1.
The Antibiotic Thermorubin Inhibits Protein Synthesis by Binding to Inter-Subunit Bridge B2a of the Ribosome.
Source
Department of Chemistry, Yale University, 225 Prospect Street, PO Box 208107, New Haven, CT 06520-8107, USA.
Abstract
Thermorubin is a small-molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. The structure of the complex between thermorubin and the 70S ribosome from Thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. Though it is structurally similar to tetracycline, it binds to the ribosome at an entirely different location-the interface between the small and large subunits that is formed by inter-subunit bridge B2a. This region of the ribosome is known to play a role in the initiation of translation, and thus, the binding site we observe is consistent with evidence suggesting that thermorubin inhibits the initiation stage of protein synthesis. The binding of thermorubin induces a rearrangement of two bases on helix 69 of the 23S rRNA, and presumably, this rearrangement blocks the binding of an A-site tRNA, thereby inhibiting peptide bond formation. Due in part to its low solubility in aqueous media, thermorubin has not been used clinically, although it is a potent antibacterial agent with low toxicity (Therapeutic Index>200). The interactions between thermorubin and the ribosome, as well as its adjacency to the observed binding sites of three other antibiotic classes, may enable the design of novel derivatives that share thermorubin's mode of action but possess improved pharmacodynamic properties.
Copyright © 2011. Published by Elsevier Ltd.
- PMID:
- 22240456
- [PubMed - as supplied by publisher]
Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-(TDOPA)3-flower-like gold–Fe3O4 optical nanoparticles.
Authors
Source
Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.
2011 Oct 06 [updated 2012 Jan 05].
Excerpt
Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3). Gold has not been used as an X-ray contrast agent in vivo. Gold has a higher atomic number and a higher absorption coefficient than iodine, providing 2.7-fold greater contrast/weight than iodine (4). Furthermore, imaging gold at 80–100 keV reduces interference from bone absorption and provides lower soft tissue absorption, which would reduce radiation to patients. Hainfeld et al. (4) used gold nanoparticles (AuNPs; 1.9 nm in diameter, ~50 kDa) as a computed tomography (CT) contrast agent in mice; these experiments showed enhanced CT contrast of the vasculature, kidneys, and tumors in mice. However, plasma proteins in blood adsorb onto the surface of bare AuNPs, which produces large aggregates (5) that may result in altered pharmacokinetics and biodistribution of AuNPs (6). Polyethylene glycol (PEG) is found to minimize nonspecific adsorption of proteins onto NPs and to reduce their uptake by the liver (6). PEG-AuNPs have been being studied as cancer CT imaging and photothermal agents (7). Several families of MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (8-11). MMP expression is highly regulated in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (12). The peptide Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-NH2 was found to be a MMP substrate and is cleaved between the Leu and Gly residues. Lee et al. (13) used this sequence with a Cy5.5 NIR dye molecule to attach to AuNPs to form fluorescence-quenched nanoparticles (Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-AuNPs (Cy5.5-MMP-AuNPs or GANPs). The Cy5.5 molecules are in close proximity, resulting in fluorescence quenching because of efficient fluorescence resonance energy transfer to Au. NIR fluorescence signal will increase when the Leu-Gly bond is cleaved by MMPs, releasing Cy5.5-containing fragments. Cy5.5 is a NIR fluorescent dye with an absorbance maximum at 675 nm, an emission maximum at 694 nm, and a high extinction coefficient of 250,000 M−1cm−1. Cy5.5-MMP-AuNPs are being developed for NIR fluorescence imaging of MMPs expressed in tumors, atherosclerosis, myocardial infarction, and other diseases. Xie et al. (14) replaced the AuNP with Au-Fe3O4, a composite NP shaped like a flower, to induce a fluorescently quenched state to the overall nanostructure. There were three iron oxide “petals” on each AuNP. The MMP peptide was covalently linked to an anchoring unit, Lys-tridihydrophenylalanine (Lys-TDOPA) on the surface of the iron oxide NP. The gold surface was passivated with a thiolated PEG (SH-PEG5000). Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-(Lys-TDOPA)3-flower-like gold–Fe3O4 optical NPs (FANPs) have been evaluated for imaging protease expression in vivo.
