Dissertations / Theses on the topic 'Peptides – Analysis'

Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.
Committee Chair: Dr. Sheldon W. May; Committee Member: Dr. James C. Powers; Committee Member: Dr. Nicholas Hud; Committee Member: Dr. Niren Murthy; Committee Member: Dr. Stanley H. Pollock. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Gas phase fragmentations of cationic radical peptides provide important fundamental information that forms the basis for peptide sequencing by using mass spectrometry. Presenting results from low-energy collision-induced dissociation (CID) experiments and theoretical density functional theory (DFT) calculations in conjunction with Rice–Ramsperger–Kassel–Marcus modeling, this thesis describes some of the chemical properties, including the locations of the charge and radical sites that determine the gas-phase chemistry of peptide radical cations. The first Section (3.1) documents the dissociations of two isomeric glycylglycylarginine methyl ester radical cations, [G•GR–OMe]+ and [GG•R–OMe]+, with well-defined initial radical sites at the N-terminal and middle α-carbon atoms, respectively. These two isomers undergo similar fragmentations to form the y2+ ion and protonated allylguanidine; their identical CID spectra suggest that isomerization occurs prior to dissociation. DFT calculations at the B3LYP/6-31++G(d,p) level revealed that the proton is sequestered on the guanidine group of the side chain in the presence of a highly basic arginine residue, thereby decreasing the isomerization barriers among the α-carbon–centered radicals to approximately 36 kcal mol–1 (cf. 45 kcal mol–1 for the non-basic [GGG]•+ analogues) and facilitating the radical migration along the peptide backbone and subsequent dissociation reactions. The second section (3.2) describes an investigation into the specific effect of the N-terminal basic residue on selective Cα–C bond cleavage of aromatic-containing radical cationic peptides. Upon replacing the arginine residue of [R(G)n–2X(G)7–n]•+ by a less-basic lysine residue, forming [K(G)n–2X(G)7–n]•+ (X = Phe or Tyr; n = 2–7) analogues, the selective Cα–C peptide bond cleavage no longer occurs. The dissociations of the prototypical radical cationic tripeptides [RFG]•+ and [KFG]•+ at the second Cα–C peptide bonds of α-radical intermediates proceed with comparable barriers (ca. 33 and 35 kcal mol–1, respectively); the generation of the competitive [b2 – H]•+ fragment from [RFG]•+ (ca. 40 kcal mol–1) is much higher in energy than that from [KFG]•+ (ca. 27 kcal mol–1). Thus, the selective Cα–C bond cleavage product from [KFG]•+ can be overridden by the [b2 – H]•+ species in the absence of a basic N-terminal residue. Section (3.3) further examines the mechanistic roles of various α- andβ-carbon–centered radicals prior to Cα–C bond cleavage, leading to the observation of novel x-type radical fragments. DFT calculations and RRKM modeling of a prototypical π-radical cationic system, [AY]•+, suggested that direct Cα–C bond cleavage leading to the formation of the [x1 + H]•+ species is thermodynamically comparable (ca. 16 kcal mol–1) with, but kinetically at least three-fold more favorable than, the well-characterized competitive formation of [c1 + 2H]+ and [z1 – H]•+ species. This finding agrees well with the experimental yield of the [x1 + H]•+ radical cation being higher than that of the minor [c1 + 2H]+ species.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

The thesis discusses protein targeting within cells, and in particular targeting to the mitochondrion. The synthesis and conformational analysis by CD and NMR of a mitochondrial targeting presequence peptide, and a non-functional deletion peptide are described. The intramitochondrial sorting of proteins is also discussed and investigated. Ch. 1 reviews the currently available knowledge of the mechanisms and factors involved in protein targeting. Ch. 2 describes the synthesis and purification of the 25 residue peptide corresponding to the mitochondrial targeting presequence of subunit IV of yeast cytochrome oxidase. Ch. 3 describes the conformational analysis by CD and NMR studies of the synthetic peptide and discusses the structural information that can be drawn from the data. Both sets of data indicate that the peptide appears to adopt a partially helical structure. Ch. 4 describes the synthesis and purification of a 23 residue peptide corresponding to a non-functional deletion mutation of the cytochrome oxidase subunit IV targeting peptide. The CD and NNR studies carried out on this peptide, the preliminary results of which indicate an absence of any regular structural motifs, are discussed. Ch. 5 reviews the current understanding of the mechanisms of intramitochondrial sorting and their evolutionary relationship to protein secretion. E.Coli cells expressing cytochrome b2 with various portions of the presequence removed were subfractioned to determine whether any form of the presequence showed a strong tendency to target the protein to the cell membranes. Conclusive results were not obtained due to the formation of inclusion bodies within the cells.

Understanding the dissociation of odd-electron peptide radical cations is of great importance for the analytical applications of biological mass spectrometry because their diverse array of fragmentation pathways provides structural information to supplement that from even-electron protonated peptides—allowing peptide sequencing and, ultimately, protein identification. Nevertheless, the mechanisms of peptide radical formation and dissociation remain largely unexplored. In the studies reported in this Thesis, peptide radical cations (M?+) were generated through one-electron transfer (ET) in collision-induced dissociation (CID) of [CuII(L)M]?2+ (L = auxiliary ligand; M = peptide) complexes. Competitive dissociative pathways were circumvented experimentally through judicious selection of the macrocyclic auxiliary ligand, allowing the formation of a broad range of M?+ species. Chapter 3.1 examines the competition between proton transfer (PT) and ET within [CuII(L)His]?2+ complexes with L = dien (an open-chain ligand), or L = 9-aneN3 (the macrocyclic analogue of dien). Density functional theory (DFT) calculations revealed that macrocyclic ligand (9-aneN3) facilitates M?+ formation by maintaining similar ET barriers with open-chain ligand (dien), but substantially increasing PT barriers. Studying and understanding the fragmentations of M?+ species is fundamentally important and a formidable challenge—both charge-directed and radical-driven fragmentations play important roles, in a competitive manner, in the dissociations of M?+ species. Chapters 3.2-3.4 were built upon successful gas phase syntheses of a wide variety of M?+ species. Chapter 3.2 reports the novel Cβ–Cγ bond cleavage of tryptophan residues in the dissociations of various tryptophan-containing M?+ species, resulting in a neutral 116-Da loss; this process is an α-radical–induced fragmentation. Substitution of the tryptophan residue by a 1-methyltryptophan residue revealed that the 116-Da neutral species is a radical with an unpaired electron on the indole nitrogen atom. Chapter 3.3 describes a systematic examination of tryptophan-containing model systems, both with and without basic residues, to unveil the mechanisms of Cβ–Cγ bond cleavages. M?+ species containing non-basic residues undergo protonation of the γ-carbon atom of the tryptophan residue, thereby weakening the Cβ–Cγ bond and facilitating its cleavage. The formation of [1H-indole]?+ (m/z 117) or [M – CO2 – 116]+ ions is a competition between two incipient fragments for the proton in a dissociating proton-bound dimer. In basic residue containing M?+ species, the proton is tightly sequestered by the basic side chain, resulting in more accessible radical migration barriers prior to subsequent bond cleavages; DFT calculations supported the notion that the charge-remote radical-driven pathway is more favorable than the proton-driven process by 6.2 kcal/mol. Selective radical-induced fragmentations were then used to investigate the radical propagation processes occurring via hydrogen atom transfers—in particular, for Cα–C bond cleavages leading to the formation of an+ ions. The energetics and kinetics of the dissociations of [RGn–2FG7–n – CO2]?+ (n = 2–6) with well-defined C-terminal α-radicals were determined by RRKM modeling of surface-induced dissociation experiments and DFT calculations, revealing that radical propagations in peptide radical cations are not necessarily stepwise processes.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

