Structural insights into and activity analysis of the antimicrobial peptide myxinidin.

The marine environment has been poorly explored in terms of potential new molecules possessing antibacterial activity. Antimicrobial peptides (AMPs) offer a new potential class of pharmaceuticals; however, further optimization is needed if AMPs are to find broad use as antibiotics. We focused our studies on a peptide derived from the epidermal mucus of hagfish (Myxine glutinosa L.), which was previously characterized and showed high antimicrobial activity against human and fish pathogens. In the present work, the activities of myxinidin peptide analogues were analyzed with the aim of widening the original spectrum of action of myxinidin by suitable changes in the peptide primary structure. The analysis of key residues by alanine scanning allowed for the design of novel peptides with increased activity. We identified the amino acids that are of the utmost importance for the observed antimicrobial activities against a set of pathogens comprising both Gram-negative and Gram-positive bacteria. Overall, optimized bactericidal potency was achieved by adding a tryptophan residue at the N terminus and by the simultaneous substitution of residues present in positions 3, 4, and 11 with arginine. These results indicate that the myxinidin analogues emerge as an attractive alternative for treating drug-resistant infectious diseases and provide key insights into a rational design for novel agents against these pathogens.

Binding ability of a thymine-functionalized oligolysine towards nucleic acids.

In this Letter, we investigated the binding properties towards nucleic acids of a thymine-functionalized oligolysine, composed of nucleobase-bearing amino acid moieties and underivatized l-lysine residues alternate in the backbone. The basic nucleopeptide proved to be well soluble in water and able to interact with both DNA and RNA, as suggested by circular dichroism, UV and surface plasmon resonance studies performed on the thymine-containing oligomer with both adenine-containing DNA (dA12) and RNA (rA12 and poly rA) molecules. In both cases the thymine-functionalized oligolysine was proven to form complexes characterized by a 1:1 T/A stoichiometric ratio, as evidenced by CD titration. UV melting experiments revealed that the complex formed between the homothymine oligolysine and rA12 RNA was more stable than the complex with dA12 DNA probably due to the additional H-bonding of the 2'-OH groups in RNA, that reinforces the overall interaction with the nucleopeptide. Finally, human serum stability assays were conducted on the thymine-bearing nucleopeptide which showed a half-life of 45min.

X-ray structure of the first `extremo-α-carbonic anhydrase', a dimeric enzyme from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1.

SspCA, a novel `extremo-α-carbonic anhydrase' isolated from the thermophilic bacterium Sulfurihydrogenibium yellowstonense YO3AOP1, is an efficient catalyst for the hydration of CO2 and presents exceptional thermostability. Indeed, SspCA retains a high catalytic activity even after being heated to 343-373 K for several hours. Here, the crystallographic structure of this α-carbonic anhydrase (α-CA) is reported and the factors responsible for its function at high temperature are elucidated. In particular, the study suggests that increased structural compactness, together with an increased number of charged residues on the protein surface and a greater number of ionic networks, seem to be the key factors involved in the higher thermostability of this enzyme with respect to its mesophilic homologues. These findings are of extreme importance, since they provide a structural basis for the understanding of the mechanisms responsible for thermal stability in the α-CA family for the first time. The data obtained offer a tool that can be exploited to engineer α-CAs in order to obtain enzymes with enhanced thermostability for use in the harsh conditions of the CO2 capture and sequestration processes.

Chimeric beta-defensin analogs, including the novel 3NI analog, display salt-resistant antimicrobial activity and lack toxicity in human epithelial cell lines.

