Regulatory peptides and control of food intake in non-mammalian vertebrates

The current view of the control of food intake involves a central feeding system in the hypothalamus receiving input from peripheral systems. The presence of food in the gut stimulates the release of several regulatory peptides that control gut motility and secretion. Some of these peptides also act as feedback satiety signals, responsible for termination of a meal. Among the regulatory peptides suggested as peripheral satiety signals are cholecystokinin and gastrin releasing peptide. A more long-term peripheral regulation of food intake has also been postulated and leptin has been suggested as a regulator of food intake. Several regulatory peptides mediate orexigenic or anorexigenic effects in the central feeding system. Neuropeptide Y and galanin both act centrally and stimulate the intake of food, while corticotropin releasing factor reduces food intake. At present, most information about the regulation of food intake is gained from mammalian studies and these findings are used as a base for a discussion on the current knowledge of how regulatory peptides control appetite in non-mammalian vertebrates.     

Biochemical and metabolic mechanisms by which dietary whey protein may combat obesity and Type 2 diabetes

Consumption of milk and dairy products has been associated with reduced risk of metabolic disorders and cardiovascular disease. Milk contains two primary sources of protein, casein (80%) and whey (20%). Recently, the beneficial physiological effects of whey protein on the control of food intake and glucose metabolism have been reported. Studies have shown an insulinotropic and glucose-lowering properties of whey protein in healthy and Type 2 diabetes subjects. Whey protein seems to induce these effects via bioactive peptides and amino acids generated during its gastrointestinal digestion. These amino acids and peptides stimulate the release of several gut hormones, such as cholecystokinin, peptide YY and the incretins gastric inhibitory peptide and glucagon-like peptide 1 that potentiate insulin secretion from β-cells and are associated with regulation of food intake. The bioactive peptides generated from whey protein may also serve as endogenous inhibitors of dipeptidyl peptidase-4 (DPP-4) in the proximal gut, preventing incretin degradation. Indeed, recently, DPP-4 inhibitors were identified in whey protein hydrolysates. This review will focus on the emerging properties of whey protein and its potential clinical application for obesity and Type 2 diabetes.     

Study of peptides inhibiting the angiotensin-converting enzyme of human seminal plasma.

Several peptides were investigated for their inhibitory capacity against dipeptidyl carboxypeptidase (angiotensin-converting enzyme) from human seminal fluid. The strongest inhibitor was the nonapeptide SQ 20881. A marked inhibition was also shown by the compounds Phe-Ala-Pro and Boc-Phe-Ala-Pro, which behaved as competitive inhibitors. Among the peptides related to angiotensin, angiotensin III was the strongest inhibitor, followed by angiotensin II and the C-terminal hexapeptide of angiotensin II. The results indicate the dipeptidyl carboxypeptidase of human semen is very similar to pulmonary dipeptidyl carboxypeptidase in its susceptibility to peptide inhibitors. In view of these and other previously reported similarities, it is possible that both enzymes are identical.     

A cleavable signal peptide is required for the full function of the polytopic inner membrane protein FliP of Escherichia coli

FliP is a rare bacterial polytopic membrane protein synthesized with a cleavable highly hydrophobic signal peptide. It is essential for flagellum assembly and for bacterial motility. In this study, we assessed specificity of signal peptide for the FliP function. Like the wild type FliP, two altered FliPs with more hydrophilic Tat- or Sec-dependent signal peptides were both able to restore the motility of the DeltafliP mutant. Therefore, the Tat- and the Sec-dependent signal peptides seemed to be compatible with the FliP function. Moreover, deletion of the FliP signal peptide or replacing it with the transmembrane segment of MotA severely impaired the FliP function. Together these results showed that a cleavable signal peptide is required for the full function of FliP.     

Potential of Plant’s Dipeptidyl Peptidase I & II Homologs in Generation of ACE Inhibitory Peptides

Synthetic inhibitors of angiotensin converting enzyme (ACE) are commonly used in the treatment of hypertension and other cardiovascular diseases. But their diverse side effects stipulated nontoxic safer and economic inhibitors which can be accomplished by using peptidyl inhibitors from natural sources or functional food ingradients. Dipeptidyl peptidases cleaved dipeptide moieties from amino terminus of oligopepides so, they may be used to generate effective dipeptidyl ACE inhibitors. In present study, role of purified DPP-I and II in generation of ACE inhibitors have been explored. Results showed that collagen alpha-1(III) from chicken showed highest ACE inhibitory potential. Both dipeptidyl peptidases hydrolysed various potent inhibitory dipeptides from their oligopeptide precursors. In addition, sequential digestion of proteins with trypsin, DPP-I and II released approximate 15 % of total inhibitory peptides. Furthermore, inhibitory peptide concentration can be increased up to 30 % or more by using more proteases in presence of DPP-I and II. Results revealed that both DPP-I and II possesses the ability to generate ACE dipeptide inhibitors from oligopeptides. Still various dipeptidyl inhibitory peptides remained in generating oligopeptides, which required study of other endopeptidases with broad specificities.     

