Fat3 acts through independent cytoskeletal effectors to coordinate asymmetric cell behaviors during polarized circuit assembly.

The polarized flow of information through neural circuits depends on the orderly arrangement of neurons, their processes, and their synapses. This polarity emerges sequentially in development, starting with the directed migration of neuronal precursors, which subsequently elaborate neurites that form synapses in specific locations. In other organs, Fat cadherins sense the position and then polarize individual cells by inducing localized changes in the cytoskeleton that are coordinated across the tissue. Here, we show that the Fat-related protein Fat3 plays an analogous role during the assembly of polarized circuits in the murine retina. We find that the Fat3 intracellular domain (ICD) binds to cytoskeletal regulators and synaptic proteins, with discrete motifs required for amacrine cell migration and neurite retraction. Moreover, upon ICD deletion, extra neurites form but do not make ectopic synapses, suggesting that Fat3 independently regulates synapse localization. Thus, Fat3 serves as a molecular node to coordinate asymmetric cell behaviors across development.

Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations.

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.

Unraveling the structural and molecular properties of 34-residue levans with various branching degrees by replica exchange molecular dynamics simulations.

Levan has various potential applications in the pharmaceutical and food industries, such as cholesterol-lowering agents and prebiotics, due to its beneficial properties, which depend on its length and branching degree. A previous study also found that the branching degree of levan affected anti-tumor activities against SNU-1 and HepG2 tumor cell lines. Despite its promising potential, the properties of levans with different branching degrees are not well understood at the molecular level. In two models of the generalized Born implicit solvent (GBHCT and GBOBC1), we employed replica-exchange molecular dynamics simulations to explore conformational spaces of 34-residue levans (L34) with branching degrees of zero (LFO34B0), one (LFO34B1), three (LFO34B3) and five (LFO34B5), as well as to elucidate their structural and molecular properties. To ensure a fair comparison of the effects of branching degree on these properties, we focused on analyzing the properties of the central 21-residue of the main chains of all systems. Our results show that all major representative conformations tend to form helix-like structures with kinks, where two-kink helix-like structures have the highest population. As branching degree increases, the population of helix-like structures with zero or one kink tends to increase slightly. As the number of kinks in the structures with the same branching degree increases, the average values of the lengths and angles among centers of masses of three consecutive turns of residue i, i+3, and i+6 tended to decrease. Due to its highest occurring frequencies, the O6 (i)-H3O (i+1) hydrogen bond could be important for helix-like structure formation. Moreover, hydrogen bonds forming among the branching residue (br), branching position (bp) and other residues of L34B1, L34B3 and L34B5 were identified. The O1(bp)-H3O(br), O1(br)-H3O(br) and O5(br)-H1O(br) hydrogen bonds were found in the first-, second- and third-highest occurrence frequencies, respectively. Our study provides novel and important insights into conformational spaces and the structural and molecular properties of 34-residue levans with various branching degrees, which tend to form helix-like structures with kinks.

Effects of amphipathic profile regularization on structural order and interaction with membrane models of two highly cationic branched peptides with ß-sheet propensity.

Antimicrobial peptides attracted increasing interest in last decades due to the rising concern of multi-drug resistant pathogens. Dendrimeric peptides are branched molecules with multiple copies of one peptide functional unit bound to the central core. Compared to linear analogues, they usually show improved activity and lower susceptibility to proteases. Knowledge of structure-function relationship is fundamental to tailor their properties. This work is focused on SB056, the smallest example of dendrimeric peptide, whose amino acid sequence is WKKIRVRLSA. Two copies are bound to the α- and ε- nitrogen of one lysine core. An 8-aminooctanamide was added at the C-terminus to improve membrane affinity. Its propensity for ß-type structures is also interesting, since helical peptides were already thoroughly studied. Moreover, SB056 maintains activity at physiological osmolarity, a typical limitation of natural peptides. An optimized analogue with improved performance was designed, ß-SB056, which differs only in the relative position of the first two residues (KWKIRVRLSA). This produced remarkable differences. Structure order and aggregation behavior were characterized by using complementary techniques and membrane models with different negative charge. Infrared spectroscopy showed different propensity for ordered ß-sheets. Lipid monolayers' surface pressure was measured to estimate the area/peptide and the ability to perturb lipid packing. Fluorescence spectroscopy was applied to compare peptide insertion into the lipid bilayer. Such small change in primary structure produced fundamental differences in their aggregation behavior. A regular amphipathic peptide's primary structure was responsible for ordered ß-sheets in a charge independent fashion, in contrast to unordered aggregates formed by the former analogue.