Human beta-defensins (hBDs) are crucial peptides for the innate immune response and are thus prime candidates as therapeutic agents directed against infective diseases. Based on the properties of wild-type hBD1 and hBD3 and of previously synthesized analogs (1C, 3I, and 3N), we have designed a new analog, 3NI, and investigated its potential as an antimicrobial drug. Specifically, we evaluated the antimicrobial activities of 3NI versus those of hBD1, hBD3, 1C, 3I, and 3N. Our results show that 3NI exerted greater antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis than did hBD1 and hBD3, even with elevated salt concentrations. Moreover, its antiviral activity against herpes simplex virus 1 was greater than that of hBD1 and similar to that of hBD3. Subsequently, we investigated the cytotoxic effects of all peptides in three human epithelial carcinoma cell lines: A549 from lung, CaCo-2 from colon, and Capan-1 from pancreas. None of the analogs significantly reduced cell viability versus wild-type hBD1 and hBD3. They did not induce genotoxicity or cause an increase in the number of apoptotic cells. Using confocal microscopy, we also investigated the localization of the peptides during their incubation with epithelial cells and found that they were distributed on the cell surface, from which they were internalized. Finally, we show that hBD1 and hBD3 are characterized by high resistance to serum degradation. In conclusion, the new analog 3NI seems to be a promising anti-infective agent, particularly given its high salt resistance--a feature that is relevant in diseases such as cystic fibrosis.

Synthesis of a novel benzodifuran derivative and its molecular recognition of poly rA RNA.

In this manuscript, we describe a synthetic approach to a novel benzodifuran derivative as well as CD studies regarding its ability to interact with DNA and RNA. After the chemical synthesis and ESI-MS and NMR characterization, this heteroaromatic molecule was investigated by CD spectroscopy in order to evaluate it as a potential nucleic acid binder. Interestingly, the benzodifuran compound was found to be able to induce conformational changes in both DNA and RNA homoadenine molecules forming in the latter case a complex with a 6:1 benzodifuran/nucleobase stoichiometric ratio, as evidenced by CD titration experiments.

Synthesis of a thymine-functionalized nucleoamino acid for the solid phase assembly of cationic nucleopeptides.

In this work, we report the synthesis of a thymine-functionalized nucleoamino acid suitable for the solid phase synthesis of nucleopeptides. The monomer was obtained in solution starting from commercial compounds and after NMR ((1)H and (13)C) and ESIMS (positive ions) characterization it was used for the assembly of a cationic nucleopeptide obtained by sequentially introducing underivatized L-lysine units and nucleoamino acid monomers. After detachment from the resin, performed in acidic conditions, the oligomer was purified by HPLC and characterized by LC-ESIMS (positive ions) which confirmed the identity of the thymine-based nucleopeptide. The cationic nucleobase-containing peptide, well soluble in water, was studied by CD spectroscopy which allowed us to exclude any helical pre-organization of the nucleopeptide in the experimental conditions used. Furthermore, CD behavior of the oligomer at different temperatures was also studied as described in this work.

Corrigendum to: ß-Barrel Membrane Bacterial Proteins: Structure, Function, Assembly and Interaction with Lipids.

The authors declare after the publication of the article entitled 'ß-Barrel Membrane Bacterial Proteins: Structure, Function, Assembly and Interaction with Lipids'', published in Current Protein and Peptide Science, 2007, 8, 63-82 [1], that a reference by Koebnik was inadvertently omitted. The missing reference has now been included as: Original: [1] Rosenbusch, J.P. (1988) Zentralbl. Bakteriol., 17, 259-266. Corrected: [1] (a) Rosenbusch, J.P. (1988) Zentralbl. Bakteriol., 17, 259-266. (b) Koebnik, R.; Locher, K.P.; Gelder, P.V. Structure and function of bacterial outer membrane proteins: Barrels in a nutshell. Mol. Biol., 2000, 37(2), 239-53. The original article can be found online at: https://www.eurekaselect.com/article/22780

Levels of 24-hydroxycholesteryl esters in cerebrospinal fluid and plasma from patients with amyotrophic lateral sclerosis.

OBJECTIVE: In this context, our study aimed to ascertain whether the esterification of 24-hydroxycholesterol, a process heavily affected by oxidative stress, is altered in ALS. METHODS: The study examined the level of 24-hydroxycholesteryl esters in cerebrospinal fluid and plasma of 18 ALS patients by spectroscopic technique as Ultra-high performance liquid chromatography mass spectrometry (UPLC-MS). RESULTS: The level of 24-hydroxycholesteryl esters in cerebrospinal fluid was found to be lower as the brain-blood barrier was damaged. Such a level was positively correlated with the level of esters in plasma. Both cerebrospinal fluid (CSF) level and plasma level were lower in ALS patients (60.05 ± 4.24 % and 54.07 ± 20.37 % respectively) than in controls (79.51 ± 2.47 % and 80.07 ± 10.02 % respectively). CONCLUSIONS: The data suggest that the level 24-hydroxycholesteryl esters might be a new biomarker of ALS and can be measured for monitoring the disease progression.