Sections
Investigation of some biologically relevant redox reactions using electrochemical mass spectrometry interfaced by desorption electrospray ionization.
Source
Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Abstract
Recently we have shown that, as a versatile ionization technique, desorption electrospray ionization (DESI) can serve as a useful interface to combine electrochemistry (EC) with mass spectrometry (MS). In this study, the EC/DESI-MS method has been further applied to investigate some aqueous phase redox reactions of biological significance, including the reduction of peptide disulfide bonds and nitroaromatics as well as the oxidation of phenothiazines. It was found that knotted/enclosed disulfide bonds in the peptides apamin and endothelin could be electrochemically cleaved. Subsequent tandem MS analysis of the resulting reduced peptide ions using collision-induced dissociation (CID) and electron-capture dissociation (ECD) gave rise to extensive fragment ions, providing a fast protocol for sequencingpeptides with complicated disulfide bond linkages. Flunitrazepam and clonazepam, a class of nitroaromatic drugs, are known to undergo reduction into amines which was proposed to involve nitroso and N-hydroxyl intermediates. Now in this study, these corresponding intermediate ions were successfully intercepted and their structures were confirmed by CID. This provides mass spectrometric evidence for the mechanism of the nitro to amine conversion process during nitroreduction, an important redox reaction involved in carcinogenesis. In addition, the well-known oxidation reaction of chlorpromazine was also examined. The putative transient one-electron transfer product, the chlorpromazine radical cation (m/z 318), was captured by MS, for the first time, and its structure was also verified by CID. In addition to these observations, some features of the DESI-interfaced electrochemical mass spectrometry were discussed, such as simple instrumentation and the lack of background signal. These results further demonstrate the feasibility of EC/DESI-MS for the study of the biology-relevant redox chemistry and would find applications in proteomics and drug development research.
Structural studies on bovine heart cytochrome c oxidase.
Abstract
Among the X-ray structures of bovine heart cytochrome c oxidase (CcO), reported thus far, the highest resolution is 1.8Å. CcO includes 13 different protein subunits, 7 species of phospholipids, 7 species of triglycerides, 4 redox-active metal sites (Cu(A), heme a (Fe(a)), Cu(B), heme a(3) (Fe(a3))) and 3 redox-inactive metal sites (Mg(2+), Zn(2+) and Na(+)). The effects of various O(2) analogs on the X-ray structure suggest that O(2) molecules are transiently trapped at the Cu(B) site before binding to Fe(a3)(2+) to provide O(2)(-). This provides three possible electron transfer pathways from Cu(B), Fe(a3) and Tyr244 via a water molecule. These pathways facilitate non-sequential 3 electron reduction of the bound O(2)(-) to break the OO bond without releasing active oxygen species. Bovine heart CcO has a proton conducting pathway that includes a hydrogen-bond network and a water-channel which, in tandem, connect the positive side phase with the negative side phase. The hydrogen-bond network forms two additional hydrogen-bonds with the formyl and propionate groups of heme a. Thus, upon oxidation of heme a, the positive charge created on Fe(a) is readily delocalized to the heme peripheral groups to drive proton-transport through the hydrogen-bond network. A peptide bond in the hydrogen-bond network and a redox-coupled conformational change in the water channel are expected to effectively block reverse proton transfer through the H-pathway. These functions of the pathway have been confirmed by site-directed mutagenesis of bovine CcO expressed in HeLa cells. This article is part of a Special Issue entitled: Respiratory Oxidases.
Copyright © 2011. Published by Elsevier B.V.
Constrained α/γ-peptides: a new stable extended structure in solution without any hydrogen bond and characterized by a four-fold symmetry.
Source
Univ Paris-Sud, Laboratoire de Chimie des Procédés et Substances Naturelles, ICMMO, UMR 8182, CNRS, Bât 410, Orsay, F-91405, France. valerie.alezra@u-psud.fr.
Abstract
Small α/γ-peptides alternating α-aminoisobutyric acid and cyclic γ-amino acid residues are described. NMR studies together with restrained simulated annealing revealed that an extended backbone conformation largely dominates in solution for as short as 4-residues long oligomers. This new fold type is devoid of any hydrogen bond and characterized by a four-fold symmetry.
