Unmodified Peptides Research Articles

Methods to Enhance the Metabolic Stability of Peptide-Based PET Radiopharmaceuticals.

The high affinity and specificity of peptides towards biological targets, in addition to their favorable pharmacological properties, has encouraged the development of many peptide-based pharmaceuticals, including peptide-based positron emission tomography (PET) radiopharmaceuticals. However, the poor in vivo stability of unmodified peptides against proteolysis is a major challenge that must be overcome, as it can result in an impractically short in vivo biological half-life and a subsequently poor bioavailability when used in imaging and therapeutic applications. Consequently, many biologically and pharmacologically interesting peptide-based drugs may never see application. A potential way to overcome this is using peptide analogues designed to mimic the pharmacophore of a native peptide while also containing unnatural modifications that act to maintain or improve the pharmacological properties. This review explores strategies that have been developed to increase the metabolic stability of peptide-based pharmaceuticals. It includes modifications of the C- and/or N-termini, introduction of d- or other unnatural amino acids, backbone modification, PEGylation and alkyl chain incorporation, cyclization and peptide bond substitution, and where those strategies have been, or could be, applied to PET peptide-based radiopharmaceuticals.

MetaLab 2.0 Enables Accurate Post-Translational Modifications Profiling in Metaproteomics

Studying the structure and function of microbiomes is an emerging research field. Metaproteomic approaches focusing on the characterization of expressed proteins and post-translational modifications (PTMs) provide a deeper understanding of microbial communities. Previous research has highlighted the value of examining microbiome-wide protein expression in studying the roles of the microbiome in human diseases. Nevertheless, the regulation of protein functions in complex microbiomes remains underexplored. This is mainly due to the lack of efficient bioinformatics tools to identify and quantify PTMs in the microbiome. We have developed comprehensive software termed MetaLab for the data analysis of metaproteomic data sets. Here, we build an open search workflow within MetaLab for unbiased identification and quantification of unmodified peptides as well as peptides with various PTMs from microbiome samples. This bioinformatics platform provides information about proteins, PTMs, taxa, functions, and pathways of microbial communities. The performance of the workflow was evaluated using conventional proteomics, metaproteomics from mouse and human gut microbiomes, and modification-specific enriched data sets. Superior accuracy and sensitivity were obtained simultaneously by using our method compared with the traditional closed search strategy.

Abstract A02: Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters

Abstract RAS oncoproteins are frequently mutated in pancreatic, colon and lung cancers and play a causal role in human carcinogenesis. Although it is a potentially good target for anticancer drugs development, it has been challenging to specifically target different forms of RAS using small molecules because of absence of evident target binding sites. Chemical reactivity of protein functional groups depends on the environment of a particular residue and reagent accessibility due to protein structure. We wanted to explore chemical reactivity of KRAS 4b functional groups using chemical reagents to identify novel sites for covalent and noncovalent drug development. NHS esters have been used for years for chemical modification of proteins. They are believed to selectively react with primary amino groups. However, there are reports in the literature that other nucleophilic groups such as sulfhydryl group of Cys can react with N-hydroxysuccinimide esters. It is possible that other functional groups may also react. Sulfo-NHS-acetate, sulfo-NHS-biotin and NHS-biotin were used for modification of K, HRAS proteins in GDP and GppNHp-loaded forms. To follow the extent of protein modification, MALDI-TOF MS was used. Generally, the reaction was performed until almost no unmodified protein was left. After modification the protein was digested by Glu-C and peptides were analyzed by MALDI-TOF MS and ES-MS/MS. A “modification index” (ratio of intensities of modified and unmodified peptide forms in MALDI-TOF MS spectra) was calculated to enable relative quantitation for all modified peptides. The comparison of the modification indexes for peptides derived from NHS-biotin modified KRAS and HRAS in GDP or GppNHp form suggested that GppNHp-loaded protein in general allows higher degree of modification. This suggests that there are inducible and expandable binding sites for small organic molecules that could be exploited for design of covalent and noncovalent drug leads. Acknowledgments: Supported by NCI contract HHSN261200800001E. Citation Format: Oleg Chertov, Xiaoying Ye, Timothy Waybright, Andrew G. Stephen. Probing amino acids residues chemical reactivity of KRAS 4b using N-hydroxysuccinimide esters [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A02.

Understanding the Limit of Open Search in the Identification of Peptides With Post-translational Modifications - A Simulation-Based Study.