A fundamental understanding of the isomerization and fragmentation of peptide ions forms the scientific basis underlying peptide sequencing in the gas phase—an important emerging analytical technique routinely used in proteomics applications. Gas phase dissociation of odd-electron radical peptide cations (M?+) provides an alternative and complementary analytical method for identifying peptide sequences; this fragmentation behavior is distinct from that of even-electron protonated peptides ([M+H]+). Despite recent experimental and theoretical advances in studies of radical cationic peptides, their gas phase chemistry remains poorly understood. The first part of this Thesis documents three mechanistic studies on the formation, isomerization, and dissociation of prototypical tripeptide radical cations in the gas phase using biological mass spectrometry. A combination of low-energy collision-induced dissociation (CID) experiments and density functional theory calculations at the B3LYP 6-31++G(d,p) level of theory was used to investigate the influence of the position of the radical site and the basicity of the amino acid residues in the radical cationic tripeptide analogs on their dissociation pathways. The CID spectra of two isomeric glycylglycyltryptophan radical cations—[GGW]?+ and [G?GW]+, with well-defined initial radical sites at the 3-methylindole ring and the N-terminal α-carbon atom, respectively—are significantly different. The former leads to the formation of [a3 + H]?+, [c2 + 2H]?+, and [z1 – H]?+ product ions through C–Cα and N–Cα peptide bond cleavages, while the latter leads to the predominant fragment ions of y1+, [b2 – H]?+, and [b3 – H]?+ via amide bond cleavages. After substitution of the central glycine residue of GGW with an arginine residue, however, the two isomers [G?RW]+ and [GRW]?+ produced almost identical CID spectra. The calculated energy barriers and microcanonical rate constants for isomerizations and competitive dissociations are in accordance with the perception that isomerizations between the GGW isomers could not compete with their fragmentations. For the radical cationic isomers, the presence of the highly basic arginine residue decreases the isomerization barriers (ca. 7–11 kcal/mol) and mediates facile hydrogen atom transfers—both along the peptide backbone and within the side chain residues—prior to subsequent dissociations. The effect of a basic amino acid residue on the isomerizations and dissociations of α-carbon–centered radical peptides also extends to distinctive Cβ–Cγ bond cleavages of isobaric leucine and isoleucine (Xle) residues. The CID spectra of [G?RXle]+ radical cations lead to the formation of characteristic product ions resulting from losses of ?CH(CH3)2 in [G?RL]+ and ?CH2CH3 in [G?RI]+ through Cβ–Cγ side-chain cleavages of (iso)leucine residues, allowing the two peptides to be distinguished. Finally, the first implementation of laser-induced dissociation (LID) on a hybrid quadrupole linear ion trap mass spectrometer is presented. After laser irradiation of mass-selected and -trapped ions in the quadrupole linear ion trap, LID spectra of [M+H]+ undergo both facile backbone and side-chain cleavages. These products are strikingly different from those formed in the CID spectra of [M+H]+, but are similar to those in the corresponding CID spectra of M?+. This approach provides an alternative means of identifying peptide sequence in shogun proteomic analysis.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

An extraordinary diverse components of animal toxins have emerged along with the long-term evolution of animals, which have unique functions that protect them to survive in the wild. Specifically, the granular glands of amphibians contain multiple chemical compounds, among which, peptides are one of the major types of the constituents. This thesis is divided into six chapters. Chapter 1, as general introduction, describes the detailed background on peptide-based therapeutic agents, the promising therapeutic application of animal toxins, and a brief review on anuran skins and their secretions. Chapter 2 presents the materials and methods employed in this project. In chapter 3, a novel bradykinin-related peptide was isolated and identified from the skin secretion of odorrana livida using shotgun cloning and tandem mass spectrometry fragmentation sequencing approach. This peptide exhibited a dose-dependent contractile property on rat bladder and ileum and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations. It also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the peptide was modified by substituting the penultimate amino acid in the amino terminus from phenylalanine to leucine. Theanalogue completely abolished parental peptide activity, but showed an inhibition effects on bradykinin-induced rat tail artery smooth muscle relaxation. By using specific antagonists for bradykinin B1 and B2 receptors, we found that bradykinin b2receptor is highly likely to be involved in the rat tail artery related effects caused bythis novel bradykinin-related peptide and its analogue. Chapter 4 and 5 are about thediscovery of two pairs of novel antimicrobial peptides belonging to Bombinin and VIBombinin H families, respectively, from the skin secretion of Bombina genus. In chapter 4, the sequence modification was applied on bombinin HL by replacing Lisomer-leucine to D-isomer leucine from the second position of the amino terminus. Both the wild type and modified peptides displayed well-defined α-helical structure in bacterial membrane mimicking environment. BHL-bombinin displayed broad-spectrum bactericidal activity against a wide range of microorganisms, while bombinin H only exhibited a mild bacteriostatic effect on gram positive bacteria. The synergistic antimicrobial effects were observed between BHL-bominin with bombininH and between bombinin H with ampicillin. In addition, haemolytic and cytotoxic examination exhibited a highly synergistic selectivity and low cytotoxicity on mammalian cells of these three peptides. In chapter 5, the sequence modification was employed in the BHK-bombinin by replacing the glutamic acid with lysine at the 23rdposition from amino terminus, which increased the net charge and expanded thenonpolar face of the original peptide. According to the results from in vitro function alanalysis, this modification strategy significantly improved the selectivity index of thepeptide with increased antimicrobial activity and decreased haemolysis activity. The combined antimicrobial evaluation on both natural and modified peptides showed synergistic inhibition activity against both gram positive bacteria and fungi yeast. In summary, this thesis reveals the combined strategy of using a molecular cloning technique and mass spectrometric method for novel host-defensive peptides identification from amphibian skin secretions. In vitro and ex vivo functionalevaluations were subsequently employed which not only bring us a better understanding on the diversity of natural sourced bioactive peptides but also emphasized the research value for characterizing their in depth mechanisms.