To4, the first Tityus obscurus ß-toxin fully electrophysiologically characterized on human sodium channel isoforms.

Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or ß-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked ß-type effect on different sodium channel subtypes (hNav1.1-hNav1.7) at low concentrations, and has more pronounced activity on hNav1.1, hNav1.2 and hNav1.4. By comparing To4 primary structure with other Tityus ß-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms.

Elucidating a chemical defense mechanism of Antarctic sponges: A computational study.

In 2000, a novel secondary metabolite (erebusinone, Ereb) was isolated from the Antarctic sea sponge, Isodictya erinacea. The bioactivity of Ereb was investigated, and it was found to inhibit molting when fed to the arthropod species Orchomene plebs. Xanthurenic acid (XA) is a known endogenous molt regulator present in arthropods. Experimental studies have confirmed that XA inhibits molting by binding to either (or both) of two P450 enzymes (CYP315a1 or CYP314a1) that are responsible for the final two hydroxylations in the production of the molt-inducing hormone, 20-hydroxyecdysone (20E). The lack of crystal structures and biochemical assays for CYP315a1 or CYP314a1, has prevented further experimental exploration of XA and Ereb's molt inhibition mechanisms. Herein, a wide array of computational techniques - homology modeling, molecular dynamics simulations, binding site bioinformatics, flexible receptor-flexible ligand docking, and molecular mechanics-generalized Born surface area calculations - have been employed to elucidate the structure-function relationships between the aforementioned P450s and the two described small molecule inhibitors (Ereb and XA). Results indicate that Ereb likely targets CYP315a1 by interacting with a network of aromatic residues in the binding site, while XA may inhibit both CYP315a1 and CYP314a1 because of its aromatic, as well as charged nature.

A novel bispecific peptide HIV-1 fusion inhibitor targeting the N-terminal heptad repeat and fusion peptide domains in gp41.

HIV-1 fusion with the target cell is initiated by the insertion of the gp41 fusion peptide (FP) into the target cell membrane and the interaction between the gp41 N- and C-terminal heptad repeats (NHR and CHR), followed by the formation of the six-helix bundle (6-HB) fusion core. Therefore, both FP and NHR are important targets for HIV-1 fusion inhibitors. Here, we designed and synthesized a dual-target peptidic HIV-1 fusion inhibitor, 4HR-LBD-VIRIP, in which 4HR-LBD is able to bind to the gp41 NHR domain, while VIRIP is able to interact with gp41 FP. We found that 4HR-LBD-VIRIP is about tenfold more potent than 4HR-LBD and VIRIP in inhibiting HIV-1IIIB infection and HIV-1 envelope glycoprotein (Env)-mediated cell-cell fusion, suggesting that this dual-target HIV-1 fusion inhibitor possesses a strong synergistic antiviral effect. A biophysical analysis indicates that 4HR-LBD-VIRIP can interact with N70 peptide that contains the gp41 NHR and FP domains and binds with lipid membrane. This study provides a new approach for designing novel viral fusion inhibitors against HIV and other enveloped viruses with class I membrane fusion proteins.

Specificity and mechanism of action of alpha-helical membrane-active peptides interacting with model and biological membranes by single-molecule force spectroscopy.

In this study, to systematically investigate the targeting specificity of membrane-active peptides on different types of cell membranes, we evaluated the effects of peptides on different large unilamellar vesicles mimicking prokaryotic, normal eukaryotic, and cancer cell membranes by single-molecule force spectroscopy and spectrum technology. We revealed that cationic membrane-active peptides can exclusively target negatively charged prokaryotic and cancer cell model membranes rather than normal eukaryotic cell model membranes. Using Acholeplasma laidlawii, 3T3-L1, and HeLa cells to represent prokaryotic cells, normal eukaryotic cells, and cancer cells in atomic force microscopy experiments, respectively, we further studied that the single-molecule targeting interaction between peptides and biological membranes. Antimicrobial and anticancer activities of peptides exhibited strong correlations with the interaction probability determined by single-molecule force spectroscopy, which illustrates strong correlations of peptide biological activities and peptide hydrophobicity and charge. Peptide specificity significantly depends on the lipid compositions of different cell membranes, which validates the de novo design of peptide therapeutics against bacteria and cancers.