Peptide identification from tandem mass spectrometry data is a fundamental task in computational proteomics. Traditional algorithms perform well when facing unmodified peptides. However, when peptides have post-translational modifications (PTMs), these methods cannot provide satisfactory results. Recently, open search methods have been proposed to identify peptides with PTMs. While the performance of these new methods is promising, the identification results vary greatly with respect to the quality of tandem mass spectra and the number of PTMs in peptides. This motivates us to systematically study the relationship between the performance of open search methods and the quality parameters of tandem mass spectrometry data as well as the number of PTMs in peptides. In this paper, we have proposed an analytical model derived from simulated data to describe the relationship between the probability of obtaining correct results and the spectrum quality as well as the number of PTMs. The proposed model is verified using 1,464,146 real experimental spectra. The consistent trend observed in both simulated data and real data reveals the necessary conditions to effectively apply open search methods. Source code of our study is available at http://bioinformatics.ust.hk/PST.html.

Multilevel Characterization of Antibody-Ligand Conjugates by CESI-MS

To demonstrate the capabilities of our new capillary electrophoresis - mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function. To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis. Capillary electrophoresis coupled with electrospray ionization mass spectrometry was used to analyze the antibody-ligand conjugates. We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates. The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is the small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.

A Novel Data Analysis Pipeline for Bottoms‐Up Mass Spectrometry Proteomic Analysis of Primary Tissues

Modern mass spectrometry‐based proteomic studies require reliable, and robust data analysis for a comprehensive and quantitative profile of histone post‐translational modifications (PTM). Studies involving primary tissue samples face additional challenges in comparative sample analysis, due to various protein concentrations, multiple modification sites and modification states. A novel data analysis pipeline was developed in order to streamline the analysis process and normalize samples of varying concentration. This is relevant in the proteomic analysis of addiction, where various modification isoforms can correlate with different addiction phenotypes. A self‐administration model addiction study of methamphetamine (METH) addiction was used to develop this pipeline.Following nuclear protein isolation, proteins underwent tryptic digestion and histone H3 samples were derivatized with propionic anhydride. Identification, validation, and label‐free quantification of histone modifications was performed. Protein loading of different samples was normalized, using high abundance, proteotypic unmodified peptides. To localize changes in specific site and state, peptide isoforms for each site‐ and state‐specific modification were binned. Using this approach, we were able to identify histone modifications that underwent statistically significant changes in METH self‐administrated rat brain samples. In addition, we were able to identify histone modifications that are indicative of METH controllability in tested individual subjects. This data analysis pipeline has led to robust analysis of histone PTMs. Information on changes in site‐ and state‐specific histone modifications can be integrated with RNA‐seq data to elucidate driving chromatin pathways in the formation and maintenance of pathophysiological epigenetic states.Support or Funding InformationThis project was supported in part by the University of New Hampshire Hamel Center for Undergraduate Research.

Evaluation and minimization of nonspecific tryptic cleavages in proteomic sample preparation.

In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less-complex Escherichia coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography/tandem mass spectrometry. Trypsin digestion buffers and digestion times were proved to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate. Because prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2-h protocol was developed with 45.2% less semispecific tryptic peptides but 18.5% more unmodified peptides identified than the commonly used 16-h protocol. The significant decrease in nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. For trypsin digestion, the proposed 2-h protocol can potentially be a replacement for the traditional 16-h protocol.

Glycopeptide variable window SWATH for improved data independent acquisition glycoprotein analysis

N-glycosylation plays an essential role in regulating protein folding and function in eukaryotic cells. Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH) has proven useful as a data independent acquisition (DIA) MS method for analysis of glycoproteins and their glycan modifications. By separating the entire m/z range into consecutive isolation windows, DIA-MS allows comprehensive MS data acquisition and high-sensitivity detection of molecules of interest. Variable width DIA windows allow optimal analyte measurement, as peptide ions are not evenly distributed across the full m/z range. However, the m/z distribution of glycopeptides is different to that of unmodified peptides because of their large glycan structures. Here, we improved the performance of DIA glycoproteomics by using variable width windows optimized for glycopeptides. This method allocates narrow windows at m/z ranges rich in glycopeptides, improving analytical specificity and performance. We show that related glycoforms must fall in separate windows to allow accurate glycopeptide measurement. We demonstrate the utility of the method by comparing the cell wall glycoproteomes of wild-type and N-glycan biosynthesis deficient yeast and showing improved measurement of glycopeptides with different glycan structures. Our results highlight the importance of appropriately optimized DIA methods for measurement of post-translationally modified peptides.