Due to the large gaps in the fossil record, the evolutionary history of the mammalian order Cetacea is incomplete and controversial. Increasingly researchers are utilizing molecular and biochemical procedures to supplement cetacean paleontology. One of these methods is the comparison of amino acid sequences of myoglobin among species of this order. since this method is time-consuming and expensive, an alternative procedure is desirable.

2A sequences are short 20 ~30 amino acid peptides initially characterised from foot and mouth disease virus (FMDV), but also encoded in a range of Picornaviruses and other viruses as well as non-LTR retrotransposons in a broad range of organisms. They direct a translational recoding event in which ribosomes that have reached the final codon of the 2A sequence pause and terminate translation in the absence of a stop codon and then restart translation. The effect is to separate a single ORF into 2 polypeptides, between the final 2 amino acids (glycine and proline) of 2A, ‘skipping’ a peptide bond. 2A sequences comprise 2 parts, a conserved GDV/IEXNPGP C-terminal motif, and a highly variable, but necessary, N-terminal portion. In the work described in this thesis, extensive mutagenesis studies were carried out across both the conserved and non-conserved portions of the FMDV 2A peptide, including alanine, glycine and proline scanning mutagenesis, site-directed mutagenesis screens. These data indicate that while the amino acid present at the first 2-3 positions of the peptide is not critical for activity, the residue at most other positions is important, including several positions that vary considerably between different 2A peptides. Intriguingly, results of alanine, glycine and proline scanning mutagenesis were very similar, suggesting that features of side chains, and not just the secondary structure propensity of the peptide, plays a critical role. Despite their great variability, each 2A peptide must provide a set of interactions between side chains within the peptide and with the ribosomal exit tunnel to allow the 2A reaction to proceed. Finally, the work also verified a previous finding that insertion of a stop codon in place of the final proline codon of 2A leads ribosomes to stall, unable to terminate translation. Efforts were made to establish a system for purification of such stalled ribosome-nascent chain complexes towards the eventual aim of structural characterisation. The data strongly support a model in which the interaction of the 2A peptide with the ribosome distorts ribosomal conformation.