Probing interactions of Vpu from HIV-1 with amiloride-based compounds.

Viral ion channels or viroporins are short membrane proteins that participate in wide-ranging functions including virus replication and entry, assembly, and virus release. One such viroporin is the 81 amino acid residue Vpu protein derived from HIV-1. This protein consists of one transmembrane (TM) and two cytoplasmic helical domains, the former of which oligomerises to form cation-selective ion channels. In this study, we investigate the binding properties of amiloride compounds to Vpu embedded into liposomes using surface plasmon resonance (SPR). We explore the Vpu ion channel inhibitor, hexamethylene amiloride (HMA), as a molecular tool to examine the potential interactive role of key TM residues, Trp23, Ser24, and Glu29, in terms of positioning of these residues on the channel pore and the orientation of its constituent helices. The study provides experimental support that a direct interaction between Ser24 and HMA occurs and that this residue is most likely located in the channel pore. Mutation of Trp23 does not impact HMA affinity suggesting no direct involvement in binding and that this residue is lipid facing. These findings indicate that small molecules such as amilorides are capable of specifically interacting with Vpu ion channels. Although a correlation between ion channel and functional activity cannot be dismissed, alternative mechanisms involving protein-protein interactions may play an important role in the efficacy of these compounds.

Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach.

AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE substrates have been identified by immunoprecipitation and mass spectrometry approaches, and the full range of targets is yet to be determined in mammalian cells. We describe here the first example of global chemoproteomic screening and substrate validation for HYPE-mediated AMPylation in mammalian cell lysate. Through quantitative mass-spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification, we identified a total of 25 AMPylated proteins, including the previously validated substrate endoplasmic reticulum (ER) chaperone BiP (HSPA5), and also novel substrates involved in pathways of gene expression, ATP biosynthesis, and maintenance of the cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.

Vaccine design via nonnegative lasso-based variable selection.

There are many different strains of malaria parasites, each represented by a unique sequence of amino acids. A desirable vaccine would match the amino acid sequence of the parasite antigen. Because of the three-dimensional structure of protein, not all sites in the amino acid sequence participate in the binding between the vaccine-induced antibody and the parasite antigen. Nor do all sites have equal importance. In this work, we apply a nonnegative lasso-based variable selection to identify the 'important' amino acid sites and evaluate their relative importance. We then define a metric, the functional coverage, to measure the 'effective' matching in the amino acid sequence between the vaccine and the parasite. With the variable selection procedure, development of a vaccine needs only to target the important sites, and the potential effectiveness of a vaccine candidate is reflected by the functional coverage. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Acetaminophen reactive intermediates target hepatic thioredoxin reductase.

Acetaminophen (APAP) is metabolized in the liver to N-acetyl-p-benzoquinone imine (NAPQI), an electrophilic metabolite known to bind liver proteins resulting in hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a cellular antioxidant containing selenocysteine (Sec) in its C-terminal redox center, a highly accessible target for electrophilic modification. In the present study, we determined if NAPQI targets TrxR. Hepatotoxicity induced by APAP treatment of mice (300 mg/kg, i.p.) was associated with a marked inhibition of both cytosolic TrxR1 and mitochondrial TrxR2 activity. Maximal inhibition was detected at 1 and 6 h post-APAP for TrxR1 and TrxR2, respectively. In purified rat liver TrxR1, enzyme inactivation was correlated with the metabolic activation of APAP by cytochrome P450, indicating that enzyme inhibition was due to APAP-reactive metabolites. NAPQI was also found to inhibit TrxR1. NADPH-reduced TrxR1 was significantly more sensitive to NAPQI (IC50 = 0.023 µM) than the oxidized enzyme (IC50 = 1.0 µM) or a human TrxR1 Sec498Cys mutant enzyme (IC50 = 17 µM), indicating that cysteine and selenocysteine residues in the redox motifs of TrxR are critical for enzyme inactivation. This is supported by our findings that alkylation of reduced TrxR with biotin-conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. LC-MS/MS analysis confirmed that NAPQI modified cysteine 59, cysteine 497, and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. In addition to disulfide reduction, TrxR is also known to mediate chemical redox cycling. We found that menadione redox cycling by TrxR was markedly less sensitive to NAPQI than disulfide reduction, suggesting that TrxR mediates these reactions via distinct mechanisms. These data demonstrate that APAP-reactive metabolites target TrxR, suggesting an additional mechanism by which APAP induces oxidative stress and hepatotoxicity.