ß-hairpin peptide that targets vascular endothelial growth factor (VEGF) receptors: design, NMR characterization, and biological activity.

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor ß-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.

Structure-based design of a potent artificial transactivation domain based on p53.

Malfunctions in transcriptional regulation are associated with a number of critical human diseases. As a result, there is considerable interest in designing artificial transcription activators (ATAs) that specifically control genes linked to human diseases. Like native transcriptional activator proteins, an ATA must minimally contain a DNA-binding domain (DBD) and a transactivation domain (TAD) and, although there are several reliable methods for designing artificial DBDs, designing artificial TADs has proven difficult. In this manuscript, we present a structure-based strategy for designing short peptides containing natural amino acids that function as artificial TADs. Using a segment of the TAD of p53 as the scaffolding, modifications are introduced to increase the helical propensity of the peptides. The most active artificial TAD, termed E-Cap-(LL), is a 13-mer peptide that contains four key residues from p53, an N-capping motif and a dileucine hydrophobic bridge. In vitro analysis demonstrates that E-Cap-(LL) interacts with several known p53 target proteins, while in vivo studies in a yeast model system show that it is a 20-fold more potent transcriptional activator than the native p53-13 peptide. These results demonstrate that structure-based design represents a promising approach for developing artificial TADs that can be combined with artificial DBDs to create potent and specific ATAs.

Native expression and purification of hormone-sensitive lipase from Psychrobacter sp. TA144 enhances protein stability and activity.

Psychrobacter, a micro-organism originally isolated from Antarctic sea water, expresses an extremely active hormone-sensitive lipase (HSL) which catalyzes the hydrolysis of fatty acid esters at very low temperature and is therefore of great potential industrial and pharmaceutical interest. An insoluble form of the entire enzyme has previously been cloned and expressed in Escherichia coli, subsequently refolded and shown to be active, whilst a shorter but completely inactive version, lacking the N-terminal 98 amino acids has been expressed in soluble form. In this study the entire enzyme has been expressed as a fully soluble protein in E. coli in the presence of either the osmolyte trehalose, plus high salt concentration, or the membrane fluidizer benzyl alcohol. Trehalose promotes protein mono-dispersion by increasing the viscosity of the growth medium for bacterial cells, thereby helping circumvent protein aggregation, whilst the heat-shock inducer benzyl alcohol stimulates the production of a network of endogenous chaperones which actively prevent protein misfolding, whilst also converting recombinant aggregates to native, correctly folded proteins. The resultant recombinant protein proved to be more stable than its previously expressed counterpart, as shown by CD and enzymatic activity data which proved the enzyme to be more active at a higher temperature than its refolded counterpart. By light scattering analysis it was shown that the newly expressed protein was monomeric. The stability of the full length native protein will help in understanding the structure of PsyHSL and the role of its regulatory N-terminal for eventual application in a myriad of biotechnological processes.

Apolipoprotein A-I (ApoA-I) mimetic peptide P2a by restoring cholesterol esterification unmasks ApoA-I anti-inflammatory endogenous activity in vivo.