Le passage des médicaments a travers la membrane cellulaire représente souvent une limitation majeur dans un grand nombre de thérapies (anti-cancéreuse, anti-virale par exemple). Des peptides vecteurs connus comme les CPPs (cell penetrating peptides) ont été utilises avec succès pour introduire a l’intérieur des cellules diverses molécules (protéines, peptides, siRNA, quantum dots) et présentent un fort potentiel dans l'adressage de médicaments. Parmi les différents CPPs décrits dans la littérature la plupart sont des peptides basiques ou amphiphiles.Nous nous sommes intéressés a l'utilisation d’oligomères non charges construits a partir de motifs contraints mimes de dipeptides comme vecteurs de pénétration cellulaire. L'internalisation cellulaire et leur localisation ont été établies a l'aide de dérivés fluorescents par microscopie confocale. L' étude de pénétration cellulaire par mesure de fluorescence a montre que des oligomères de (3S)-amino-5-carbonylmethyl-2,3-dihydro-1,5-benzothiazepine-4(5H)-one] (DBT) sont aussi puissants que les oligomères d'arginine (oligoArg), vecteurs de référence. Par microscopie confocale nous avons montré que ces composés sont internalisés dans les lysosomes. L’efficacité d'internalisation de nos composés a été confirmé par une méthode de quantification par spectrométrie de masse MALDI-TOF développée dans notre groupe. Cette méthode repose sur l'utilisation conjointe d'un marqueur UV-absorbant dérivé de l'acide alfa-cyano-4-hydoxycinnamique (HCCA) et d'une matrice MALDI adaptée. Un effet important de discrimination spectrale est obtenu, permettant une amplification du signal de la molécule d' intérêt dans un mélange complexe. Ainsi les faibles concentrations internalisées peuvent être détectées. Grâce a cette technique et l'utilisation d'un étalon deutéré, nous avons calculé la concentration intracellulaire de deux CPP de référence l'octa-arginine et la pénétratine. Nous avons aussi étudier l’internalisation de petits oligomères construits a partir d'acide 2-aminomethyl-phenyl-acetique (AMPA). Par microscopie confocal nous avons constaté que ces petits oligomères sont internalisés par voie endo-lysosomale.L’efficacité de la pénétration cellulaire de ces petits oligomères aromatiques (oligoAMPA et oligoDBT) offre une nouvelle classe de vecteurs qui ont la particularité d’être non-cationiques et hydrophobes. De tels composés pourraient être utilisés pour la délivrance de médicaments dans le traitement des maladies comme le cancer, les maladies lysosomales ou la maladie d'Alzheimer. Afin de montrer que cette nouvelle classe de vecteurs est capable d'internaliser des composés biologiquement actifs, nous les avons associés a un inhibiteur puissant de la Cathepsine D (CD) la pepstatine. CD est une endopeptidase lysosomale qui dans des conditions normales est localisée dans les endosomes et les lysosomes. Pour certains cancers, la CD est surexprimée et secrétée a l’extérieur de la cellule. La CD est probablement impliquée dans la prolifération des cellules cancéreuses par l'activation de certains facteurs de croissances dans les endosomes. La pepstatine est une inhibiteur puissant de la CD. Cependant son efficacité thérapeutique potentielle est limitée par une faible capacité de pénétration des membranes cellulaires et une faible solubilité nécessitant de fortes doses pour l'inactivation de la CD in vitro et in vivo. Afin d’améliorer son efficacité et sa biodisponibilité, des conjugues de la pepstatine avec nos vecteurs de pénétration cellulaire, oligo (AMPA)4 et (DBT)4, et une partie solubilisante ont été développés. Certains de ces bioconjugués ont montre une toxicité élevée (IC50 = 2.10-6) in vitro sur différentes lignées cellulaires tumorales. Des tests in vivo sur des souris sont prévus pour le futur
As a part of a program for foldamer design two ¦Â-turn mimetics (3S)-amino-5-(carboxylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one or DBT and 2-aminomethyl-phenyl-acetic acid or AMPA were selected as frameworks from a molecular modeling study for their suitability to adopt helical structure. At first we developed a highly efficient scale up synthesis of the DBT moiety protected by 9-fluorenylmetoxycarbonyl (Fmoc) group. By standard solid phase peptide synthesis (SPPS) we synthesized DBT oligomers of different lenghts and modifications were introduced at their N-terminus. Our first task was to perform structural analysis of the oligomers by NMR and X-Ray. Numerous NOE interactions in the DBT pentamer and hexamer molecules were detected by NMR 2D NOESY experiments. These data strongly suggest the organization of these DBT oligomers. Small crystals were obtained from the same molecules in DMSO but at the time being their size is not importan t enough for X-Ray crystallography studies. In a parallel study we hypothesized that short oligomers constructed by DBT or AMPA frameworks could translocate the cellular membrane and could be used as new cell penetrating non-peptides - CPNP. Even though these compounds are not charged as most cell penetrating peptides (CPP)5 or CPNP, we considered that by virtue of their aromaticity, hydrophobicity and their well-organized structure they could have a non-specific interaction with the lipid bilayer and thus be internalized into the cell. Short oligomers were synthesized on Rink amide (RA) resin following SPPS methodology and labelled at their N-terminus with fluorescein isothiocyanate (FITC). At first the cellular uptake of the (DBT)2-4 oligomers in MDA-MB-231 breast cancer cells was analyzed by fluorescence emission measurement and compared to the potent and well-studied CPP octa-arginine (Arg)8 as a positive control and carboxyfluorescein as a negative control. The highest intracellular fluorescence intensity was found for (DBT)4 with a drastic decrease (>4-times) for (DBT)3 and (DBT)2 oligomers. Thus, the cellular uptake appeared length-dependent with an increase of the internalization with the oligomer size. Moreover, the amount of (DBT)4 that was internalized was more significant than that of (Arg)8 despite the fact that it is uncharged. By confocal microscopy we determined that (DBT)4 is mainly localized in the endosomes after 3 hours of incubation and in the lysosomes after 16 hours of incubation. Altogether, these data indicate the ability of these oligomers to target the endolysosomal pathway. Although most of the initial drug delivery studies aimed to avoid lysosomal addressing to prevent subsequent drug degradation, more recent studies demonstrated the relevant clinical utility to target this compartment for drug delivery in the treatment of lysosomal storage diseases, Alzheimer¡¯s disease, and cancer.While analyzing the internalization efficiency of our CPNP we decided to straightforward evaluate their concentration inside the cells. We studied our compounds internalization by total fluorescence emission measurement and by confocal microscopy but none of these techniques gave us the possibility to determine the exact amount of compound internalized per cell. A study reported by Burlina et al. brought a great improvement in proposing a highly reproducible quantification method based on MALDI-TOF MS to measure the concentration of the internalized peptides. However, after cell lysis, this method requires the capture of the biotin-labelled CPP by streptavidin coated magnetic beads. This step is particularly critical for the accuracy of the quantification. This is the reason why we decided to develop a new general methodology based on MALDI-TOF mass spectrometry (MS) which does not require any purification or separation steps. We studied the internalization of CPP/CPNP compou nds by using an UV light-absorbing tag alpha-cyano-4-hydroxycinnamic acid (HCCA) and preparing the samples in a neutral matrix such as alpha-cyano-4-hydroxycinnamic methyl ester (HCCE). This combination (HCCA tag and HCCE matrix) enabled us to discriminate MS signals induced by peptides of interest that were present in low concentration from those of unlabelled more abundant peptides. By addition of a precise amount of deuterated-HCCA-tagged CPP/CPNP prior the MALDI TOF MS experiment, the internalized CPP/CPNP could be quantified on the basis of the ratio between the [M+H]+ peaks of the deuterated and nondeuterated HCCA-tagged CPP.Another direction for research was to synthesize bioconjugates between our newly discovered CPNP and some biologically active compounds that are unable to cross the cell membrane. We selected pepstatine which is a powerful transition state inhibitor of the Cathepsin D (CD). Pepstatine while a very potent inhibitor of the CD is unable to cross the cellular membrane. Moreover pepstatine activity in vitro or in vivo is hampered by its poor solubility in water. CD is a soluble lysosomal aspartic endopeptidase synthesized in rough endoplasmic reticulum as preprocathepsin D (pCD).12 Upon entering the acidic endosomal and lysosomal compartments proteolytic cleavages of the pCD result in the formation of the active enzymatic form of CD. Under normal physiological conditions pCD is sorted to the lysosomes and found intracellularly but in some pathological and physiological conditions like cancer pCD/CD escape the normal targeting mechanism and is secreted from the cell. Once secreted to the outside, pCD can be endocytosed via M6PR or yet unknown receptor by both cancer cells and fibroblasts. The endocytosed pCD undergoes maturation into the enzymatically active CD. An enzymatic activity of CD outside of the cell or inside the endosomes could be responsible for the activation of several growth factors and growth factor receptors. Several groups have proven that the tumour growth is not inhibited by the powerful CD inhibitor pepstatine. These results exclude the importance of the CD enzymatic activity outside of the cell but as already mentioned pepstatine is unable to penetrate into the cell thus CD activation of growth factors inside the endosomes or the lysosomes is still a possibility. Different CPNP-Pepstatine conjugates were synthesized and tested in vitro for their ability to inhibit MDA-MB-231 breast cancer cells growth. Some of these conjugates showed high cytotoxicity, probably via a Cathepsin D inhibition in the endosomes or the lysosomes. One o f the most potent tested compounds was JMV4463. This compound was obtained by the conjugation of pepstatine with a CPNP as delivery system (AMPA4) and with solubilizing moiety composed of polyethylene glycol and D-Arginine residue. The good in vitro results obtained with the vectorized pepstatine encouraged us to perform in vivo tests. We performed scale up synthesis of JMV4463 in order to obtain enough product for anti-cancer activity on mice in the near future