Monitoring the crosstalk between methylation and phosphorylation on histone peptides with host-assisted capillary electrophoresis.

Post-translational modifications (PTMs) greatly increase protein diversity and regulate their functions by changing the structures, properties, and molecular interactions of proteins. In peptide regions with high density of PTMs, PTMs can influence modification on residues in proximity or even at distal positions, adding another layer of regulation. Methods that can monitor the activities of PTM enzymes on peptides carrying multiple modifications are valuable tools for better understanding of PTM crosstalk. Herein, we developed a host-assisted capillary electrophoresis (CE) method to separate histone peptides with methylation and phosphorylation and applied it to monitor the crosstalk between serine phosphorylation and lysine methylation when they were added by Aurora B kinase and G9a lysine methyltransferase, respectively. A synthetic receptor molecule, 4-hexasulfonatocalix[6]arene (CX6), was included in the CE buffer to improve the resolution of the corresponding substrates and products. A linear polyacrylamide-coated capillary was employed to effectively reduce wall adsorption of the cationic histone peptides. The peptide substrates were labeled with fluorescein to enhance their detectability during CE separation. Our method successfully revealed that the activity of G9a methyltransferase was completely inhibited by the adjacent phosphorylation, while 25% reduction in the activity of Aurora B kinase was observed with the presence of dimethylation on the nearby residue. The PTM crosstalk was examined not only using a pure peptide substrate, but also in a competitive reaction environment, in which the modified and unmodified peptides were mixed and the enzyme actions on both peptides were monitored simultaneously. Our work demonstrates that host-assisted CE is an effective method for study of PTM crosstalk, which could offer the advantages of fast separation, high resolution, and low sample consumption. Graphical abstract.

Parathyroid Hormone Derivative with Reduced Osteoclastic Activity Promoted Bone Regeneration via Synergistic Bone Remodeling and Angiogenesis.

Osteogenesis, osteoclastogenesis, and angiogenesis are the most important processes in bone repair. Parathyroid hormone (PTH) has pro-osteogenic, pro-osteoclastogenic, and proangiogenic effects and may be a candidate for use in bone defect repair. However, the local application of PTH to bone defects is counterproductive due to its excessive osteoclastic and bone resorptive effects. In this study, a PTH derivative, PTHrP-2, is developed that can be applied to local bone defects. First, a modified peptide with a calcium-binding repeat glutamine tail undergoes controlled local release from a ceramic material and is shown to be a better fit for the repair process than the unmodified peptide. Second, the modified peptide is shown to have strong pro-osteogenic activity due to mineralization and its facilitation of serine (Ser) phosphorylation. Third, the modified peptide is shown to maintain the pro-osteoclastogenic and proangiogenic properties of the unmodified peptide, but its pro-osteoclastogenic activity is reduced compared to that of the unmodified peptide. The reduced pro-osteoclastogenic and increased pro-osteogenic properties of the modified peptide reverse the imbalance between osteoblasts and osteoclasts with local PTH application and shift bone resorption to bone regeneration.

18F-Trifluoromethanesulfinate Enables Direct C-H 18F-Trifluoromethylation of Native Aromatic Residues in Peptides.

18F labeling strategies for unmodified peptides with [18F]fluoride require 18F-labeled prosthetics for bioconjugation more often with cysteine thiols or lysine amines. Here we explore selective radical chemistry to target aromatic residues applying C–H 18F-trifluoromethylation. We report a one-step route to [18F]CF3SO2NH4 from [18F]fluoride and its application to direct [18F]CF3 incorporation at tryptophan or tyrosine residues using unmodified peptides as complex as recombinant human insulin. The fully automated radiosynthesis of octreotide[Trp(2-CF218F)] enables in vivo positron emission tomography imaging.