Disruption of δ-opioid receptor phosphorylation at threonine 161 attenuates morphine tolerance in rats with CFA-induced inflammatory hypersensitivity.

OBJECTIVE: Our previous study identified Threonine 161 (Thr-161), located in the second intracellular loop of the δ-opioid receptor (DOR), as the only consensus phosphorylation site for cyclin-dependent kinase 5 (Cdk5). The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund's adjuvant (CFA)-induced inflammatory pain and morphine tolerance. METHODS: Dorsal root ganglion (DRG) neurons of rats with CFA-induced inflammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 (pThr-161-DOR), and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. RESULTS: Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. Intrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L) increased inflammatory hypersensitivity, and inhibited DOR- but not µ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. CONCLUSION: Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr-161 attenuates morphine tolerance.

Diseño de estrategias para controlar la resistencia a los antimicrobianos utilizando herramientas de bioinformática y modelado molecular / Design of strategies to control antimicrobial resistance using bioinformatics tools and molecular modeling

El éxito del tratamiento de las enfermedades infecciosas se ha visto comprometido en los últimos años debido a la diseminación de genes de resistencia a antibióticos entre las bacterias patógenas. Estos genes de resistencia a antibióticos son transportados por plásmidos, los cuáles son transferidos de una bacteria a otra mediante el proceso de conjugación. La reducción del uso inadecuado de los antibióticos y la búsqueda de inhibidores de la conjugación bacteriana son estrategias que podrían contribuir a la solución de este grave problema de salud pública. Basándose en la primera de esta estrategias, en enero de 2006 se regulo la dispensación de un grupo de antibióticos a fin de controlar su consumo. El análisis realizado en este trabajo seǹala que esta medida ha resultado ineficaz, puesto que el consumo y la resistencia bacteriana total a estos antimicrobianos se incrementó significativamente durante el periodo posterior a su promulgación. La resistencia bacteriana a muchas de las familias de antibióticos estudiadas esta solo parcialmente influenciada por su consumo, destacando la participación de otros factores, como la transferencia de genes de resistencia a antibióticos, en la prevalencia de cepas bacterianas resistentes. La identificación de proteínas del cito-cromo P450 de estructura y ligados conocidos, que tenían una similitud significativa en su secuencia de aminoácidos con la proteína de acoplamiento TRAG de los plásmidos R27 y R478, permitió identificar a los medicamentos diclofenac y ketoprofeno como potenciales inhibidores de la transferencia por conjugación de estos plásmidos. El modelado por homología de TRAG revelo que su dominio de solo hélices alfa podría ser el blanco de estos medicamentos. El ingreso de diclofenac o ketoprofeno a una cavidad en este dominio podría interferir en la interacción con el DNA portador de genes de resistencia a antibióticos que esta siendo transferido mediante el proceso de conjugación.

Prolongation of interferon therapy for recurrent hepatitis C after living donor liver transplantation: analysis of predictive factors of sustained virological response, including amino acid sequence of the core and NS5A regions of hepatitis C virus.

OBJECTIVE: The aim of the present retrospective study was to evaluate the therapeutic efficacy and predictive factors of prolongation of treatment with peginterferon (PEGIFN) combined with ribavirin (RBV) for recurrent hepatitis C after living donor liver transplantation (LDLT). METHODS: Fifty-three patients underwent LDLT due to HCV-related end-stage liver disease. Sixteen patients were removed from the study as a result of early death (n=14), no recurrence of HCV (n=1) and refusal of antiviral therapy (n=1). Therapy is ongoing in another 10 patients. The remaining 27 patients were available to establish the efficacy of IFN therapy. HCV genotype was 1b in 24 patients. All patients with genotype 1b were treated with IFN therapy for at least 48 weeks after HCV RNA levels had become undetectable. Amino acid substitutions in the HCV core region and NS5A region were analyzed by direct sequencing before LDLT. RESULTS: The rate of sustained virological response (SVR) was 37.0% (10/27). SVR rate in patients with genotype 1 was 29.2% (7/24) and 100% (3/3) in patients with genotype 2. Most patients with genotype 1b whose HCV RNA reached undetectable levels achieved SVR (87.5%; 7/8). However, mutation of the HCV core region and number of ISDR mutations were not associated with SVR rate in LDLT in our study. CONCLUSIONS: Prolonged IFN therapy for more than 48 weeks after HCV RNA reached undetectable levels might prevent virological relapse of HCV.