Since ticks are classified as hematophagous ectoparasites, the primary feeding event involves a bloodmeal on a vertebrate host. Such activities facilitate the ingestion of microorganisms which may be detrimental to the survival of a tick. It is observed, however, that ticks are able to survive such invasion by microorganisms and in several cases, facilitate the transmission of pathogens, while themselves remaining unaffected. This phenomenon is attributed to the innate immune system of ticks. The focus of this project is on stimulus-induced immunoreactive peptides known as antimicrobial peptides. In chapter 2, an attempt was made to identify a homolog of the anti Gram-positive and bacteriostatic peptide microplusin, in the salivary glands of the argasid tick Ornithodoros savignyi. It was reported previously that tissue and life stage specific expression of this transcript occurs in the fat body of adult, fully fed, female Rhipicephalus (Boophilus) microplus ticks. The positive control used for this study was unsuccessful due to the incorrect tissue and life stage of R. (B.) microplus ticks. No significant homolog was identified due to the possible existence of stringent regulation of expression as well as differences in the induction stimuli between argasid and ixodid ticks. Lysozyme catalyzes the cleavage of the β-1,4 glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine of the peptidoglycan layer of bacterial cell walls affording the molecule antibacterial activity. In argasid ticks, lysozyme was observed to be induced by feeding. In chapter 3, an attempt was made to elucidate the O. savignyi homolog of the O. moubata lysozyme molecule. The partial sequence obtained revealed the presence of a lysozyme homolog in O. savignyi. The tissue expression profile revealed constitutive expression in the midgut and ovaries and induction of transcription in the hemolymph upon feeding. In salivary glands, upregulation was observed following ingestion of Gram-positive bacteria. In chapter 4, the tissue expression profile of O. savignyi defensin was investigated. It was found that transcription is induced following the ingestion of Gram-positive bacteria, while in the hemolymph upregulation was observed upon feeding. Furthermore, chapter 4 saw the attempts made at the RNAi mediated silencing of the lysozyme and defensin transcripts. Silencing, analysed by real time PCR, was not efficient as no statistically significant silencing was observed. Observation of the phenotype revealed mortality. However, statistical analysis of silencing revealed that the mortality observed was not due to silencing, but non-specific and possibly the result of injury during injection. Overall, the abovementioned experiments revealed the tissue specificity of expression of ixodid microplusin and that a more strategic approach is required for the elucidation of the argasid homolog. The partial O. savignyi lysozyme sequence was elucidated together with the tissue expression profile of this molecule and O. savignyi defensin. The RNAi experiments require optimization for future studies.
Dissertation (MSc)--University of Pretoria, 2010.
Biochemistry
unrestricted

Several novel polymer monoliths for the analysis of peptides and proteins were synthesized using polyethylene glycol diacrylate (PEGDA) as crosslinker. Photo-initiated copolymerization of polyethylene glycol methyl ether acrylate and PEGDA yielded an inert monolith that could be used for size exclusion liquid chromatography of peptides and proteins. This macroscopically uniform monolith did not shrink or swell in either water or tetrahydrofuran. More importantly, it was found to resist adsorption of both acidic and basic proteins in aqueous buffer without any organic solvent additives. A strong cation-exchange polymer monolith was synthesized by copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and PEGDA. A ternary porogen (water, methanol and ethyl ether) was found suitable to prepare a flow-through monolith with moderate pressure drop in aqueous buffer. The resulting monolith showed excellent ion exchange capillary liquid chromatography of peptides using a simple salt gradient. Extremely narrow peaks were obtained for the analysis of synthetic peptides, natural peptides and a protein digest. A peak capacity of 179 was achieved. Although the poly(AMPS) monolith demonstrated extraordinary performance, one main drawback of this monolith was its relatively strong hydrophobicity. A decrease in hydrophobicity was achieved by using more hydrophilic monomers (e.g., sulfoethyl methacrylate or vinyl sulfonic acid). The most hydrophilic poly(vinyl sulfonic acid) monolith provided high resolution cation-exchange liquid chromatography of protein standards and lipoproteins. Use of the new PEGDA biocompatible crosslinker over the conventional ethylene glycol dimethacrylate crosslinker for the preparation of polymer monoliths was found to be advantageous for the analysis of biological compounds in several chromatography modes.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Electron capture dissociation (ECD) and electron transfer dissociation (ETD) can generate unique fragments and preserve post-translational modifications (PTMs), enabling their detection in biological samples. They have been used to differentiate isomeric aspartic (Asp) and isoaspartic acids (isoAsp) produced upon non-enzymatic deamidation of asparagine (Asn) -- a frequently occurring PTM. IsoAsp formation was detected in amyloid-β (Aβ) peptides in the specimens of Alzheimer's disease (AD) patients, and is a potential biomarker for AD if it can be detected early in biofluids of live individuals. Synthetic isoAsp-containing Aβ fragments were studied using ECD to test the method's applicability. IsoAsp-7 and -23 were detected in top-down analysis of the 4.5kDa Aβ42 protein and in Aβ17-28 peptide. Further, a related method, electron ionization/impact dissociation (EID), was successfully applied to Asp/isoAsp differentiation for the first time. High-performance liquid chromatography (HPLC) is a powerful technique for the separation of complex mixtures. HPLC separation of Asp- and isoAsp-containing peptides revealed inconsistent elution orders, especially when isoAsp was located at the N-terminus, requiring ECD for identification. New diagnostic fragments were proposed for N-terminal isoAsp based on the ECD and ETD results. Challenges in detection of such fragments were improved by supplemental activation and chemical modifications. Furthermore, a model for retention time prediction was applied to isoAsp-containing peptides and suggested for their improved identification in HPLC-MS/MS approach. IsoAsp is a 13-amino acid, which distinctively contains an additional methylene group in the backbone, forming a Cα-Cβ bond. Cleavage of this bond provides diagnostic fragments for isoAsp by ECD. The same was proposed for other β-amino acids. However, the Cα-Cβ bond cleavages were rare due to the instability of the Cβ radical. Alternatively, in-source decay (ISD) fragmentation during matrixassisted laser desorption/ionization (MALDI) process can produce abundant ECD-like fragmentation. It was proposed that use of hydrogen-accepting matrices may lead to Cα-Cβ bond cleavage in β-amino acids, because the resulting radical would be stabilized by the carbonyl group. To test this, β-amino acid-containing peptides were analyzed by MALDI-ISD using 5-nitrosalicylic acid matrix. The Cα-Cβ bond cleavages were observed. Overall, new and improved methods were implemented allowing better characterization and differentiation of β-amino acids.