PEGylated leuprolide with improved pharmacokinetic properties

Leuprolide, a gonadotropin-releasing hormone (GnRH) agonist widely used in androgen deprivation therapy for the treatment of advanced prostate cancer, suffers from a short circulating half-life like other peptide therapeutics. As an attempt to improve its pharmacokinetic properties, two PEGylated leuprolides with different molecular weight were synthesized utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. The reaction conditions, including reaction temperature, reaction time and feed ratio of the reactants, were optimized to obtain a higher yield. Reverse-phase high performance liquid chromatography (RP-HPLC) characterization indicates a high purity of the resulting conjugates. Matrix-assisted laser desorption mass spectrometry (MALDI-MS) characterization suggests a 1:1 PEGylation. 1H NMR study reveals that the reaction occurs on the imidazolyl group on the histidine residue and the conjugates are stable in pH7.4 aqueous solutions. The in vitro bioactivity of the conjugates was evaluated using both hormone-sensitive and hormone-insensitive cell lines. It was found that the PEGylated peptides can still counteract the stimulatory action of androgens and the mitogenic action of epidermal growth factor on cell proliferation. The in vivo bioactivity of the conjugates was also tested. Like the unmodified peptide, administration of the conjugates to male rats leads to an initial testosterone surge, followed by a suppression of testosterone secretion. Pharmacokinetics of the drugs after i.v. and s.c. administrations were determined. In both cases, a prolonged circulating half-life, an increased AUC, and a decreased Cl_F were observed for the PEGylated drugs.

A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry.

Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.

Structure of spastin bound to a glutamate-rich peptide implies a hand-over-hand mechanism of substrate translocation

Many members of the AAA+ ATPase family function as hexamers that unfold their protein substrates. These AAA unfoldases include spastin, which plays a critical role in the architecture of eukaryotic cells by driving the remodeling and severing of microtubules, which are cytoskeletal polymers of tubulin subunits. Here, we demonstrate that a human spastin binds weakly to unmodified peptides from the C-terminal segment of human tubulin α1A/B. A peptide comprising alternating glutamate and tyrosine residues binds more tightly, which is consistent with the known importance of glutamylation for spastin microtubule severing activity. A cryo-EM structure of the spastin-peptide complex at 4.2 Å resolution revealed an asymmetric hexamer in which five spastin subunits adopt a helical, spiral staircase configuration that binds the peptide within the central pore, whereas the sixth subunit of the hexamer is displaced from the peptide/substrate, as if transitioning from one end of the helix to the other. This configuration differs from a recently published structure of spastin from Drosophila melanogaster, which forms a six-subunit spiral without a transitioning subunit. Our structure resembles other recently reported AAA unfoldases, including the meiotic clade relative Vps4, and supports a model in which spastin utilizes a hand-over-hand mechanism of tubulin translocation and microtubule remodeling.

Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites.

Mass spectrometry (MS)-based identification of ubiquitinated sites requires trypsin digestion prior to MS analysis, and a signature peptide was produced with a diglycine residue attached to the ubiquitinated lysine (K-ε-GG peptide). However, the missed cleavage of modified lysines by trypsin results in modified peptides with increased length and charge, whose detection by MS analysis is suppressed by the vast majority of internally unmodified peptides. LysargiNase, the mirrored trypsin, is reported to cleave before lysine and arginine residues and to be favorable for the identification of methylation and phosphorylation, but its digestive characteristics related to ubiquitination are unclear. Herein, we tested the capacity of the in-house developed acetylated LysargiNase (Ac-LysargiNase) with high activity and stability, for cleaving ubiquitinated sites in both the seven types of ubiquitin chains and their corresponding K-ε-GG peptides. Interestingly, Ac-LysargiNase could efficiently cleave the K63-linked chain but had little effect on the other types of chains. Additionally, Ac-LysargiNase had higher exopeptidase activity than trypsin. Utilizing these features of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase tandem digestion was developed for the identification of ubiquitinated proteins. Through this method, the charge states and ionization capacity of the unmodified peptides were efficiently reduced, and the identification of modified sites was consequently increased by 30% to 50%. Strikingly, approximately 15% of the modified sites were cleaved by Ac-LysargiNase, resulting in shorter K-ε-GG peptides for better identification. The enzyme Ac-LysargiNase is expected to serve as an option for increasing the efficiency of modified site identification in ubiquitome research.

Identification and analysis of characteristic tryptic peptides from porcine gelatin extracted with multi-stage batch processing

The extraction process significantly affects the characteristics and identification of gelatins. Here, three porcine gelatins were extracted using multi-stage batch processing by three extraction stages with temperatures of 50, 70, and 90 °C, respectively. Liquid chromatography-high-resolution mass spectrometry was used to investigate differences in the characteristic tryptic peptides derived from three porcine gelatins. Identification by accurate mass and tandem mass spectrometry yielded 97, 88, and 58 characteristic tryptic peptides from porcine gelatin extracted in the first, second, and third stages, respectively. Among these, 46 characteristic tryptic peptides were detected in all porcine gelatin samples. A comparison was then made of gelatins extracted by one-stage processing and those from multi-stage batch processing. Two methods included different extraction times and condition sets, as well as produced various gelatins. These analyses yielded 35 characteristic tryptic peptides, including 8 unmodified peptides from all porcine gelatins recovered from the two extraction methods. Moreover, 27 characteristic peptides showed various modifications with hydroxylation of proline residues. The resultant marker peptides can be highly useful in the accurate identification of porcine gelatins in industrial settings.

