclic β‐sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure–activity studies of de novo‐designed 14‐residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single l ‐amino acid with its corresponding d ‐enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274 , 13181]. The diastereomer with a d ‐Lys substituted at position 4 caused the greatest improvement in specificity vs. other l to d substitutions within the cyclic 14‐residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted β‐sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275 , 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a d ‐ or l ‐amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the d ‐ and l ‐substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic d ‐amino acids d ‐Lys and d ‐Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the l ‐diastereomers, which demonstrated strong hemolysis. All of the l ‐substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the d ‐substituted peptides tended to improve based on the hydrophilicity of the residue. d ‐Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic d ‐amino acid substitutions had superior antimicrobial activity vs. the l ‐enantiomers although substitution of a hydrophobic d ‐amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted β‐sheet structure on antimicrobial activity.     

β‐turn formation by a six‐residue linear peptide in solution

Abstract: A model peptide AAGDYY‐NH₂ (B1), which is found to adopt a β‐turn conformation in the TEM‐1 β‐lactamase inhibitor protein (BLIP) in the TEM‐1/BLIP co‐crystal, was synthesized to elucidate the mechanism of its β‐turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT‐IR and ¹H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT‐IR spectroscopy confirmed the formation of a β‐turn in solution by the model peptide. The dihedral angles [(φ₃, ?₃) (φ₄, ?₄)] of [(?52°, ?32°) (?38°, ?44°)] of Gly‐Asp fragment in the model peptide are consistent with those of a type III β‐turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of β‐turn formation and stability.     

β‐asarone and levodopa co‐administration protects against 6‐hydroxydopamine‐induced damage in parkinsonian rat mesencephalon by regulating autophagy: down‐expression Beclin‐1 and light chain 3B and up‐expression P62

In this study, we investigated Beclin‐1, light chain (LC)3B, and p62 expression in 6‐hydroxydopamine (6‐OHDA)‐induced parkinsonian rats after β‐asarone and levodopa (l ‐dopa) co‐administration. Unilateral 6‐OHDA injection into the medial forebrain bundle was used to create the models, except in sham‐operated rats. Rats were divided into eight groups: sham‐operated group; 6‐OHDA model group; madopar group (75 mg/kg, per os (p.o.)); l ‐dopa group (60 mg/kg, p.o.); β‐asarone group (15 mg/kg, p.o.); β‐asarone + l ‐dopa co‐administered group (15 mg/kg + 60 mg/kg, p.o.); 3‐methyladenine group (500 nmol, intraperitoneal injection); and rapamycin group (1 mg/kg, intraperitoneal injection). Then, Beclin‐1, LC3B, and p62 expression in the mesencephalon were detected. The mesencephalon was also observed by transmission electron microscope. The results showed that Beclin‐1 and LC3B expression decreased and that p62 expression increased significantly in the madopar, l ‐dopa, β‐asarone, and co‐administered groups when compared with the 6‐OHDA model. Beclin‐1 and LC3B expression in the β‐asarone and co‐administered groups were less than in the madopar or l ‐dopa groups, whereas p62 expression in the β‐asarone and co‐administered groups was higher than in the madopar or l ‐dopa groups. In addition, a significant decrease in autophagosome was exhibited in the β‐asarone and co‐administered groups when compared with the 6‐OHDA group. Our findings indicate that Beclin‐1 and LC3B expression decreased, whereas p62 expression increased after co‐administration treatment. In sum, all data suggest that the co‐administration of β‐asarone and l ‐dopa may contribute to the treatment of 6‐OHDA‐induced damage in rats by inhibiting autophagy activity.     

Studies on the conformational properties of CP‐1042−55, the hinge region of CP‐10, using circular dichroism and RP‐HPLC

Abstract: The conformational properties of CP‐10^42?55, a peptide corresponding to the hinge region of CP‐10, were investigated using circular dichroism spectroscopy and reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The circular dichroism studies indicated that CP‐10^42?55 formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mm sodium dodecyl sulfate, which comprised a mixture of α‐helix and β‐sheet. The effect of temperature on the conformation of CP‐10^42?55 was investigated between 5 and 40°C, with very small changes in the spectra being observed.RP‐HPLC was then used to investigate the effect of temperature on the conformation of CP‐10^42?55 in the presence of a hydrophobic surface. Using a C₁₈‐adsorbent, CP‐10^42?55 exhibited a conformational transition at 25°C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near‐planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25°C in theRP‐HPLC system most likely corresponds to the unfolding of an α‐helical and/or β‐sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Improved solid‐phase synthesis of α,α‐dialkylated amino acid‐rich peptides with antimicrobial activity*