The intrinsic plasticity of biological systems provides opportunities for rational design of selective and potent ligands. Increasingly, computational methods are being applied to predict biomolecular flexibility. However, the motions involved in these processes can be large and occur on time scales generally difficult to achieve with standard simulation methods. In order to overcome the intrinsic limitations of classical molecular dynamics, this Ph.D. project focuses on the application of advanced sampling computational techniques to capture the plasticity of diverse biological systems. The first of these applications involved the evaluation of the secondary structure of the N-terminal portion of p53 and its inverse, reverse and retro-inverso sequences by using replica exchange molecular dynamics simulations in implicit solvent. In this study, we also evaluated the effects of reversal of sequence and stereochemistry in mimicking an inhibitory pharmacophoric conformation. The results showed how the ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids) and side-chain orientation (for reversed sequences). Moreover, the structural information obtained from simulations showed good agreement with NMR and circular dichroism studies, confirming the validity of the combination of replica exchange molecular dynamics with the ff99SB force field and Generalized Born solvent model for computational modelling of D-peptide conformations.In a second work, we explored conformations of the DFG motif of the p38α mitogen-activated protein (MAP) kinase. To achieve this, we employed an advanced sampling simulation method that has been developed in-house, called swarm-enhanced sampling molecular dynamics (sesMD). In contrast to multiple independent MD simulations, swarm-coupled sesMD trajectories were able to sample a wide range of DFG conformations, some of which map onto existing crystal structures. Simulated structures intermediate between DFG-in and DFG-out conformations were predicted to have druggable pockets of interest for structure-based ligand design. Overall, sesMD shows promise as a useful tool for enhanced sampling of complex conformational landscapes. Finally, we used microsecond MD simulations to evaluate the molecular plasticity of R-spondins, a class of proteins involved in the activation of the Wnt pathway. The unbound R-spondin 1 is characterised by a closed conformation, while, when complexed to proteins LGR and RNF43/ZNRF3, assumes an open and more extended arrangement. This is true also for R-spondin 2, in both its unbound or bound forms. From our simulation, we find that the closed R-spondin 1 conformation is stable, whilst, R-spondin 1 and 2 from their open conformation explore several intermediate structures. In addition, we evaluated the druggability of a potential binding site located at the interface between the second and the third β-hairpin moiety of the first furin domain. The computational screening with small molecular fragments provided interesting insights about the druggability and the pharmacophoric features of the potential binding pockets identified, outlining promising future perspectives of structure-based design of Wnt pathway inhibitors.

In order to identify Leishmania proteins which may be immunologically relevant or may play a role in interactions between Leishmania and its mammalian host, a Leishmania major genomic DNA library was constructed in the vector λgt11 and screened with antibodies raised to Leishmania major promastigote membranes. Two recombinant DNA clones were identified which encoded repetitive sequences (Clone 20 and Clone 39). Clone 20 encoded a repetitive peptide of 14 amino acids and clone 39 encoded an unrelated repetitive peptide of 10 amino acids. Analysis of one of these clones, Clone 20, indicated that there were two RNA transcripts of 9500 and 5200 nucleotides expressed which corresponded to this clone in Leishmania major and Leishmania donovani and this expression was not stage-specific. The results of genomic DNA analysis and isolation of additional clones encoding Clone 20 sequences indicated that there were two genes which corresponded to Clone 20 in both Leishmania major and Leishmania donovani and that these genes differed from one another with respect to the number of repeats which they contained. Antibodies against the fusion protein produced by Clone 20 recognized a series of Leishmania major proteins of apparent mol wt 250,000. Analysis of Clone 39 indicated that there was a single transcript of 7500 nucleotides expressed which corresponded to this clone in both Leishmania major and Leishmania donovani and that there was a single gene (or two identical genes) which encoded this transcript. The genomes of many protozoan parasites exhibit a high degree of plasticity with respect to chromosome size and number. The presence of highly repetitive regions within their DNA may be involved in maintaining this plasticity, allowing the parasite to evolve rapidly under selective pressure. Repetitive regions have been identified within many Plasmodia antigens and have been implicated in the ability of this parasite to evade the host immune system. The presence of Leishmania genes encoding proteins containing tandemly repeating peptides may indicate that these proteins play a similar role in evading the host immune system during the course of Leishmania infections. The possible evolution and functions of repetitive proteins in protozoan parasites is discussed.
Medicine, Faculty of
Medical Genetics, Department of
Graduate

This research describes the synthesis of nucleoside analogues and peptides, and their application to nanopore technology and cell biology. Nanopore technology is a technique in which analytes are detected by disturbances in ionic current caused as they traverse a pore, at a fixed applied potential. Here potential applications of the technology are explored. A nanopore-based method to detect enzymatic activity was investigated. The technique involved the detection of peptide fragments proteolytically released from a solid-support. The released fragments gave rise to distinct electrical signals which facilitated their identification and characterisation. The frequency with which they traversed the pore was indicative of enzyme activity. The technique was successfully applied to the detection of the protease enzyme, renin, in the presence of human serum. An approach to count nucleotide repeat sequences, which are the basis of forensic DNA fingerprinting, was also investigated. It was hypothesised that chemically modified copies of the repeat regions could be generated using primer extension and nucleoside analogues. The copying could be performed such that a single analogue was incorporated per repeat. Ensuing nanopore analysis would then give rise to electrical signals in which the analogues could be identified from unique signals. The number of repeat sequences could simply be determined by counting the number of signals. It was envisaged that this could be achieved using adamantane-modified nucleosides. However, these did not give rise to the expected result. Inspired by the adamantane project, analogues of the drug, azacytidine were synthesised. Azacytidine is used to treat cancers that arise due to epigenetic modifications of DNA. It was hypothesised that its side-effects could be mitigated using photocaged-derivatives of the nucleoside, as these would enable greater temporal and spatial control of drug release. Analogues of azacytidine were successfully synthesised and shown to be uncaged back to the parent nucleoside.