A dataset describing a suite of novel antibody reagents for the RAS signaling network

RAS genes are frequently mutated in cancer and have for decades eluded effective therapeutic attack. The National Cancer Institute’s RAS Initiative has a focus on understanding pathways and discovering therapies for RAS-driven cancers. Part of these efforts is the generation of novel reagents to enable the quantification of RAS network proteins. Here we present a dataset describing the development, validation (following consensus principles developed by the broader research community), and distribution of 104 monoclonal antibodies (mAbs) enabling detection of 27 phosphopeptides and 69 unmodified peptides from 20 proteins in the RAS network. The dataset characterizes the utility of the antibodies in a variety of applications, including Western blotting, immunoprecipitation, protein array, immunohistochemistry, and targeted mass spectrometry. All antibodies and characterization data are publicly available through the CPTAC Antibody Portal, Panorama Public Repository, and/or PRIDE databases. These reagents will aid researchers in discerning pathways and measuring expression changes in the RAS signaling network.

O-GlcNAc Engineering of GPCR Peptide-Agonists Improves Their Stability and in Vivo Activity.

Peptide agonists of GPCRs and other receptors are powerful signaling molecules with high potential as biological tools and therapeutics, but they are typically plagued by instability and short half-lives in vivo. Nature uses protein glycosylation to increase the serum stability of secreted proteins. However, these extracellular modifications are complex and heterogeneous in structure, making them an impractical solution. In contrast, intracellular proteins are subjected to a simple version of glycosylation termed O-GlcNAc modification. In our studies of this modification, we found that O-GlcNAcylation inhibits proteolysis, and strikingly, this stabilization occurs despite large distances in primary sequence (10-15 amino acids) between the O-GlcNAc and the site of cleavage. We therefore hypothesized that this "remote stabilization" concept could be useful to engineer the stability and potentially additional properties of peptide or protein therapeutics. Here, we describe the application of O-GlcNAcylation to two clinically important peptides: glucagon-like peptide-1 (GLP-1) and the parathyroid hormone (PTH), which respectively help control glucose and calcium levels in the blood. For both peptides, we found O-GlcNAcylated analogs that are equipotent to unmodified peptide in cell-based activation assays, while several GLP-1 analogs were biased agonists relative to GLP-1. As we predicted, O-GlcNAcylation can improve the stability of both GLP-1 and PTH in serum despite the fact that the O-GlcNAc can be quite remote from characterized sites of peptide cleavage. The O-GlcNAcylated GLP-1 and PTH also displayed significantly improved in vivo activity. Finally, we employed structure-based molecular modeling and receptor mutagenesis to predict how O-GlcNAcylation can be accommodated by the receptors and the potential interactions that contribute to peptide activity. This approach demonstrates the potential of O-GlcNAcylation for generating analogs of therapeutic peptides with enhanced proteolytic stability.

The case for class II bacteriocins: A biophysical approach using “suicide probes” in receptor-free hosts to study their mechanism of action

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called “suicide probes”. The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.

Pharmacological Characterization and Investigation of N-Terminal Loop Amino Acids of Adrenomedullin 2 That Are Important for Receptor Activation.

Adrenomedullin 2 (AM2) is a peptide hormone with potent effects in the cardiovascular system. The N-terminal disulfide loop of AM2 is thought to be important for interacting with its receptors to initiate a signaling response. However, the relative contribution of each amino acid within this region is currently unknown. Thus, the region was investigated using an alanine scanning approach. Two AM2 peptides (AM2-47 and AM2-40) were directly compared at the CGRP, AM1, and AM2 receptors in transfected Cos7 cells and found to have equivalent activity. Analogues of AM2-40 were then synthesized, substituting each individual amino acid within the disulfide loop with alanine. The ability of these analogues to stimulate a cAMP response was evaluated at the CGRP, AM1, and AM2 receptors. AM2-40 L12A and T14A were less able to elicit cAMP responses through all tested receptors. In contrast, AM2-40 G13A was slightly more potent than the unmodified peptide at all tested receptors. Thus, it appears that residues within the disulfide loop region play differential roles in the ability of AM2 to stimulate cAMP production. The data provide the first structure-function investigation of AM2 agonism.