Receptor and tissue specificity of the effects of peptides corresponding to intracellular regions of the serpentine type receptors

Proximal regions of the third intracellular loop (ICL-3) are responsible for the interaction with heterotrimeric G proteins in most of the serpentine type receptors. The peptides corresponding to these regions are able to activate G proteins in the absence of hormone and to alter the transduction of hormonal signal via the respective homologous receptor. However, the molecular mechanisms of action of the peptides, their specificity to receptors and target tissues are currently not well understood. The goal of this work was to study the receptor and tissue specificity of peptides-derivatives of C-terminal regions of the ICL-3 of luteinizing hormone receptor (LHR), type 1 relaxin receptor (RXFP1), somatostatin receptors of types 1 and 2 (Som₁R and Som₂R), and 5-hydroxytryptamine receptors of subtype 1B and type 6 (5-HT_1BR and 5-HT₆R) on the functional activity of adenylyl cyclase (AC) and GppNHp-binding of G proteins in the brain, myocardium, and testis of rats. It was shown that the influence of peptides on AC and G proteins is well detected in tissues enriched in homologous receptors. The effects stimulating AC and GppNHp-binding were most pronounced in the testes for LHR peptide, in the brain for peptide 5-HT₆R, and in all of the tested tissues (but mainly in the myocardium) for the RXFP1 peptide. The AC-inhibiting effects of peptides Som₁R, Som₂R and 5-HT_1BR, as well as the stimulation of GppNHp binding induced by these peptides, were most pronounced in the brain. In the presence of the peptides, the AC effects of hormones acting via homologous receptors were significantly attenuated, while the AC effects of other hormones changed insignificantly. The findings suggest that biological activity of the peptides depends on their interaction with complementary regions of homologous receptors, which should be taken into account when developing highly selective regulators of hormonal signaling systems on the basis of these peptides.     

Characterization of the mechanisms involved in the gastric antisecretory effect of TLQP-21, a vgf-derived peptide,in rats

TLQP-21, a vgf-derived peptide modulates gastric emptying and prevents ethanol-induced gastric lesions in rats. However, it remains to be studied whether or not TLQP-21 affects gastric acid secretion. In this study, we evaluated the effects of central (0.8–8 nmol/rat) or peripheral (48–240 nmol/kg, intraperitoneally) TLQP-21 administration on gastric acid secretion in pylorus-ligated rats. The mechanisms involved in such activity were also examined. Central TLQP-21 injection significantly reduced gastric acid volume and dose-dependently inhibited total acid output (ED₅₀ = 2.71 nmol), while peripheral TLQP-21 administration had no effect. The TLQP-21 antisecretory activity was prevented by cysteamine (300 mg/kg, subcutaneously), a depletor of somatostatin, by indomethacin (0.25 mg/rat, intracerebroventricularly), a non-selective cyclooxygenase inhibitor, and by functional ablation of sensory nerves by capsaicin. We conclude that TLQP-21 could be considered a new member of the large group of regulatory peptides affecting gastric acid secretion. The central inhibitory effect of TLQP-21 on gastric acid secretion is mediated by endogenous somatostatin and prostaglandins and requires the integrity of sensory nerve fibres.     

鸭疫里氏杆菌B739＿RS00825基因缺失株抗血红素毒性和氧化应激损伤以及定殖能力分析

【目的】血红素可作为细菌重要的铁离子来源,然而转运过多的血红素也会对细菌造成毒性。细菌通过调节、外排、螯合等多种方式减轻血红素毒性作用。鸭疫里氏杆菌(Riemerella anatipestifer, RA)是一种感染鸭及其他禽类的革兰氏阴性病原菌。前期研究表明,该菌编码血红素转运系统,且能够从血红蛋白获取血红素,然而该菌是否编码血红素解毒蛋白未知。本研究以编码一氧化氮合成酶的基因B739＿RS00825为研究对象,分析其在抗血红素毒性和氧化应激损伤以及定殖能力中的功能。【方法】构建B739＿RS00825缺失株,并通过测定生长曲线、细菌存活率、毒力及定殖等试验方法鉴定其在抗血红素毒性、抗氧化应激损伤、宿主致病中的功能。【结果】与RA CH-1相比,RA CH-1ΔB739＿RS00825在添加过量血红素的培养基中生长不受影响;然而与RACH-1Δfur相比,RACH-1ΔfurΔB739＿RS00825在含血红素培养基中的生长明显受到抑制且对H₂O₂的抵抗力降低;B739＿RS00825基因在氧化应激条件下及fur缺失株中明显上调;与RA ...     