Abstract: A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid‐phase synthetic techniques. Each nonapeptide was rich in α,α‐dialkylated amino acids [one 4‐aminopiperidine‐4‐carboxylic acid (Api) and six α‐aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9‐fluorenylmethyloxycarbonyl (Fmoc)‐Aib‐Aib‐OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3₁₀‐helical, amphipathic design of these peptides was born out most prominently in the N‐terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations ≤100 μm ) and the acetylated peptides (concentrations ≤200 μm ) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Synthetic protein design: construction of a four‐stranded β‐sheet structure and evaluation of its integrity in methanol–water systems

Abstract: The characterization of a four‐stranded β‐sheet structure in a designed 26‐residue peptide Beta‐4 is described. The sequence of Beta‐4 (Arg‐Gly‐Thr‐Ile‐Lys‐^Dpro‐Gly‐Ile‐Thr‐Phe‐Ala‐^DPro‐Ala‐Thr‐Val‐Leu‐Phe‐Ala‐Val‐^DPro‐Gly‐Lys‐Thr‐Leu‐Tyr‐Arg) was chosen such that three strategically positioned ^DPro‐Xxx segments nucleate type II′β‐turns, which facilitate hairpin extension. A four‐stranded β‐sheet structure is determined in methanol from 500 MHz ¹H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal J_CαH‐NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four‐stranded sheet and a nonpolar cluster involving Ile⁸, Phe¹⁰, Val¹⁵ and Phe¹⁷. Circular dichroism studies in water–methanol mixtures provide evidence for melting of the β‐sheet structure at high water concentrations. NMR analysis establishes that the four‐stranded sheet in Beta‐4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three β‐turn nuclei appear to be maintained.     

Labedipinedilol‐A: A vanilloid‐based α/β‐adrenoceptor blocker with calcium entry blocking and long‐acting antihypertensive properties

The combination of β‐adrenoceptor blockade and vasodilator action have proved highly useful in antihypertensive therapy. Studies of the mechanisms of action of labedipinedilol‐A that combine these effects within a single molecule are described in this report. Intravenous labedipinedilol‐A (0.1–1.0 mg/kg) produced dose‐dependent hypotensive and bradycardia responses for above 1.0 h, significantly different from nifedipine (0.5 mg/kg, i.v.)‐induced hypotensive and reflex tachycardia activities in pentobarbital‐anesthetized Wistar rats. Pretreatment with labedipinedilol‐A also inhibited phenylephrine (20 μg/kg, i.v.)‐induced hypertensive and (‐)isoprenaline (0.5 μg/kg, i.v.)‐induced tachycardia effects. Oral administration of labedipinedilol‐A (5–50 mg/kg) in spontaneously hypertensive rats (SHR) reduced the blood pressure and heart rate for 24 h but did not increase heart rate. Labedipinedilol‐A (10^–7–10^–5 M) competitively antagonized (‐)isoprenaline (10^–10–10^–4M)‐induced positive chronotropic and inotropic effects of the isolated rat atria and tracheal relaxation responses of the isolated guinea pig tissues. Labedipinedilol‐A also prevented the rate‐increasing effects of increased extracellular Ca²⁺ (3.0–9.0 mM) in a concentration‐dependent manner. In the isolated rat aorta, labedipinedilol‐A competitively antagonized CaCl₂ and norepinephrine‐induced contractions with pKCa^–1 and pA₂ values of 8.46 ± 0.05 and 8.28 ± 0.03 and had a potent effect of inhibiting high K⁺‐induced vasocontraction. Furthermore, labedipinedilol‐A, in an equal antagonist activity, inhibited norepinephrine‐induced phasic and tonic contraction. In the cultured blood vessel smooth muscle cell (A7r5 cell line), KCl, norepinephrine, and Bay K 8644‐induced intracellular calcium changes were decreased after application of labedipinedilol‐A (10^–9–10^–6 M). The binding characteristics of labedipinedilol‐A were evaluated in [³H]CGP‐12177 binding to ventricle and lung and [³H]nitrendipine and [³H]prazosin binding to brain membranes in rats. The ‐logIC₅₀ values of labedipinedilol‐A for β₁‐, β₂‐, and α₁‐adrenoceptor and calcium channel, were 8.17 × 10^–7 M, 8.20 × 10^–7 M, 2.20 × 10^–8 M, and 2.46 × 10^–8 M, respectively. Labedipinedilol‐A‐induced sustained depressor effect was mainly attributed to its calcium entry and α‐adrenoceptor blocking activities in the blood vessel. Sustained bradycardia effect resulted from β‐adrenoceptor and calcium entry blocking, which deleted the sympathetic activation‐associated reflex tachycardia in the heart. Drug Dev. Res. 49:94–108, 2000. © 2000 Wiley‐Liss, Inc.     

Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*

Abstract: During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a β‐Ala insertion mutant of the mother peptide. The source of the β‐Ala was identified as contamination of the Fmoc‐Ala‐OH raw material with Fmoc‐β‐Ala‐Ala‐OH. Further studies also demonstrated the presence of β‐Ala in other Fmoc‐amino acids, particularly in Fmoc‐Arg(Pbf)‐OH. In this case, it was due to the presence of both Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐Arg(Pbf)‐OH. It is concluded that β‐Ala contamination of Fmoc‐amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc‐amino acid derivatives. The β‐Ala is often present as Fmoc‐β‐Ala‐OH and/or as a dipeptide, Fmoc‐β‐Ala‐amino acid‐OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of β‐Ala‐related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated β‐Ala contamination as a problem in the manufacture of the drug substance.     

Reactivity toward deamidation of asparagine residues in β‐turn structures

Mimetics of β‐turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a β‐turn and in a random coil. N‐Acetyl‐Asn‐Gly‐6‐aminocaproic acid, an acyclic analog of a β‐turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N‐acetyl‐Asp‐N‐Gly‐6‐aminocaproic acid (6‐aminocaproic acid, hereafter Aca) and N‐acetyl‐l ‐iso‐aspartyl (isoAsp)‐Gly‐Aca (pH 8.8, 37 °C) ≈ 3‐fold faster than does the cyclic β‐turn mimic cyclo‐[L‐Asn‐Gly‐Aca] with asparagine at position 2 of the β‐turn. The latter compound, in turn, undergoes deamidation ≈ 30‐fold faster than its positional isomer cyclo‐[Gly‐Asn‐Aca] with asparagine at position 3 of the β‐turn. Both cyclic peptides assume predominantly β‐turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open‐chain compound and its isomer N‐acetyl‐Gly‐Asn‐Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2‐fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil ≈ 3× asparagine in position 2 of a β‐turn ≈ 30× asparagine in position 3 of a β‐turn.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

Involvement of NF‐κB in the upregulation of cystathionine‐γ‐lyase,a hydrogen sulfide‐forming enzyme,and bladder pain accompanying cystitis in mice

Hydrogen sulfide (H₂S) is generated from l ‐cysteine by multiple enzymes including cystathionine‐γ‐lyase (CSE), and promotes nociception by targeting multiple molecules such as Ca_v3.2 T‐type Ca²⁺ channels. Bladder pain accompanying cyclophosphamide (CPA)‐induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H₂S/Ca_v3.2 pathway. Therefore, we investigated whether NF‐κB, as an upstream signal of the CSE/H₂S system, contributes to bladder pain in mice with CPA‐induced cystitis. Bladder pain‐like nociceptive behaviour was observed in CPA‐treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β‐Cyano‐l ‐alanine, a reversible selective CSE inhibitor, prevented CPA‐induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF‐κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF‐κB inhibitor. PDTC and curcumin, which inhibits NF‐κB signals, abolished CPA‐induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA‐induced activation of NF‐κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H₂S/Ca_v3.2 signaling. Therefore, NF‐κB‐inhibiting compounds including curcumin may be useful for the treatment of cystitis‐related bladder pain.     

‘100 years of peptide synthesis’: ligation methods for peptide and protein synthesis with applications to β‐peptide assemblies*

Abstract: A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, ‘ Synthesis of Proteins by Chemical Ligation ’ a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo‐prolines, ‘unrestricted’ amino‐acid residues (using the Staudinger reaction), as well as solid‐phase segment coupling by thioligation of unprotected peptides. In another section, ‘ Synthesis of β‐peptides by Thioligation ’, couplings involving β²‐ and β³‐peptides are described (with experimental details).     

Determination of stereochemistry stability coefficients of amino acid side‐chains in an amphipathic α‐helix

Abstract: We describe here a systematic study to determine the effect on secondary structure of d ‐amino acid substitutions in the nonpolar face of an amphipathic α‐helical peptide. The helix‐destabilizing ability of 19 d ‐amino acid residues in an amphipathic α‐helical model peptide was evaluated by reversed‐phase HPLC and CD spectroscopy. l ‐Amino acid and d ‐amino acid residues show a wide range of helix‐destabilizing effects relative to Gly, as evidenced in melting temperatures (ΔT_m) ranging from ?8.5°C to 30.5°C for the l ‐amino acids and ?9.5°C to 9.0°C for the d ‐amino acids. Helix stereochemistry stability coefficients defined as the difference in T_m values for the l ‐ and d ‐amino acid substitutions [(ΔT_m′ = T_mL and T_mD)] ranging from 1°C to 34.5°C. HPLC retention times [Δt_R(X_L?X_D)] also had values ranging from ?0.52 to 7.31 min at pH 7.0. The helix‐destabilizing ability of a specific d ‐amino acid is highly dependent on its side‐chain, with no clear relationship to the helical propensity of its corresponding l ‐enantiomers. In both CD and reversed‐phase HPLC studies, d ‐amino acids with β‐branched side‐chains destabilize α‐helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure‐activity studies and de novo design of novel biologically active peptides.