José R. Pires

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single α-helical globular domain, microplusin consists of five α-helices: α1 (residues Gly-9 to Arg-21), α2 (residues Glu-27 to Asn-40), α3 (residues Arg-44 to Thr-54), α4 (residues Leu-57 to Tyr-64), and α5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Quality of Recombinant Protein Determines the Amount of Autoreactivity Detected against the Tumor-Associated Epithelial Cell Adhesion Molecule Antigen: Low Frequency of Antibodies against the Natural Protein

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. 1H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.

Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50μM of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized.

Solution NMR analysis of the interaction between the actinoporin Sticholysin I and DHPC micelles—Correlation with backbone dynamics

Sticholysin I (StI), an actinoporin expressed as a water‐soluble protein by the sea anemone Stichodactyla helianthus, binds to natural and model membranes, forming oligomeric pores. It is proposed that the first event of a multistep pore formation mechanism consists of the monomeric protein attachment to the lipid bilayer. To date there is no high‐resolution structure of the actinoporin pore or other membrane‐bound form available. Here we evaluated StI:micelle complexes of variable lipid composition to look for a suitable model for NMR studies. Micelles of pure or mixed lysophospholipids and of dihexanoyl phosphatidylcholine (DHPC) were examined. The StI:DHPC micelle was found to be the best system, yielding a stable sample and good quality spectra. A comprehensive chemical shift perturbation analysis was performed to map the StI membrane recognition site in the presence of DHPC micelles. The region mapped (residues F51, R52, S53 in loop 3; F107, D108, Y109, W111, Y112, W115 in loop 7; Q129, Y132, D134, M135, Y136, Y137, G138 in helix‐α2) is in agreement with previously reported data, but additional residues were found to interact, especially residues V81, A82, T83, G84 in loop 5, and A85, A87 in strand‐β5. Backbone dynamics measurements of StI free in solution and bound to micelles highlighted the relevance of protein flexibility for membrane binding and suggested that a conformer selection process may take place during protein–membrane interaction. We conclude that the StI:DHPC micelles system is a suitable model for further characterization of an actinoporin membrane‐bound form by solution NMR. Proteins 2014; 82:1022–1034.

(1)H, (15)N and (13)C resonance assignments and secondary structure prediction of Q4D059, a conserved and kinetoplastid-specific hypothetical protein from Trypanosoma cruzi.

Trypanosoma cruzi is a human parasite that causes Chagas disease, an illness affecting millions of people and without an efficient treatment available. Sequencing the pathogen genome has revealed that near half of protein-coding genes correspond to hypothetical proteins of unknown function, increasing the possibilities for novel target discovery. Q4D059 is a putative essential hypothetical protein from T. cruzi and it is specific and conserved among the trypanosomatid genomes. Here, we report the sequential backbone and side chain resonance assignments and secondary structure analysis of Q4D059, as first step for protein structure determination, function elucidation and drug screening.

1H, 15N and 13C assignments of the Rhipicephalus (Boophilus) microplus anti-microbial peptide microplusin

Microplusin, a Rhipicephalus (Boophilus) microplus anti-microbial peptide (AMP) is the first member of a new family of cysteine-rich AMPs with histidine-rich regions at the N- and C-termini, which is being fully characterized by biophysical and biochemical methods. Here we report the NMR resonance assignments for 1H, 15N, and 13C nuclei in the backbone and side chains of the microplusin as basis for further studies of structure, backbone dynamics and interactions mapping.

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions.

Sabellastarte magnifica Carboxypeptidase Inhibitor: The first Kunitz inhibitor simultaneously interacting with carboxypeptidases and serine proteases

Multi-domain inhibitors capable to block the activity of different classes of proteases are not very common in nature. However, these kinds of molecules are attractive systems for biomedical or biotechnological applications, where two or more different targets need to be neutralized. SmCI, the Sabellastarte magnifica Carboxypeptidase Inhibitor, is a tri-domain BPTI-Kunitz inhibitor capable to inhibit serine proteases and A-like metallocarboxypeptidases. The BPTI-Kunitz family of proteins includes voltage gated channel blockers and inhibitors of serine proteases. SmCI is therefore, the only BPTI-Kunitz protein capable of inhibiting metallocarboxypeptidases. The X-ray structure of the SmCI-carboxypeptidase A complex previously obtained by us, revealed that this enzyme interacts with SmCI N-tail. In the complex, the reactive loops for serine protease inhibition remain fully exposed to the solvent in each domain, suggesting SmCI can simultaneously interact with multiple serine proteases. The twofold goals of this study were: i) to establish serine proteases-SmCI binding stoichiometry, given that the inhibitor is comprised of three potential binding domains; and ii) to determine whether or not SmCI can simultaneously bind both classes of enzymes, to which it binds individually. Our experimental approach included a variety of techniques for the study of protein-protein interactions, using as model enzymes pancreatic trypsin, elastase and carboxypeptidase A. In particular, we combined information obtained from gel filtration chromatography, denaturing electrophoresis, nuclear magnetic resonance spectroscopy and enzyme inhibition assays. Our results show that SmCI is able to bind three trypsin molecules under saturating conditions, but only one elastase interacts with the inhibitor. Additionally, we demonstrated that SmCI can bind serine proteases and carboxypeptidases at the same time (at least in the ratio 1:1:1), becoming the first protease inhibitor that simultaneously blocks these two mechanistic classes of enzymes.

Combining Nuclear Magnetic Resonance Spectroscopy and Density Functional Theory Calculations to Characterize Carvedilol Polymorphs

The experiments of carvedilol form II, form III, and hydrate by (13)C and (15)N cross-polarization magic-angle spinning (CP MAS) are reported. The GIPAW (gauge-including projector-augmented wave) method from DFT (density functional theory) calculations was used to simulate (13)C and (15)N chemical shifts. A very good agreement was found for the comparison between the global results of experimental and calculated nuclear magnetic resonance (NMR) chemical shifts for carvedilol polymorphs. This work aims a comprehensive understanding of carvedilol crystalline forms employing solution and solid-state NMR as well as DFT calculations.

1H, 13C and 15N resonance assignments and secondary structure analysis of CmPI-II, a serine protease inhibitor isolated from marine snail Cenchritis muricatus

A protease inhibitor (CmPI-II) (UNIPROT: IPK2_CENMR) from the marine mollusc Cenchritis muricatus, has been isolated and characterized. It is the first member of a new group (group 3) of non-classical Kazal-type inhibitors. CmPI-II is a tight-binding inhibitor of serine proteases: trypsin, human neutrophil elastase (HNE), subtilisin A and pancreatic elastase. This specificity is exceptional in the members of Kazal-type inhibitor family. Several models of three-dimensional structure of CmPI-II have been constructed by homology with other inhibitors of the family but its structure has not yet been solved experimentally. Here we report the 1H, 15N and 13C chemical shift assignments of CmPI-II as basis for NMR structure determination and interaction studies. Secondary structure analyses deduced from the NMR chemical shift data have identified three β-strands β1: residues 14–19, β2: 23–35 and β3: 43–45 and one helix α1: 28–37 arranged in the sequential order β1–β2–α1–β3. These secondary structure elements suggest that CmPI-II adopts the typical scaffold of a Kazal-type inhibitor.