Analysis of the heterogeneity of human collagens by two-dimensional polyacrylamide-gel electrophoresis.

The heterogeneity of the CNBr-cleavage peptides of human types I, II, III and V collagens were studied by using two-dimensional electrophoresis combining non-equilibrium pH-gradient-gel electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Specific 'maps' were produced by the peptides obtained from the chains of each type of collagen, and most peptides had at least three charged forms of the same molecular weight. Specific 'maps' were also produced by the peptides of types I, III and V collagens from insoluble dermis and the peptides of types I and V collagens from decalcified bone. The alpha 1(I) CB7 and alpha 1(I) CB8 and the alpha 2 CB4 peptides obtained from the type I collagens of these tissues contained the same number of charged components, but there was a relative increase in the more basic components in bone. Some aspects of the involvement of the alpha 1(I) CB6 and the alpha 1(III) CB9 peptides in cross-linkages were also studied. The recovery of the alpha 1(I) CB6 peptide from bone and dermis was decreased and the alpha 1(III) CB9 peptide was not detected in dermis. Additional peptides, which were probably cross-linked peptides involving the alpha 1(I) CB6 peptide, were also observed.     

Peptides derived from the C2 domain of protein kinase C epsilon (epsilon PKC) modulate epsilon PKC activity and identify potential protein-protein interaction surfaces

Peptides derived from protein kinase C (PKC) modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919-924). We previously demonstrated that the C2 domain of PKC plays a critical role in these interactions. By focusing on epsilonPKC and using a rational approach, we then identified one C2-derived peptide that acts as an isozyme-selective activator and another that acts as a selective inhibitor of epsilonPKC. These peptides were used to identify the role of epsilonPKC in protection from cardiac and brain ischemic damage, in prevention of complications from diabetes, in reducing pain, and in protecting transplanted hearts. The efficacy of these two peptides led us to search for additional C2-derived peptides with PKC-modulating activities. Here we report on the activity of a series of 5-9-residue peptides that are derived from regions that span the length of the C2 domain of epsilonPKC. These peptides were tested for their effect on PKC activity in cells in vivo and in an ex vivo model of acute ischemic heart disease. Most of the peptides acted as activators of PKC, and a few peptides acted as inhibitors. PKC-dependent myristoylated alanine-rich C kinase substrate phosphorylation in epsilonPKC knock-out cells revealed that only a subset of the peptides were selective for epsilonPKC over other PKC isozymes. These epsilonPKC-selective peptides were also protective of the myocardium from ischemic injury, an epsilonPKC-dependent function (Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell. Cardiol. 31, 1937-1948), and caused selective translocation of epsilonPKC over other isozymes when injected systemically into mice. Examination of the structure of the C2 domain from epsilonPKC revealed that peptides with similar activities clustered into discrete regions within the domain. We propose that these regions represent surfaces of protein-protein interactions within epsilonPKC and/or between epsilonPKC and other partner proteins; some of these interactions are unique to epsilonPKC, and others are common to other PKC isozymes.     

Potent and selective inhibition of zinc aminopeptidase A (EC 3.4.11.7, APA) by glutamyl aminophosphinic peptides: importance of glutamyl aminophosphinic residue in the P1 position