The acute-phase protein haptoglobin (Hpt) binds apolipoprotein A-I (ApoA-I) and impairs its action on lecithin-cholesterol acyltransferase, an enzyme that plays a key role in reverse cholesterol transport. We have previously shown that an ApoA-I mimetic peptide, P2a, displaces Hpt from ApoA-I, restoring the enzyme activity in vitro. The aim of this study was to evaluate whether P2a displaces Hpt from ApoA-I in vivo and whether this event leads to anti-inflammatory activity. Mice received subplantar injections of carrageenan. Paw volume was measured before the injection and 2, 4, 6, 24, 48, 72, and 96 h thereafter. At the same time points, concentrations of HDL cholesterol (C) and cholesterol esters (CEs) were measured by high-performance liquid chromatography, and Hpt and ApoA-I plasma levels were evaluated by enzyme-linked immunosorbent assay. Western blotting analysis for nitric-oxide synthase and cyclooxygenase (COX) isoforms was also performed on paw homogenates. CEs significantly decreased in carrageenan-treated mice during edema development and negatively correlated with the Hpt/ApoA-I ratio. P2a administration significantly restored the CE/C ratio. In addition, P2a displayed an anti-inflammatory effect on the late phase of edema with a significant reduction in COX2 expression coupled to an inhibition of prostaglandin E(2) synthesis, implying that, in the presence of P2a, CE/C ratio rescue and edema inhibition were strictly related. In conclusion, the P2a effect is due to its binding to Hpt with consequent displacement of ApoA-I that exerts anti-inflammatory activity. Therefore, it is feasible to design drugs that, by enhancing the physiological endogenous protective role of ApoA-I, may be useful in inflammation-based diseases.

Gold nanoparticles capped by a GC-containing peptide functionalized with an RGD motif for integrin targeting.

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)(2) peptide as stabilizer. As comparison, the behavior of the (GC)(2) peptide has been studied. The (GC)(2) and RGD-(GC)(2) peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV-visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)(2) and Au-(GC)(2) stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)(2) capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)(2) gold nanoparticles into the nucleus.

Synthesis of a diaminopropanoic acid-based nucleoamino acid and assembly of cationic nucleopeptides for biomedical applications.

In this work, we report a synthetic approach to a Fmoc-protected nucleoamino acid, based on L-diaminopropanoic acid, carrying the DNA nucleobase on the alpha-amino group by means of an amide bond, suitable for the solid-phase synthesis of novel nucleopeptides of potential interest in biomedicine. After ESI-MS and NMR characterization this building block was used for the assembly of a thymine-functionalized nucleopeptide, composed of nucleobase-containing L-diaminopropanoic acid moieties and underivatized L-lysine residues alternated in the backbone. Circular dichroism studies performed on the cationic nucleopeptide and adenine-containing DNA and RNA molecules suggested that the thymine-containing peptide is able to interact with both DNA and RNA. In particular, a significant conformational variation in the RNA structure was suggested by CD studies. Human serum stability assays were also conducted on the cationic nucleopeptide, which was found to be highly resistant to enzymatic degradation.

Dakin-West reaction on 1-thyminyl acetic acid for the synthesis of 1,3-bis(1-thyminyl)-2-propanone, a heteroaromatic compound with nucleopeptide-binding properties.

This work deals with the Dakin-West synthesis, starting from the nucleoamino acid 1-thyminyl acetic acid, as well the NMR, ESI MS, and X-ray characterization of a heteroaromatic compound denominated by us T(2)CO, comprising two thymine moieties anchored to a 2-propanonic unit, the spectroscopic properties of which were studied by UV as a function of temperature and ionic strength. Preliminary binding-studies with molecules of biomedical interest such as nucleic acids and proteins, performed on samples containing T(2)CO, suggested that this molecule is able to interact very weakly with double-stranded RNA, whereas it does not seem to bind other nucleic acids or proteins. Moreover, by studies with fresh human serum we found that T(2)CO is resistant to enzymatic degradation till 24 h, whereas UV metal binding-studies, performed using solutions of copper (II) chloride dihydrate and nickel (II) chloride hexahydrate, revealed a certain ability of T(2)CO to bind copper (II) cation. Finally, by CD spectroscopy we investigated the influence of T(2)CO on the already described supramolecular networks based on L-serine-containing nucleopeptides. More particularly, we found that T(2)CO is able to increase the level of structuration of the non-covalent supramolecular assembly of the chiral nucleopeptides, which is a feature of remarkable interest for the development of innovative drug delivery tools.

Synthesis and aggregation properties of a novel enzymatically resistant nucleoamino acid.