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles.
Source
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University.
Abstract
More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality (1). Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released (2-5). Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect (6). Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells (7). In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.(8) Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells (9). The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes (10). In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells (11). Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.
Cis-trans peptide variations in structurally similar proteins.
Source
INSERM UMR-S 665, Dynamique des Structures et Interactions des Macromolécules Biologiques (DSIMB), Université Denis Diderot-Paris 7, INTS, 6, rue Alexandre Cabanel, 75739, Paris Cedex 15, France.
Abstract
The presence of energetically less favourable cis peptides in protein structures has been observed to be strongly associated with its structural integrity and function. Inter-conversion between the cis and trans conformations also has an important role in the folding process. In this study, we analyse the extent of conservation of cis peptides among similar folds. We look at both the amino acid preferences and local structural changes associated with such variations. Nearly 34% of the Xaa-Proline cis bonds are not conserved in structural relatives; Proline also has a high tendency to get replaced by another amino acid in the trans conformer. At both positions bounding the peptide bond, Glycine has a higher tendency to lose the cis conformation. The cis conformation of more than 30% of β turns of type VIb and IV are not found to be conserved in similar structures. A different view using Protein Block-based description of backbone conformation, suggests that many of the local conformational changes are highly different from the general local structural variations observed among structurally similar proteins. Changes between cis and trans conformations are found to be associated with the evolution of new functions facilitated by local structural changes. This is most frequent in enzymes where new catalytic activity emerges with local changes in the active site. Cis-trans changes are also seen to facilitate inter-domain and inter-protein interactions. As in the case of folding, cis-trans conversions have been used as an important driving factor in evolution.
Conformational preferences of proline derivatives incorporated into vasopressin analogues: NMR and molecular modelling studies.
Source
Faculty of Chemistry, University of Gdańsk, Sobieskiego 18, 80-952 Gdańsk.
Abstract
In this study, AVP analogues modified with proline derivatives: indoline-2-carboxylic acid, Ica; (2S,4R)-4-(naphthalene-2-ylmethyl)pyrrolidine-2-carboxylic acid, Nmp; (2S,4S)-4- aminopyroglutamic acid, APy and (2R,4S)-4-aminopyroglutamic acid, Apy, were examined using NMR spectroscopy and molecular modelling methods. The results have shown that Ica is involved in the formation of the cis peptide bond. Moreover, it reduces to a great extent the conformational flexibility of the peptide. In turn, incorporation of (2S,4R)-Nmp stabilizes the backbone conformation, which is heavily influenced by the pyrrolidine ring. However, the aromatic part of the Nmp side chain exhibits a high degree of conformational freedom. With analogues IV and V, introduction of the 4-aminopyroglumatic acid reduces locally conformational space of thepeptides, but it results also in weaker interactions with the DPC/SDS micelle. Admittedly, both analogues are adsorbed on the micelle's surface but they do not penetrate into its core. With analogue V, the interactions between the peptideand the micelle seem to be so weak that conformational equilibrium is established between different bound states. © 2012 John Wiley & Sons A/S.
© 2012 John Wiley & Sons A/S.
Solid state NMR studies of oligourea foldamers: Interaction of (15)N-labelled amphiphilic helices with oriented lipid membranes.
Source
Université de Strasbourg/CNRS, UMR7177, Institut de Chimie, Faculté de chimie, 4 rue Blaise Pascal, 67070 Strasbourg, France. bechinger@unistra.fr.
Abstract
Synthetic oligomers that are derived from natural polypeptide sequences, albeit with unnatural building blocks, have attracted considerable interest in mimicking bioactive peptides and proteins. Many of those compounds adopt stable folds in aqueous environments that resemble protein structural elements. Here we have chemically prepared aliphatic oligoureas and labeled them at selected positions with (15)N for structural investigations using solid-state NMR spectroscopy. In the first step, the main tensor elements and the molecular alignment of the (15)N chemical shift tensor were analyzed. This was possible by using a two-dimensional heteronuclear chemical shift/dipolar coupling correlation experiment on a model compound that represents the chemical, and thereby also the chemical shift characteristics, of the urea bond. In the next step (15)N labeled versions of an amphipathic oligourea, that exert potent antimicrobial activities and that adopt stable helical structures in aqueous environments, were prepared. These compounds were reconstituted into oriented phospholipid bilayers and the (15)N chemical shift and (1)H-(15)N dipolar couplings of two labeled sites were determined by solid-state NMR spectroscopy. The data are indicative of an alignment of this helix parallel to the membrane surface in excellent agreement with the amphipathic character of the foldamer and consistent with previous models explaining the antimicrobial activities of α-peptides.