Through the development of a new chemical strategy, aminophosphinic peptides containing a pseudoglutamyl residue (Glu Psi(PO2-CH2)Leu-Xaa) in the N-terminal position were synthesized and evaluated as inhibitors of aminopeptidase A (APA). The most potent inhibitor developed in this study, Glu Psi(PO2-CH2)Leu-Ala, displayed a Ki value of 0.8 nM for APA, but was much less effective in blocking aminopeptidase N (APN) (Ki = 31 microM). The critical role of the glutamyl residue in this phosphinic peptide, both in potency and selectivity, is exemplified by the P1 position analogue, Ala Psi(PO2-CH2)Leu-Ala, which exhibited a Ki value of 0.9 microM toward APA but behaved as a rather potent inhibitor of APN (Ki = 25 nM). Glu Psi(PO2-CH2)Leu-Xaa peptides are poor inhibitors of angiotensin converting enzyme (Ki values higher than 1 microM). Depending on the nature of the Xaa residue, the potency of these phosphinic peptides toward neutral endopeptidase 24-11 varied from 50 nM to 3 microM. In view of the in vivo role of APA in the formation of brain angiotensin III, one of the main effector peptides of the renin angiotensin system in the central nervous system, highly potent and selective inhibitors of APA may find important therapeutic applications soon.     

Ceruletide acts in the abdomen, not in the brain, to produce satiety

Ceruletide (caerulein), a decapeptide extracted from the skin of the frog, Hyla caerulea, is very similar in structure to the C-terminal octapeptide of cholecystokinin (CCK-8). Although ceruletide and CCK-8 act through similar or identical receptors to produce the same visceral effects, previous studies in the rat suggested that peripherally administered ceruletide acted directly on the ventromedial hypothalamic (VMH) area to decrease food intake, but peripherally administered CCK-8 acted at a vagally innervated abdominal site to decrease food intake. Since it is unprecedented for these two peptides to produce the same effect by acting at different sites, we investigated the site of action of ceruletide's satiety effect in the rat and compared it to the site of action of CCK-8. The major results were: (1) intraperitoneal administration of ceruletide and CCK-8 inhibited food intake, but intraventricular administration did not; (2) the satiety effect of ceruletide and CCK-8 was not changed by bilateral lesions of the VMH; and (3) the satiety effect of ceruletide and CCK-8 was abolished or markedly reduced by bilateral abdominal vagotomy. We conclude that ceruletide acts at the same vagally innervated abdominal site to produce satiety as CCK-8 does and that neither peptide acts directly on the VMH area.     

Effect of synthetic cationic peptides on activation of the adenylyl cyclase signaling system by biogenic amines in muscle tissue of molluscs and rats

The hormone-sensitive adenylyl cyclase signaling system (ACS), made of serpentine receptor, heterotrimeric G-protein and enzyme adenylyl cyclase (AC), regulates a wide spectrum of growth and metabolic processes in the cell. Molecular mechanisms of functional coupling of ACS components still remain obscure. We examined the influence of synthetic cationic peptides Ac-Ala-His(Ala)2-His-Ala-NH2 (I), Ac-Ala-His-(Ala)3-His-(Ala)2-His-Ala-NH2 (II), and Ac-(Pro)2-His-(Ala)2-His-(Ala)3-His-(Ala)2-His-Ala-NH2 (III) on the basal AC activity and that stimulated by nonhormonal (NaF) and hormonal reagents (serotonin--molluscs, beta-isoproterenol--rats) in smooth muscles of the freshwater bivalve molluscs Anodonta cygnea and in skeletal muscles of rats. Peptides II and III (the latter more effective) were shown to decrease hormone-stimulated AC activity in both tissues, in a dose-dependent manner. Peptide III strongly reduced NaF stimulating effect to AC, which suggests the involvement of this peptide in the functional coupling of both receptors with G-proteins, and of G-proteins with AC. A correlation was found between the efficacy of peptide action on the functional activity of ACS components and peptide length. As shown by IR-spectroscopy, in water all peptides can form helical structures. However, alpha-helicity of peptides I and II was higher than that of peptide III, which does not conform to a power series in efficacy of these peptides. Thus, it is the length of cationic peptides that plays a key role in hormonal regulation of the functional activity of ACS, especially on the step of receptor-G-protein coupling.     

下丘脑弓状核PYY对大鼠摄食、胃运动和能量代谢的影响及机制

目的:探究YY肽(PYY)对雄性Wistar大鼠的摄食、胃运动和能量代谢的影响及潜在机制。方法:采用免疫组织化学实验方法观察大鼠下丘脑弓状核(ARC)中Y2受体的表达;通过ARC微量注射PYY,观察其对下丘脑中编码摄食相关代谢激素的m RNA表达以及ARC中PYY反应性神经元的放电频率、食物摄入量及水摄入量、氧气消耗(VO_2)、CO_2产生(VCO_2)及能量代谢的影响。结果:免疫组化结果显示大鼠ARC内存在Y2受体;大鼠ARC注射PYY能够兴奋PYY反应性神经元,上调可卡因-苯丙胺调节转录肽(CART)及促肾上腺皮质释放激素(CRH)等抑食肽m RNA的表达,下调神经肽Y(NPY)及下丘脑泌素(HCRT)等促食肽m RNA的表达;且抑制大鼠食物摄入量,并参与调控大鼠呼吸、能量代谢及胃运动的改变。结论:ARC微量注射PYY可减少食物摄入并调节全身能量平衡,PYY可能是一种新型代谢肽。     