NUTRITIONAL PEPTIDOMICS: DISCOVERY, QUANTIFICATION, AND FUNCTIONAL ANALYSIS OF PLANT PROTEIN DERIVED PEPTIDES Sector CHIM/10 - Food chemistry Introduction and aims of thesis The study of bioactive peptides is a central issue in the development of innovative therapies. The increased attention for fresher and ‘greener’ foods and nutraceuticals possessing health-preventing or health-promoting properties makes bioactive peptides suitable candidates for a new era of pharmaceutical products. Analysing and understanding nature and bioactivity of nutritional peptides, typically delivered from parent food proteins, means comprehending an important level of environmental regulation of the human genome: diet is the environmental factor having the most profound life-long influence on health. Although a remarkable progress has been done in protein analysis, as a consequence of proteomic research, and in small molecule analysis, as a consequence of drug discovery/development initiatives, the field of nutritional peptidomic is still quite unexplored and some drawbacks should be addressed. The pharmacological applications of bioactive peptides depend primarily on their ability to be absorbed in order to exert their bioactivity. In addition, it is very likely that peptide sequences are subjected to structural alterations before performing their final activity in vivo due to different events, such as the attack of gastrointestinal enzymes, brush border peptidases, absorption through the intestinal barrier, and attack of intracellular peptidases in the intracellular absorption. Therefore, all of these different aspects about the bioavailability have attracted a growing interest in the last years. In addition, the possibility of the peptides breakdown during the gastrointestinal digestion is one of the most important factor to be considered when evaluating food-derived peptides for the promotion of human health. Chemical stability is also crucial for proper assay development, since these peptides could lose stability when placed in solution or in biological fluids or even before absorption. Once they are delivered, the biodistribution of bioactive peptides may be sometimes hampered as a result of proteolytic attack, primarily due to the action of brush border peptidases overexpressed at the microvilli surface of intestinal cells. However, before evaluating aspects such as the bioavailability, the optimization of hydrolytic conditions and the chemical identification of protein hydrolysates are other important aspects to be highlighted. The broad variety of physiological activities attributed to protein hydrolysates are determined by the type, number, position, and properties of amino acids present in the sequence of bioactive peptides. The optimization of the hydrolytic conditions for the obtainment of bioactive hydrolysates was also addressed in this work. Since peptides derive from proteins, the integration of peptidomics and proteomics methodologies permitted the enlargement of proteomic databank, which may facilitate the improvement of peptidomics platform libraries. In this context, advanced analytical techniques such as those based on mass spectrometry (MS) have emerged as indispensable and irreplaceable tools in the discovery, identification, quantification and functional analysis of bioactive peptides arising from proteolysis. Among the toolkit of techniques developed to investigate proteins at the proteome-wide scale, MS has gained popularity especially because of its ability to handle the hierarchical complexity associated with the biological systems. In addition, MS-based approaches coupled to cell culturing and bioinformatics tools set a new standard in peptide research. Based on these premises, the aim of my PhD project was to set-up and apply MS strategies in order to evaluate: I. The absorption at intestinal level of peptic and tryptic hydrolysates from lupin protein using an in vitro model based on Caco-2 cells, providing for the first time this kind of data on peptides from this seed. II. The modulation of protein-protein interaction (PPI) of proprotein convertase subtilisin/kexin type 9 (PCSK9) with the low density lipoprotein receptor (LDL-R) in HepG2 cells induced by lupin peptides as well as the quantification by MS of the absorbed peptides that had been predicted to be the best inhibitors of the PCSK9-LDLR PPI by molecular modeling. III. The absorption and metabolism of authentic samples of peptides from soy proteins using Caco-2 cells. IV. The extensive investigation of the hempseed proteome including the identification of minor protein components by CPLL methodology. V. The production of some hydrolysates from hempseed protein endowed of hypocholesterolemic properties using different proteases. VI. The enlargement of apricot seed protein databank through an extensive proteome characterization followed by an in silico driven approach for the prediction of the peptides released by simulated gastrointestinal digestion. General conclusion. The results obtained in this work allowed the enlargement of the bioactive-peptide library platform adding a new dimension to the potential health benefits derived from protein from the seeds of different plants of agronomic importance. In details, a growing body of reports on novel peptide sequences and function-structure relationships contributed to the improvement of plant protein and peptide knowledge. Information obtained from characterizing structural components of plant hydrolysates offers useful technological and functional implications for food ingredient formulation or pharmacological use.

Thesis (M.S.)--Worcester Polytechnic Institute.
Keywords: solution 13C-NMR study; olid phase peptide synthesis; bicyclic peptides; ammonium ionophores; valinomycin; ion selective electrode. Includes bibliographical references (p. 63-65).