In this work, we describe the synthesis, evaluation of some biological properties, such as DNA- and RNA-binding ability and in sero stability, as well as the supramolecular assembly of a novel nucleoamino acid based on L-spinacine. More particularly, a thymine-containing L-spinacine derivative was synthesized in liquid phase by a simple peptide-coupling procedure. Subsequently, nucleic acid and Cu(2+)-binding ability, as well as self-assembly properties of the novel nucleoamino acid, were investigated by spectroscopy (CD and UV) and laser light scattering which furnished interesting information on the assembly of supramolecular networks based on the peptidyl nucleoside analog. Finally, nucleoamino acid enzymatic stability was studied and a half life of about 7 days was found in the presence of fresh human serum.

Critical lysine residues within the overlooked N-terminal domain of human APE1 regulate its biological functions.

Apurinic/apyrimidinic endonuclease 1 (APE1), an essential protein in mammals, is involved in base excision DNA repair (BER) and in regulation of gene expression, acting as a redox co-activator of several transcription factors. Recent findings highlight a novel role for APE1 in RNA metabolism, which is modulated by nucleophosmin (NPM1). The results reported in this article show that five lysine residues (K24, K25, K27, K31 and K32), located in the APE1 N-terminal unstructured domain, are involved in the interaction of APE1 with both RNA and NPM1, thus supporting a competitive binding mechanism. Data from kinetic experiments demonstrate that the APE1 N-terminal domain also serves as a device for fine regulation of protein catalytic activity on abasic DNA. Interestingly, some of these critical lysine residues undergo acetylation in vivo. These results suggest that protein-protein interactions and/or post-translational modifications involving APE1 N-terminal domain may play important in vivo roles, in better coordinating and fine-tuning protein BER activity and function on RNA metabolism.

Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX.

Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the alpha-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical alpha-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the three-dimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO(2) hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated alpha-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.

The presence of a single N-terminal histidine residue enhances the fusogenic properties of a Membranotropic peptide derived from herpes simplex virus type 1 glycoprotein H.

Herpes simplex virus type 1 (HSV-1)-induced membrane fusion remains one of the most elusive mechanisms to be deciphered in viral entry. The structure resolution of glycoprotein gB has revealed the presence of fusogenic domains in this protein and pointed out the key role of gB in the entry mechanism of HSV-1. A second putative fusogenic glycoprotein is represented by the heterodimer comprising the membrane-anchored glycoprotein H (gH) and the small secreted glycoprotein L, which remains on the viral envelope in virtue of its non-covalent interaction with gH. Different domains scattered on the ectodomain of HSV-1 gH have been demonstrated to display membranotropic characteristics. The segment from amino acid 626 to 644 represents the most fusogenic region identified by studies with synthetic peptides and model membranes. Herein we have identified the minimal fusogenic sequence present on gH. An enlongation at the N terminus of a single histidine (His) has proved to profoundly increase the fusogenic activity of the original sequence. Nuclear magnetic resonance (NMR) studies have shown that the addition of the N-terminal His contributes to the formation and stabilization of an alpha-helical domain with high fusion propensity.

Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process.

The entry of enveloped viruses involves attachment followed by close apposition of the viral and plasma membranes. Then, either on the cell surface or in an endocytotic vesicle, the two membranes fuse by an energetically unfavourable process requiring the destabilisation of membrane microenvironment in order to release the viral nucleocapsid into the cytoplasm. The core fusion machinery, conserved throughout the herpesvirus family, involves glycoprotein B (gB) and the non-covalently associated complex of glycoproteins H and L (gH/gL). Both gB and gH possess several hydrophobic domains necessary for efficient induction of fusion, and synthetic peptides corresponding to these regions are able to associate to membranes and induce fusion of artificial liposomes. Here, we describe the first application of surface plasmon resonance (SPR) to the study of the interaction of viral membranotropic peptides with model membranes in order to enhance our molecular understanding of the mechanism of membrane fusion. SPR spectroscopy data are supported by tryptophan fluorescence, circular dichroism and electron spin resonance spectroscopy (ESR). We selected peptides from gB and gH and also analysed the behaviour of HIV gp41 fusion peptide and the cationic antimicrobial peptide melittin. The combined results of SPR and ESR showed a marked difference between the mode of action of the HSV peptides and the HIV fusion peptide compared to melittin, suggesting that viral-derived membrane interacting peptides all act via a similar mechanism, which is substantially different from that of the non-cell selective lytic peptide melittin.