Structure and Catalytic Mechanism of Nicotinate (Vitamin B(3)) Degradative Enzyme Maleamate Amidohydrolase from Bordetella bronchiseptica RB50.
Source
Department of Chemistry, The College of Wooster , Wooster, Ohio 44691, United States.
Abstract
The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k(cat) and K(M) values (pH 7.5 and 25 °C) of 11.7 ± 0.2 s(-1) and 128 ± 6 μM, respectively. The observed K(D) of the NicF·maleate (E·P) complex, also measured by ITC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lys117, and Cys150) observed in the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nucleophilic addition-elimination sequence involving the thiolate side chain of Cys150.
The Thorny Way to the Mechanism of Ribosomal Peptide-Bond Formation.
Source
Department, AG Ribosomen, Abteilung Vingron, Max-Planck-Insitut für Molekulare Genetik, Ihnestrasse 73, 14195 Berlin (Germany).
Abstract
Per aspera ad astra: Kinetic isotope effects have indicated a new and comprehensive picture of the central ribosomal enzymatic activity, peptide-bond formation. To this end, isotopes were incorporated in the positions highlighted in red.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Local Unfolding of Cu, Zn Superoxide Dismutase Monomer Determines the Morphology of Fibrillar Aggregates.
Source
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Abstract
Aggregation of Cu, Zn superoxide dismutase (SOD1) is often found in amyotrophic lateral sclerosis patients. The fibrillar aggregates formed by wild type and various disease-associated mutants have recently been found to have distinct cores and morphologies. Previous computational and experimental studies of wild-type SOD1 suggest that the apo-monomer, highly aggregation prone, displays substantial local unfolding dynamics. The residual folded structure of locally unfolded apoSOD1 corresponds to peptide segments forming the aggregation core as identified by a combination of proteolysis and mass spectroscopy. Therefore, we hypothesize that the destabilization of apoSOD1 caused by various mutations leads to distinct local unfolding dynamics. The partially unfolded structure, exposing the hydrophobic core and backbone hydrogen bond donors and acceptors, is prone to aggregate. The peptide segments in the residual folded structures form the "building block" for aggregation, which in turn determines the morphology of the aggregates. To test this hypothesis, we apply a multiscale simulation approach to study the aggregation of three typical SOD1 variants: wild type, G37R, and I149T. Each of these SOD1 variants has distinct peptide segments forming the core structure and features different aggregate morphologies. We perform atomistic molecular dynamics simulations to study the conformational dynamics of apoSOD1 monomer and coarse-grained molecular dynamics simulations to study the aggregation of partially unfolded SOD1 monomers. Our computational studies of monomer local unfolding and the aggregation of different SOD1 variants are consistent with experiments, supporting the hypothesis of the formation of aggregation "building blocks" via apo-monomer local unfolding as the mechanism of SOD1 fibrillar aggregation.
Copyright © 2011 Elsevier Ltd. All rights reserved.
The Plasmodium falciparum Malaria M1 Alanyl Aminopeptidase (PfA-M1): Insights of Catalytic Mechanism and Function from MD Simulations.
Source
School of Medical and Molecular Biosciences, Sydney, New South Wales, Australia.
Abstract
Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of thepeptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 Å-long channel and at the opening of the 30 Å-long C-terminal internal chamber that facilitates entry ofpeptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.
Circular micro-proteins and mechanisms of cyclization.
Source
Department of Biochemistry, La Trobe University, Melbourne, Victoria 3086, Australia. m.anderson@latrobe.edu.au.