Pseudosubstrate peptides inhibit Akt and induce cell growth inhibition

We have designed peptide inhibitors that potently inhibit Akt both in vitro and inside cells. These peptide inhibitors are selective for Akt versus other closely related kinases. The peptides inhibit the in vitro phosphorylation of a biotinylated Bad peptide by Akt with potency up to 100 nM. We have shown that the binding between Akt1 and these peptide inhibitors requires MgATP. Mutating the two putative Akt phosphorylation sites to Ala (nonsubstrate) in these peptides increases the inhibitory potency while mutating the sites to aspartic acid (phosphorylation mimetic) reduces the potency. When delivered into cells, these peptide inhibitors can inhibit cellular Akt activity and cell growth. Thus, these Akt-specific peptide inhibitors provide prototypes for peptide mimetic drugs as well as very useful tools to dissect cellular functions of Akt.     

Human and rat dipeptidyl peptidase III: biochemical and mass spectrometric arguments for similarities and differences

Dipeptidyl peptidase (DPP III) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same molecular size and pI for both enzymes. The molecular mass of the human enzyme, measured by matrix-assisted laser desorption/ionization MS, was 82500+/-60 Da. Its tryptic peptide mass profile was determined using the same technique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high similarity between the human erythrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micromolar Co2+, differing in their pH optima and kcat/Km. Zn2+ ions, sulfhydryl reagents, and aminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently. The two enzymes showed the highest affinity for angiotensin III (Ki < 1 microM) and a preference for ahydrophobic residue at the P1' site. However, significant differences in the binding constants for several peptides indicated non-identity in the active site topography of human and rat erythrocyte DPP III.     

Covalent structure of collagen: amino acid sequence of five consecutive CNBr peptides from type III collagen of human liver

Type III collagen was solubilized from human liver by limited pepsin digestion and purified by differential salt precipitation and carboxymethylcellulose chromatography. Digestion with cyanogen bromide yielded the nine distinct peptides previously described and an additional tripeptide not recognized in earlier studies. Five of these peptides, alpha1 (III)-CB1, 2, 4, 8, and 10, were further purified by molecular sieve and/or ion exchange chromatography. They contained 12, 40, 149, 125 and 3 amino acid residues, respectively. The amino acid sequence of these peptides was determined by automated Edman degradation of tryptic (before and after maleylation), chymotryptic, thermolytic or hydroxylamine-derived peptide fragments as well as the intact peptides. The alignment of these five peptides within the collagen chain is deduced to be 1-8-10-2-4 by homology with known alpha1 (I) sequences. The known CNBr peptide alignment of the NH2-terminal portion of type III collagen so far would, therefore, be alpha1 (III)-CB3-7-6-1-8-10-2-4 and correspond to the homologous region of alpha1 (I)-CB0-1-2-4-5-8-3 or residues 11-567 of the alpha1 (III) collagen chain.     

Identification of the first Rho-GEF inhibitor,TRIPalpha, which targets the RhoA-specific GEF domain of Trio

The Rho-guanine nucleotide exchange factors (Rho-GEFs) remodel the actin cytoskeleton via their Rho-GTPase targets and affect numerous physiological processes such as transformation and cell motility. They are therefore attractive targets to design specific inhibitors that may have therapeutic applications. Trio contains two Rho-GEF domains, GEFD1 and GEFD2, which activate the Rac and RhoA pathways, respectively. Here we have used a genetic screen in yeast to select in vivo peptides coupled to thioredoxin, called aptamers, that could inhibit GEFD2 activity. One aptamer, TRIAPalpha (TRio Inhibitory APtamer), specifically blocks GEFD2-exchange activity on RhoA in vitro. The corresponding peptide sequence, TRIPalpha, inhibits TrioGEFD2-mediated activation of RhoA in intact cells and specifically reverts the neurite retraction phenotype induced by TrioGEFD2 in PC12 cells. Thus TRIPalpha is the first Rho-GEF inhibitor isolated so far, and represents an important step in the design of inhibitors for the expanding family of Rho-GEFs.     