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT_1BR) and 6 (5-HT₆R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT_1BR-peptide ARERKATKTL^307–316K-amide mimicking agonist-activated 5-HT_1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT_1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT_1BR-antagonists. The 5-HT₆R-peptides mimicking 5-HT₆R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT₆R-agonists on AC and G_s proteins, but in the presence of 5-HT₆R-antagonists their action was blocked. Of all the 5-HT₆R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL^258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT₆R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT_1BR and 5-HT₆R selectively activate G_i and G_s proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.     

Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH₂]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the ^99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH₂ with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH₂ in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with the fac-[^99mTc(CO)₃]⁺ moiety. The ^99mTc(CO)₃-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic ^99mTc(CO)₃-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear ^99mTc(CO)₃-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle⁴,dPhe⁷)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively).     

Properties and mechanism of the action of the synthetic peptide octarphin

The peptide TPLVTLFK, whose amino acid sequence corresponds to the 12–19 fragment of β-endorphin (the author’s name for the peptide octarphin), and its analogues (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, and TPLVTLFL) have been synthesized. Tritium-labeled octarphin (specific activity of 28 Ci/mol) has been obtained, and its binding to murine peritoneal macrophages has been studied. It was found that [³H]octarphin binds to macrophages with a high affinity (K _d 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin to macrophages was inhibited by the unlabeled β-endorphin and the selective agonist of the nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met5]enkephalin (K _i > 10 μM). The inhibitory activity of the octarphin analogues was more than 100 times lower than that of octarphin. It was shown that octarphin stimulates the activity of mouse immunocompetent cells in vitro and in vivo; at a concentration of 1–10 nM, it increased the adhesion and spreading of peritoneal macrophages and their ability to digest the bacteria of the Salmonella typhimurium virulent strain 415 in vitro. The intraperitoneal injection of the peptide at a dose of 20 μg/animal on day 7, 3, and 1 prior to the isolation of cells led to an increase in the activity of the peritoneal macrophages and the Tand B lymphocytes of the spleen.     

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH₂] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH₂ analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr⁵ and/or to Ser¹⁰. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe⁴, Thr⁵, Ala⁷ and Arg⁸ are crucial residues for OP₄ receptor activation. Manipulation of Phe¹ yielded peptides endowed with antagonist activity but [Nphe¹] NC(1–13)-NH₂ acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe¹] NC(1–13)-NH₂ sequence, abolished any residual agonist activity and [Nphe¹, βAla⁷] NC(1–13)-NH₂ and [Nphe¹, βAla¹¹] NC(1–13)-NH₂ acted as competitive antagonists only. Modification of both Ala⁷ and Ala¹¹ abolished the antagonist activity of [Nphe¹]NC(1–13)-NH₂ probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)¹⁰] NC(1–13)-NH₂ displayed an activity comparable with that of NC(1–13)-NH₂, [Ser(βGalNAc)¹⁰] NC(1–13)-NH₂ and [βAla¹¹] NC(1–13)-NH₂ were five and 10 times less active, respectively.The α-amino acid residues are of the l-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomeclature (1984), Eur. J. Biochem. 138, 9–37. Abbreviations listed in the guide published in (2003), J. Peptide Sci. 9, 1–8 are used without explanation.     

The synthetic peptide octraphin TPLVTLFK is a selective agonist of nonopioid β-endorphin receptor

The peptide TPLVTLFK (coined by the authors “octarphin”), corresponding to the amino acid sequence of β-endorphin fragment 12–19, and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL) were synthesized. The peptide octarphin was labeled with tritium (specific activity, 28 Ci/mol) and its binding to the rat brain cortex membranes and mouse peritoneal macrophages was studied. [³H]Octarphin was found to bind to brain membranes and macrophages with high affinity (K _d = 2.6 ±0.2 and 2.3 +0.2 nM, respectively) and specificity. The specific binding of [³H]octarphin with rat brain membranes and mouse macrophages was inhibited by unlabeled β-endorphin (K _i = 2.4 +0.2 and 2.7 +0.2 nM, respectively) and selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.9 +0.2 and 2.4 +0.2 nM, respectively) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met⁵]enkephalin (K _i > 10 mM). Inhibiting activity of unlabeled analogs of octarphin was more than 100 times lower than that of the unlabeled octarphin. Octarphin was shown to stimulate activity of mouse immunocompetent cells in vitro: at the concentration of 1 nM it enhanced the capacity of peritoneal macrophages to digest bacteria Salmonella typhimurium virulent strain 415 in vitro. Thus, octarphin is a selective agonist of nonopioid (insensitive to the opioid antagonist naloxone) β-endorphin receptor of rat brain cortex membranes and mouse peritoneal macrophages.     