Abstract
Transpeptidation reactions result in the formation of new peptide bonds and this can occur between two separatepeptides or within the one peptide. These reactions are catalyzed by enzymes and when the N- and C-terminus of the one peptide are joined it results in the formation of cyclic proteins. Cyclization via head-to-tail linkage of the termini of apeptide chain occurs in only a small percentage of proteins but gives the resultant cyclic proteins exceptional stability. The mechanisms are not well understood and this review documents what is known of the events that lead to cyclization. Gene encoded cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the θ-defensins and retrocyclins. Small cyclic proteins are also found in fungi and a large range of cyclic proteins are expressed in cyanobacteria. Three types of cyclic proteins have been found in plants, the small cyclic proteins of 5-12 amino acids, the cyclic proteins from sunflower which are made up of 12-14 amino acids, and the larger group known as cyclotides which contain 28-37 amino acids. Three classes of enzymes are able to catalyse transpeptidation reactions, these include the cysteine, serine and threonine proteases. However only cysteine and serine proteases have been documented to cyclize proteins. The cyclotides from Oldenlandia affinis, the plant in which cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack of the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide.
Rethinking amide bond synthesis.
Source
Laboratorium für Organische Chemie, ETH Zürich, 8093 Zürich, Switzerland.
Abstract
One of the most important reactions in organic chemistry--amide bond formation--is often overlooked as a contemporary challenge because of the widespread occurrence of amides in modern pharmaceuticals and biologically active compounds. But existing methods are reaching their inherent limits, and concerns about their waste and expense are becoming sharper. Novel chemical approaches to amide formation are therefore being developed. Here we review and summarize a new generation of amide-forming reactions that may contribute to solving these problems. We also consider their potential application to current synthetic challenges, including the development of catalytic amide formation, the synthesis of therapeutic peptides and the preparation of modified peptides and proteins.
An Exit Cavity Was Crucial to the Polymerase Activity of the Early Ribosome.
Source
1 Department of Biology and Biochemistry, University of Houston , Houston, Texas, USA .
Abstract
Abstract The emergence of an RNA entity capable of synthesizing peptides was a key prebiotic development. It is hypothesized that a precursor of the modern ribosomal exit tunnel was associated with this RNA entity (e.g., "protoribosome" or "bonding entity") from the earliest time and played an essential role. Various compounds that can bind and activate amino acids, including extremely short RNA chains carrying amino acids, and possibly di- or tripeptides, would have associated with the internal cavity of the protoribosome. This cavity hosts the site for peptidebond formation and adjacent to it a relatively elongated feature that could have evolved to the modern ribosomal exit tunnel, as it is wide enough to allow passage of an oligopeptide. When two of the compounds carrying amino acids or di- or tripeptides (to which we refer, for simplicity, as small aminoacylated RNAs) were in proximity within the heart of the protoribosome, a peptide bond could form spontaneously. The growing peptide would enter the nearby cavity and would not disrupt the attachment of the substrates to the protoribosome or interfere with the subsequent attachment of additional small aminoacylated RNAs. Additionally, the presence of the peptide in the cavity would increase the lifetime of the oligopeptide in the protoribosome. Thus, subsequent addition of another amino acid would be more likely than detachment from the protoribosome, and synthesis could continue. The early ability to synthesize peptides may have resulted in an abbreviated RNA World. Key Words: Ribosome-RNA World-Prebiotic synthesis-Chirality-Ribosomal RNA. Astrobiology 12, xxx-xxx.
The Early Life of a Peptide Cation-Radical. Ground and Excited-State Trajectories of Electron-Based Peptide Dissociations During the First 330 Femtoseconds.
Source
Department of Chemistry, University of Washington, Bagley Hall, Box 351700, Seattle, WA, USA.
