Probing protein sequences as sources for encrypted antimicrobial peptides

Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources

Interaction between the Rev1 C-Terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis

Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of “inserter” to “extender” DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the “inserter” Polη, Polι, or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit “extender” Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits

Conformational and functional effects induced by D- and L-amino acid epimerization on a single gene encoded peptide from the skin secretion of Hypsiboas punctatus.

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways

S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity.

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo

Hydration and Conformational Equilibrium in Yeast Thioredoxin 1: Implication for H+ Exchange

Made available in DSpace on 2015-06-08T14:01:53Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) francisco_netoetal_IOC_2014.pdf: 5780156 bytes, checksum: 1328f475e6d2ab079ec5f20184583aea (MD5) Previous issue date: 2014Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxonologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas. Rio de Janeiro, RJ, Brasil.Faculdades Integradas de Três Lagoas - AEMS. Três Lagoas, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Química. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas. Rio de Janeiro, RJ, Brasil.One of the ancestral features of thioredoxins is the presence of a water cavity. Here, we report that a largely hydrated, conserved, buried aspartic acid in the water cavity modulates the dynamics of the interacting loops of yeast thioredoxin 1 (yTrx1). It is well-established that the aspartic acid, Asp24 for yTrx1, works as a proton acceptor in the reduction of the target protein. We propose a complementary role for Asp24 of coupling hydration and conformational motion of the water cavity and interacting loops. The intimate contact between the water cavity and the interacting loops means that motion at the water cavity will affect the interacting loops and vice versa. The D24N mutation alters the conformational equilibrium for both the oxidized and reduced states, quenching the conformational motion in the water cavity. By measuring the hydration and molecular dynamics simulation of wild-type yTrx1 and the D24N mutant, we showed that Asn24 is more exposed to water than Asp24 and the water cavity is smaller in the mutant, closing the inner part of the water cavity. We discuss how the conformational equilibrium contributes to the mechanism of catalysis and H+ exchange

<i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor binds to elastase and impairs neutrophil function and inflammation

<div><p>Protease inhibitors have important function during homeostasis, inflammation and tissue injury. In this study, we described the role of <i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor in parasite development and as a molecule capable of regulating different models of inflammatory diseases. First, we determine that recombinant (r) <i>Sm</i>KI-1 and its Kunitz domain but not the C-terminal region possess inhibitory activity against trypsin and neutrophil elastase (NE). To better understand the molecular basis of NE inhibition by S<i>m</i>KI-1, molecular docking studies were also conducted. Docking results suggest a complete blockage of NE active site by <i>Sm</i>KI-1 Kunitz domain. Additionally, r<i>Sm</i>KI-1 markedly inhibited the capacity of NE to kill schistosomes. In order to further investigate the role of <i>Sm</i>KI-1 in the parasite, we designed specific siRNA to knockdown <i>Sm</i>KI-1 in <i>S</i>. <i>mansoni</i>. <i>SmKI-1</i> gene suppression in larval stage of <i>S</i>. <i>mansoni</i> robustly impact in parasite development <i>in vitro</i> and <i>in vivo</i>. To determine the ability of <i>Sm</i>KI-1 to interfere with neutrophil migration and function, we tested <i>Sm</i>KI-1 anti-inflammatory potential in different murine models of inflammatory diseases. Treatment with <i>Sm</i>KI-1 rescued acetaminophen (APAP)-mediated liver damage, with a significant reduction in both neutrophil recruitment and elastase activity. In the model of gout arthritis, this protein reduced neutrophil accumulation, IL-1β secretion, hypernociception, and overall pathological score. Finally, we demonstrated the ability of <i>Sm</i>KI-1 to inhibit early events that trigger neutrophil recruitment in pleural cavities of mice in response to carrageenan. In conclusion, <i>Sm</i>KI-1 is a key protein in <i>S</i>. <i>mansoni</i> survival and it has the ability to inhibit neutrophil function as a promising therapeutic molecule against inflammatory diseases.</p></div

Structural studies on L- and D- Phenylseptin peptide isomers by Ion Mobility Mass Spectrometry (IM-MS) and Nuclear Magnetic Resonance (NMR).

<p>(A) L-Phes and D-Phes were individually analyzed showing that each one (M+3H⁺ = 652.04 m/z) can assume at least two major conformations with distinct amounts of each type. L-Phes conformations at 10.45 and 12.89 ms and D-Phes conformations at 10.80 and 12.72 ms. D-Phes has its major conformation at 10.80 ms. Experiments were performed on a Synapt HDMS instrument (Quadrupole Ion Mobility High-Definition mass spectrometry – Waters Co. MA, USA) equipped with nano-electrospray ionization. All spectra were acquired with a direct infusion of 1 µL·min-1 of in a range m/z 300 up to 2000. Precursor charge state: 3. Tolerance: 0.1 Da. (B) and (C) The 20 lowest-energy structures for both peptides. The hydrophobic residues are represented in gold yellow, the hydrophilic residues in green. (D) The lowest-energy Phenylseptins showing Phenylalanine enatiomerization, the aromatic phenylalanine are in dark red and (E) The alignment of lowest-energy L- and D-Phes structure in the presence of 60% TFE viewed along the helix axis and from the side.</p

UFLC analysis and molecular mass determination of Phenylseptin mixture.

<p>(A) The accurate molecular masses and purity of Phes peptides were determined by MALDI-TOF/MS and the observed molecular mass was 1954.2 Da for both molecules. (B) Analytical chromatographic profile of natural (black line) and synthetic L-Phes (red dash line) and D-Phes (blue dash line). The two peptides were mixed in similar molar concentrations and load into an Ultra Fast Liquid Chromatography using a Shimpack-XR-ODS column under a linear gradient of acetonitrile at a flow rate of 0.4 mL·min-1. The two distinct fractions eluted around 11 and 12 minutes corresponded to L-Phes and D-Phes, respectively.</p

One single gene encoding Phes peptide.

<p>(A) Nucleotide sequences of clone encoding precursor of selected Phenylseptin peptides. The putative signal peptide (in box), acidic spacer (dash underline) and mature peptide (bold underline), C-terminal codon for Glycine (blue underline) and stop codon (asterisk) are indicated. The nucleotide sequences were deposited in the NCBI Nucleotide Sequence Database under HQ012497 annotated accession code. (B) Predicted amino acid sequence alignment of Phenylseptin with previously sequenced Hylidae peptides aurein, ranateurin, brevinin and gaegurin. Sequence alignments were done using CLUSTAL W software and were edited with the BIOEDIT software.</p