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-endorphin fragment 12–19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [³H]Octarphin was found to bind to macrophages with high affinity (K _d = 2.3 ± 0.2 nM) and specificity. The specific binding of [³H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K _i = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met⁵]enkephalin (K _i > 10 μM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immuno-competent cells in vitro and in vivo: at concentration of 1–10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 μg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.     

CD8+ T cell recognition of polymorphic wild-type sequence p5365–73 peptides in squamous cell carcinoma of the head and neck

The TP53 tumor suppressor gene contains a well-studied polymorphism that encodes either proline (P) or arginine (R) at codon 72, and over half of the world’s population is homozygous for R at this codon. The wild-type sequence (wt) p53 peptide, p53_65–73, has been identified as a CD8+ T cell-defined tumor antigen for use in broadly applicable cancer vaccines. However, depending on the TP53 codon 72 polymorphism of the recipient, the induced responses to the peptides incorporating R (p53_72R) or P (p53_72P) can be “self” or “non-self.” Thus, we sought to determine which wt p53_65–73 peptide should be used in wt p53-based cancer vaccines. Despite similar predicted HLA-A2-binding affinities, the p53_72P peptide was more efficient than the p53_72R peptide in HLA-A2 stabilization assays. In vitro stimulation (IVS) of CD8+ T cells obtained from healthy HLA-A2⁺ donors with these two peptides led to the generation of CD8+ T cell effectors in one-third of the samples tested, at a frequency similar to the responsiveness to other wt p53 peptides. Interestingly, regardless of their p53 codon 72 genotype, CD8+ T cells stimulated with either p53_72P or p53_72R peptide were cross-reactive against T2 cells pulsed with either peptide, as well as HLA-A2⁺ head and neck cancer (HNC) cell lines presenting p53_72P and/or p53_72R peptides for T cell recognition. Therefore, the cross-reactivity of CD8+ T cells for the polymorphic wt p53_65–73 peptides, irrespective of their p53 codon 72 polymorphism, suggests that employing either peptide in wt p53-based vaccines can result in efficient targeting of this epitope.     

Opioid Peptides: Synthesis and Biological Activity of New Endomorphin Analogues

Summary Syntheses are described of new endomorphin 1 and 2 peptoid–peptide hybrids in which Tyr¹ and either one or both Phe³ and Phe⁴ have been replaced by N-substituted-glycine. The preparation is also described of two glycosylated Hyp²-endomorphin 2 analogues in which either 2,3,4,6-tetra-O-acetyl glucose or glucose are β-O-glycosidically linked to the hydroxyproline residue. The Hyp²-endomorphin sequences have also been elongate by adding a C-terminal β-alanine residue and several linear dimers have been prepared by coupling either the native peptides or the modified analogues. The cyclo endomorphin 2 has also been synthesized. Preliminary pharmacological experiments on isolated organ preparations showed that the agonist activities of both endomorphin 1 and 2 are not significantly affected by the Pro/Hyp substitution. Phe⁴/Nphe substitution in the endomorphin 1 reduced the potency on guinea pig ileum (GPI) by about 100 times and abolished the agonist activity on mouse vas deferens (MVD) preparation. The decrease of the agonist activity induced by modification of one phenylalanine residue only, either Phe³ or Phe⁴, is lower on endomorphin 2. Either modification of both Phe³ and Phe⁴ or glycosylation of the Hyp²-endomorphin 2 cancelled any agonist activity on both preparations. The linear peptide dimers [endomorphin 1]₂, [endomorphin 2]₂, [Hyp²-endomorphin 1]₂, [Hyp²-endomorphin 2]₂, [Hyp²-endomorphin 1-Hyp²-endomorphin 2]₂ or [Hyp²-endomorphin 2-Hyp²-endomorphin 1]₂, are 7–19 times less potent than endomorphin 1 on GPI and significantly less active than endomorphins 1 and 2 on MVD. The other afforded modifications significantly affected or abolished the agonist activity of the resulting endomorphin analogues on both GPI and MVD preparations.The α-amino acid residues are of the L-configuration. Standard abbreviations for amino acid derivatives and peptides are according to the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1984) Eur. J. Biochem., 138, 9–37. Abbreviations listed in the guide published in (2003) J. Peptide Sci., 9, 1–8 are used without explanation.     

Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide

Type II collagen (CII) in joint cartilage is known to be a major auto-antigen in human rheumatoid arthritis. Several animal model- and clinical-studies on tolerance-based immunotherapy for the arthritis have been conducted by administrating synthetic immunodominant peptides through an oral route. In the present study, to produce a tolerogenic peptide with therapeutic potential in transgenic rice plants, a gene construct producing glutelin fusion protein with tandem four repeats of a CII_250–270 peptide (residues 250–270) (GluA-4XCII_250–270) containing a human T-cell epitope was introduced with a selection marker, hygromycin phosphotransferase gene (hygromycin-resistance gene) (hph), by co-transformation. Several transgenic plants with high and stable expression of gluA-4XCII _250–270 , but no hph, were selected based on both DNA and protein analyses. The GluA-4XCII_250–270 fusion proteins were detected as both precursor and processed forms mainly in a glutelin fraction of rice endosperm protein extracts and in protein-body rich fractions prepared by density gradient ultracentrifugation. The amount of accumulated CII_250–270 peptide was immunochemically estimated to be about 1 μg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 μg of the peptide per mouse a day) for 2 weeks showed tendencies lowering and delaying serum specific-IgG2a response against subsequent and repeated intraperitoneal-injection of type II collagen. Taken these together, the CII-immunodominant peptide could effectively be produced and accumulated as a glutelin-fusion protein in the transgenic rice seeds, which might be useful as pharmaceutical materials and functional food for prevention and therapy for anti-CII autoimmune diseases like human rheumatoid arthritis.     

Joint composite-rotation adiabatic-sweep isotope filtration

Joint composite-rotation adiabatic-sweep isotope filters are derived by combining the composite-rotation [Stuart AC et al. (1999) J Am Chem Soc 121: 5346–5347] and adiabatic-sweep [Zwahlen C et al. (1997) J Am Chem Soc 119:6711–6721; Kupče E, Freeman R (1997) J Magn Reson 127:36–48] approaches. The joint isotope filters have improved broadband filtration performance, even for extreme values of the one-bond ¹H–¹³C scalar coupling constants in proteins and RNA molecules. An average Hamiltonian analysis is used to describe evolution of the heteronuclear scalar coupling interaction during the adiabatic sweeps within the isotope filter sequences. The new isotope filter elements permit improved selective detection of NMR resonance signals originating from ¹H spins attached to an unlabeled natural abundance component of a complex in which the other components are labeled with ¹³C and ¹⁵N isotopes.     

Low resolution structure of subunit b (b (22-156)) of Escherichia coli F(1)F(O) ATP synthase in solution and the b-delta assembly

The first low resolution solution structure of the soluble domain of subunit b (b _22–156) of the Escherichia coli F₁F_O ATPsynthase was determined from small-angle X-ray scattering data. The dimeric protein has a boomerang-like shape with a total length of 16.2 ± 0.3 nm. Fluorescence correlation spectroscopy (FCS) shows that the protein binds effectively to the subunit δ, confirming their described neighborhood. Using the recombinant C-terminal domain (δ_91–177) of subunit δ and the C-terminal peptides of subunit b, b _120–140 and b _140–156, FCS titration experiments were performed to assign the segments involved in δ–b assembly. These data identify the very C-terminal tail b _140–156 to interact with δ_91–177. The novel 3D structure of this peptide has been determined by NMR spectroscopy. The molecule adopts a stable helix formation in solution with a flexible tail between amino acid 140 to 145.     

Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Summary Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF) (AT). Model peptides with truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic parameters of enteropeptidase hydrolysis for these substrates were determined.K _m values for all substrates with truncated linker (≈10⁻³ M) are an order of magnitude higher than corresponding values for typical enteropeptidase artificial peptide or fusion protein substrates with full enteropeptidase linker-DDDDK-(K _m ≈10⁻⁴ M).k _cat values for AT, Hb (2–8), WDDRG and WDDKG are ≈30–40 min⁻¹. But one additional amino acid residue at both N-and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency:k _cat value for Hb (1–9) is 1510 min⁻¹. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active peptidesin vitro along with its unique natural substrate trypsinogen was demonstrated.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group

The metalloprotease activity of lethal factor (LF) from Bacillus anthracis (B. anthracis) is a main source of toxicity in the lethality of anthrax infection. Thus, the understanding of the enzymatic activity and inhibition of B. anthracis LF is of scientific and clinical interests. We have designed, synthesized, and studied a peptide inhibitor of LF, R9LF-1, with the structure NH₂–(d-Arg)₉–Val–Leu–Arg–CO–NHOH in which the C-terminal hydroxamic acid is commonly used in the inhibitors of metalloproteases to chelate the active-site zinc. This inhibitor was shown to be very stable in solution and effectively inhibited LF in kinetic assays. However, its protection on murine macrophages against lethal toxin’s lysis activity was relatively weak in longer assays. We further observed that the hydroxamic acid group in R9LF-1 was hydrolyzed by LF, and the hydrolytic product of this inhibitor is considerably weaker in inhibition of potency. To resist this unique hydrolytic activity of LF, we further designed a new inhibitor R9LF-2 which contained the same structure as R9LF-1 except replacing the hydroxamic acid group with N,O-dimethyl hydroxamic acid (DMHA), –N(CH₃)–O–CH₃. R9LF-2 was not hydrolyzed by LF in long-term incubation. It has a high inhibitory potency vs. LF with an inhibition constant of 6.4 nM had a better protection of macrophages against LF toxicity than R9LF-1. These results suggest that in the development of new LF inhibitors, the stability of the chelating group should be carefully examined and that DMHA is a potentially useful moiety to be used in new LF inhibitors.     

Induction of human cytotoxic T lymphocytes that preferentially recognise tumour cells bearing a conformational p53 mutant

 The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212–217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193–237 and peptides 186–194, 187–197, 188–197, 263–272, 264–272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio>1.5) at 26 °C, and five (187–197, 193–200, 217–224, 263–272 and 264–272) also stabilised the complexes at 37 °C. Peptides 188–197, 196–203 and 217–225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217–225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217–225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264–272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217–225; and CTL-264 using peptide 264–272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2⁺ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R → H) or 273 (R → H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy. Received: 23 March 2000 / Accepted: 22 June 2000     

First selective agonist of the neuropeptide Y1‐receptor with reduced size

Selective NPY analogues are potent tools for tumour targeting. Their Y₁‐receptors are significantly over‐expressed in human breast tumours, whereas normal breast tissue only expresses Y₂‐receptors. The endogenous peptide consists of 36 amino acids, whereas smaller peptides are preferred because of better labelling efficiencies. As Y₁‐receptor agonists enhance the tumour to background ratio compared to Y₁‐receptor antagonists, we were interested in the development of Y₁‐receptor selective agonists. We designed 19 peptides containing the C‐terminus of NPY (28–36) with several modifications. By using competition receptor binding affinity assays, we identified three NPY analogues with high Y₁‐receptor affinity and selectivity. Metabolic stability studies in human blood plasma of the N‐terminally 5(6)‐carboxyfluorescein (CF) labelled peptides resulted in half‐lives of several hours. Furthermore, the degradation pattern revealed proteolytic degradation of the peptides by amino peptidases. The most promising peptide was further investigated in receptor activation and internalization studies. Signal transduction assays revealed clear agonistic properties, which could be confirmed by microscopy studies that showed clear Y₁‐receptor internalization. For the first time, here we show the design and characterization of a small Y₁‐receptor selective agonist. This agonist might be a useful novel ligand for NPY‐mediated tumour diagnostics and therapeutics. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Design and bioactivities of melanotropic peptide agonists and antagonists: Design based on a conformationally constrained somatostatin template

Summary α-Melanotropin and ACTH, POMC peptides, initiate biological activity by interaction with the classical pigment cell (α-MSH receptor, MC1R) and adrenal gland (ACTH receptor, MC2R) melanocortin receptors, respectively. The recently discovered MC3R, MC4R and MC5R receptors provide new targets and new biological functions for POMC peptides. We have developed conformationally constrained α-melanotropin peptides that interact with all of these receptors as agonists and antagonists and are examining new approaches to obtain highly selective ligands for each of these melanocortin receptors. Previously, we had converted somatostatin-derived peptides into potent and highly selective analogues that act as antagonists at the μ opioid receptors. Using the reverse turn template that came out of these studies, we have designed, de novo, agonist and antagonist peptide analogues that interact with melanocortin receptors.