Abstract
We report a new approach to investigating the mechanisms of fast peptide cation-radical dissociations based on an analysis of time-resolved reaction progress by Ehrenfest dynamics, as applied to an Ala-Arg cation-radical model system. Calculations of stationary points on the ground electronic state that were carried out with effective CCSD(T)/6-311++G(3df,2p) could not explain the experimental branching ratios for loss of a hydrogen atom, ammonia, and N-C(α)bond dissociation in (AR + 2H)(+[Symbol: see text]). The Ehrenfest dynamics results indicate that the ground and low-lying excited electronic states of (AR + 2H)(+[Symbol: see text]) follow different reaction courses in the first 330 femtoseconds after electron attachment. The ground (X) state undergoes competing loss of N-terminal ammonia and isomerization to an aminoketyl radical intermediate that depend on the vibrational energy of the charge-reduced ion. The A and B excited states involve electron capture in the Arg guanidine and carboxyl groups and are non-reactive on the short time scale. The C state is dissociative and progresses to a fast loss of an H atom from the Arg guanidine group. Analogous results were obtained by using the B3LYP and CAM-B3LYP density functionals for the excited state dynamics and including the universal M06-2X functional for ground electronic state calculations. The results of this Ehrenfest dynamics study indicate that reaction pathway branching into the various dissociation channels occurs in the early stages of electron attachment and is primarily determined by the electronic states being accessed. This represents a new paradigm for the discussion of peptide dissociations in electron based methods of mass spectrometry.
Microbial degradation of physiologically active peptides by strain B-9.
Source
Department of Pharmacology, Aichi Medical University, Nagakute, Aichi, 480-1195, Japan, fumio@ipc-tokai.or.jp.
Abstract
The reaction of some physiologically active peptides with bacterial strain B-9 has been investigated. Bradykinin, β-endorphin, and [Leu(5)]enkephalin were quickly degraded, with half-lives of <5 min. Somatostatin, substance P, and angiotensin I were degraded relatively smoothly, with half-lives of 10 min to 1 h, whereas oxytocin and insulin were slowly degraded, with half-lives of 1 and 4 days, respectively. Vasopressin was barely degraded, with a half-life of >7 days. Linearized vasopressin, prepared by the reductive cleavage of the disulfide bond followed by alkylation with iodoacetamide, was degraded significantly faster than intact vasopressin, with a half-life of 2.5 h. A loop formed by disulfide bond formation was regarded as one of the degradation-resistant factors. Hydrolysis of the peptides in this study took place through cleavage of various peptide bonds, and the strain B-9 may bear similarities to the neutral endopeptidase in terms of its broad selectivity.
Baupain, a plant cysteine proteinase that hinders thrombin-induced human platelet aggregation.
Source
Universidade Federal de São Paulo Departamento de Bioquímica Rua Três de Maio 100, 04044-020 São Paulo, SP, Brazil. olivaml.bioq@epm.br.
Abstract
Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.
- PMID:
- 22185503
- [PubMed - as supplied by publisher]
Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity.
Abstract
pH-sensitive PEGylated (with PEG-PE) long-circulating liposomes (HSPC:cholesterol and Doxil®), modified with cell-penetrating TAT peptide (TATp) moieties and cancer-specific mAb 2C5 were prepared. A degradable pH-sensitive hydrazone bond between a long shielding PEG chains and PE (PEG(2k)-Hz-PE) was introduced. TATp was conjugated with a short PEG(1k)-PE spacer and mAb 2C5 was attached to a long PEG chain (2C5-PEG(3.4k)-PE). The "shielding" effect of TATp by long PEG chains was investigated using three liposomal models. At normal pH, surface TATp moieties are "hidden" by the long PEG chains. Upon the exposure to lowered pH, this multifunctional carrier exposes TATp moieties after the degradation of the hydrazone bond and removal of the long PEG chains. Enhanced cellular uptake of the TATp-containing immunoliposomes was observed in vitro after pre-treatment at lowered pH (using flow cytometry and fluorescence microscopy techniques). The presence of mAb 2C5 on the liposome surface further enhanced the interaction between the carrier and tumor cells but not normal cells. Furthermore, multifunctional immuno-Doxil® preparation showed increased cellular cytotoxicity of B16-F10, HeLa and MCF-7 cells when pre-incubated at lower pH, indicating TATp exposure and activity. In conclusion, a multifunctional immunoliposomal nanocarrier containing a pH-sensitive PEG-PE component, TATp, and the cancer cell-specific mAb 2C5 promotes enhanced cytotoxicity and carrier internalization by cancer cells and demonstrates the potential for intracellular drug delivery after exposure to lowered pH environment, typical of solid tumors.
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