Une étude récente sur le transfert peptidique pH dépendant effectuée avec divers ARNt aminoacyles a révélé la dépendance au pH du transfert peptidique. L’instabilité hydrolytique rend impossible l’obtention de la valeur expérimentale du pKa de l’eau donné pour le groupement α-amino des esters 3'-aminoacyladenosine. Comme les analogues de la puromycine sont les analogues les plus proches du 3’-terminal des ARNt aminoacyles et qu’ils contiennent une liaison amide stable en position 3’, il est intéressant de déterminer la valeur du pKa du groupement α-amino de différents analogues de la puromycine mais aussi de corréler ces valeurs de pKa aux valeurs de pKa des groupements ARNt aminoacyles correspondants obtenues par le transfert peptidique pH dépendant. Le premier chapitre de la thèse se concentre sur la synthèse de différents analogues de la puromycine et sur la détermination de leur basicité par une analyse RMN pH dépendante. Ce chapitre discutera aussi la conformation intrinsèque des analogues de la puromycine mesurée par la pH dépendance de leur constante de couplage J1’-2’. Les synthèses d’analogues dinucléotidiques, d’un analogue xylo-puromycine et d’un analogue de désoxyxylopuromycine seront aussi décrites. Les conjugués peptidyl-ARN miment des fragments importants d’intermédiaires de la transduction. Ces analogues peuvent être utilisés comme outils expérimentaux pour comprendre l’évolution de la synthèse codée des peptides. L’innovation dans le concept de ‘négoce moleculaire’ entre les peptides, les oligonucléotides et les bicouches lipidiques, qui pourrait être à la base de l’évolution de la synthèse peptidique contrôlée par l’ARN, nous a poussé à synthétiser des conjugués peptidyl-ARN amphiphiliques et à étudier leurs interactions avec les bicouches lipidiques. Dans le deuxième chapitre les stratégies de synthèse sur support solide utilisant des analogues de puromycine comme élément constitutif seront discutées
A recent pH dependent peptidyl transfer assay in the ribosome with various aminoacyl tRNAs revealed the pH dependence of the peptidyl transfer. Hydrolytic instability makes impossible to obtain the experimental bulk water pKa data for the α-amino groups of 3'-aminoacyladenosine esters. Since puromycin analogues are the most similar analogues of the 3’-end of the aminoacyl tRNAs and they contain a stable amide bond in 3’-position, the determination of the pKa value of the α-amino groups of different puromycin analogues and correlation of these pKa values with those of α-amino groups of the corresponding aminoacyl tRNAs obtained by pH dependent peptidyl transfer deserves attention. Chapter 1 of the thesis focuses on the synthesis of different puromycin analogues and on the determination of their basicities by a pH dependent NMR analysis. This chapter also analyses the intrinsic conformations accessed by the puromycin analogues, as measured by the pH dependence of their J1’-2’ coupling constants. The synthesis of dinucleotide analogues, a xylo-puromycin analogue and a deoxyxylopuromycin analogue will also be described. Peptidyl-RNA conjugates mimic important fragments of natural intermediates of translation. These analogues can be used as an experimental tool to understand the evolution of the coded synthesis of peptides. The novelty in the concept of a ‘molecular deal’ between peptides, oligonucleotides and lipidic bilayers, which may be the basis for the evolution of RNA controlled peptide synthesis, prompted us to synthesize amphiphilic peptidyl-RNA conjugates and to study their interactions with lipidic bilayers. In chapter 2 the solid support synthetic strategies using puromycin analogues as the building blocks will be discussed

Sample preparation as an essential step in mass spectrometry-based analysis, plays a critical role in proteomics studies. Magnetic nanoparticles (MNPs) have been widely used in protein and peptide sample preparation due to their magnetic properties, biocompatibility, easy synthesis and surface functionalization. MNPs loaded with analyte or analyte modification reagent can be easily separated from the reaction medium by an externally applied magnetic field. The small size of MNPs provides high analyte loading and extraction capacity. Additionally, MNP can be decorated with different functional groups to achieve selective modification or extraction of analyte. In this study we have utilized silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) for protein and peptide sample preparation. Fluorescence-based methods were utilized for quantitative and qualitative characterization of N-hydrosucccinimidyl (NHS) ester groups on the surface of Fe3O4@SiO2 MNPs. Fluorophore Dansylcadaverine was conjugated to NHS ester functional groups. Fluorometric measurement of cleaved dansylcadaveine was employed to determine the number of NHS ester groups per MNPs that was found to be 2.6 × 102 and 3.4 × 103for 20 nm and 100 nm Fe3O4@SiO2 MNPrespectively. The efficiency of labeling native bovine serum albumin (BSA) by NHS ester coated Fe3O4@SiO2 MNPs was also explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each MNP. Lysine residues of apolipoprotein B-100 (apoB-100) on the surface of intact human low density lipoprotein (LDL) were labeled by NHS ester modified Fe3O4@SiO2 MNPs in aqueous solvents at room temperature. The MNP labeledapoB-100 was treated by SDS to remove lipids and then digested using trypsin. Tryptic peptides were eluted from MNPs by cleaving disulfide linkage between labeled peptides and MNPs. LC-MS/MS analysis found 28 peptides containing labeled lysine residues. These lysine residues should be on the solvent exposed surface of LDL since the large size of MNPs prevents contact of the labeling reagent to those lysines embedded inside the structure of LDL. TCEP- immobilized Fe3O4@SiO2MNPs were fabricated and utilized for reduction of disulfide bonds in bovine pancreas insulin and two different cyclic peptides. Disulfide bonds were efficiently cleaved at room temperature in both organic and aqueous solvents confirmed by LC-MS/MS analysis of reduced/alkylated protein and peptides. Disulfide reduction and alkylation reactions was performed in one step and the reducing agent was simply separated from peptide and protein solution by magnetic separation.

Mestrado em Biotecnologia Alimentar
O molecular pharming permite a produção de proteinas terapêuticas recombinantes a larga escala, de forma segura e a baixo custo. No presente trabalho, é proposta a produção heteróloga de quatro péptidos inibidores da ACE em dois emergentes sistemas de expressão vegetal, Lactuca sativa (alface) e Medicago truncatula (luz cortada). A utilização da alface, uma planta comestível, pode proporcionar um meio para a administração oral de péptidos anti-hipertensivos, criando um novo alimento funcional. Por outro lado, a utilização de M. truncatula, uma leguminosa modelo, garante não só a facilidade de transformação mas também a extrapolação processual para outras leguminosas. No contexto actual de demanda por terapias alternativas para a hipertensão e de processos mais eficientes de produção de péptidos inibidores da ACE, este trabalho assume particular importância.
Molecular pharming is a cost-effective, scalable and safe system to produce high-quality and biologically active recombinant therapeutic proteins. In the present work the heterologous production of four ACE inhibitory peptides in two emerging plant expression hosts, Lactuca sativa (lettuce) and Medicago truncatula is proposed. The use of lettuce, an edible plant, can provide a means for oral delivery of antihypertensive peptides, thus creating a novel functional food. On another hand, the use of M. truncatula, a model legume, ensures not only the simple transformation process but also the procedural extrapolation to other legume species. In the current scenario of global demand for alternative hypertension therapies and easier ACE inhibitory peptide manufacturing processes, this work assumes particular importance.