High-performance liquid chromatographic separation of renin-angiotensin system peptides and most of their metabolic fragments

We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-L-Ala-L-Ala-L-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients> or =0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin-angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.     

Differential actions of phosphodiesterase inhibitors on secretin- and vasoactive intestinal peptide-induced increases in chief cell cAMP

The actions of three different phosphodiesterase inhibitors, theophylline, 3-isobutyl-1-methylxanthine (IBMX) and Ro 20-1724 (Ro), on cellular cAMP and pepsinogen secretion from dispersed chief cells prepared from guinea pig stomach were examined. The relative order of potency for increasing cAMP and pepsinogen secretion was Ro greater than IBMX greater than theophylline. Ro, the most efficacious agent, caused a 17-fold increase in basal cAMP and a similar augmentation of the increase in cAMP caused by secretin or vasoactive intestinal peptide (VIP). Differential actions of these agents on the dose-response curves for secretin- and VIP-induced increases in cAMP suggest that chief cell receptors for these peptides are coupled to pools of cAMP that are acted upon by heterogeneous phosphodiesterases with varying sensitivities to inhibitors. Moreover, Ro, a selective inhibitor of low Km cAMP-specific phosphodiesterases, is the most potent and efficacious agent tested in this cell system.     

Identification of peptide inhibitors of Pseudomonas aeruginosa exotoxin A function using a yeast two-hybrid approach

The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin. A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A. From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain. The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein. In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity. These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.     

Selection of peptide inhibitors for double-stranded RNA-dependent protein kinase PKR

Protein kinase inhibitors have been developed and applied as antitumor drugs. The majority of these inhibitors are derived from ATP analogs with limited specificity towards the kinase target. Here we present our proof-of-principle study on peptide inhibitors for kinases. Two peptides were selected by phage display against double-stranded RNA-dependent protein kinase (PKR). In vitro assay revealed that these peptides exhibit an inhibitory effect on PKR-catalyzed phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). The peptides also interrupt PKR activity in cells infected by viruses, as PKR activation is one of the hallmarks of host response to viral infection. Kinetic study revealed that one of the peptides, named P1, is a competitive inhibitor for PKR, while the other, named P2, exhibits a more complicated pattern of inhibition on PKR activity. Fragment-based docking of the PKR-peptide complex suggests that P1 occupies the substrate pocket of PKR and thus inhibits the binding between PKR and eIF2α, whereas P2 sits near the substrate pocket. The computational model of PKR-peptide complex agrees with their kinetic behavior. We surmise that peptide inhibitors for kinases have higher specificity than ATP analogs, and that they provide promising leads for the optimization of kinase inhibitors.     

Comparison of the susceptibility of porcine and human dipeptidyl-peptidase IV to inhibition by protein-derived peptides

The enzyme dipeptidyl-peptidase IV (DPP-IV) is recognized to be a promising target for the management of type 2 diabetes. Over the last decade, numerous synthetic molecules and more recently, peptides from dietary proteins, have been reported to be able to inhibit DPP-IV activity. Most studies that have investigated the in vitro effect of these inhibitors have used porcine or human DPP-IV. Although structurally alike, it is unclear whether these two species display similar inhibition patterns. Therefore, the objective of this study was to compare the effects of protein-derived peptides on the activity of porcine and recombinant human DPP-IV. The two species showed different inhibition susceptibility to 43 of the 62 peptide sequences investigated. While 37 protein-derived peptides were more effective at inhibiting the porcine DPP-IV, only six caused a stronger inhibition of the activity of the human enzyme. Although the peptides WR, IPIQY and WCKDDQNPHS were found to be among the most potent inhibitors of both species, the inhibitory effect was greater on the porcine enzyme than on human DPP-IV (αK_i or K_i = 11.5, 13.4, 13.3 μM and 31.4, 28.2, 75.0 μM for porcine and human DPP-IV, respectively). Investigation into the mode of action of the most effective inhibitory peptides revealed that both species were inhibited in a similar manner by short fragments (≤5 amino acid residues), but that some of the longer peptides acted differently on the enzymes. This study shows that porcine DPP-IV is generally inhibited with greater potency by protein-derived peptides than is the human enzyme.