Residue 826 in the calcium-sensing receptor is implicated in the response to calcium and to R-568 calcimimetic compound.

Within the extracellular loops of the seven-transmembrane domain of the calcium-sensing receptor (CaR) there is a region (I819-E837) relevant for calcimimetic activity. As the naturally occurring variant Ala826Thr is within this important region, it may be postulated that this change may influence the CaR response to calcium and R-568. Human embryonic kidney (HEK-293) cells transiently transfected with three different human CaRs (wild-type [A826], variant allele [T826], and artificial mutant [W826]) were used to test the ability of calcium alone or in combination with the calcimimetic R-568 to modulate CaR activity. CaR activation was detected by flow cytometry using a fluorescent probe. Intracellular calcium changes were measured in response to changes in extracellular calcium alone or with different R-568 concentrations. The change of the alanine in the 826 position (A826) for threonine (T826) worsened calcium sensitivity, increasing the EC(50) value from 2.34 +/- 0.48 mM (A826, wild-type) to 2.96 +/- 0.75 mM (T826) (P < 0.05). The T826 receptor reached a similar response with 1 muM R-568 compared with the wild-type receptor. On the contrary, the artificial introduction of a tryptophan in the same position (W826) did not affect calcium sensitivity (EC(50) = 2.64 +/- 0.81 mM) but reduced the ability of the receptor to respond to R-568. The results demonstrate the importance of the 826 residue in the CaR response to calcium and calcimimetics. Since the A826T change was described as a natural variant, the differences in the calcium and calcimimetic responses observed between the alleles could have potential clinical impact.

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal.

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that alpha- and beta-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly alpha-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential beta-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-beta-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.

Structure and function of Plasmodium falciparum malate dehydrogenase: role of critical amino acids in co-substrate binding pocket.

The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms a characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining the co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining the co-substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD, a synthetic analog of NAD), since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in catalytic activity, and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials.

Peptide derived from the lipid binding domain of Group IB human pancreatic phospholipase A(2) possesses antibacterial activity.

Group 1B human pancreatic secretory phospholipase A(2) (hp-sPLA(2)), a digestive enzyme synthesized by pancreatic acinar cells and present in pancreatic juice, do not have antibacterial activity towards Escherichia coli. Our earlier results suggest that the N-terminal first ten amino acid residues of hp-sPLA(2) constitute major portion of the membrane binding domain of full-length enzyme and is responsible for the precise orientation of enzyme on the membrane surface by inserting into the lipid bilayers (Pande et al. (2006) Biochemistry, 45,12436-12447). In this study we report the antibacterial properties of a peptide (AVWQFRKMIK-CONH(2); N10 peptide), which corresponds to the N-terminal first ten amino acid residues of hp-sPLA(2), against E. coli. Full-length hp-sPLA(2), which contains this peptide sequence as N-terminal alpha-helix, did not showed detectable antibacterial activity. Presence of physiological concentration of salt or preincubation of N10 peptide with soluble anionic polymer inhibits the antibacterial activity indicating the importance of electrostatic interaction in binding of peptide to bacterial membrane. Addition of peptide resulted in destabilization of outer as well as inner cytoplasmic membrane of E. coli suggesting bacterial membranes to be the main target of action. N10 peptide exhibits strong synergism with lysozyme and potentiates the antibacterial activity of lysozyme. The peptide was inactive against human erythrocyte. Our result shows for the first time that a peptide fragment of hp-sPLA(2) possesses antibacterial activity towards E. coli and at subinhibitory concentration and can potentiate the antibacterial activity of membrane active enzyme. These observations suggest that N10 peptide may play an important role in the antimicrobial activity of pancreatic juice.

Inhibition of tau fibrillization by oleocanthal via reaction with the amino groups of tau.

Tau is a microtubule-associated protein that promotes microtubule assembly and stability. In Alzheimer's disease and related tauopathies, tau fibrillizes and aggregates into neurofibrillary tangles. Recently, oleocanthal isolated from extra virgin olive oil was found to display non-steroidal anti-inflammatory activity similar to ibuprofen. As our unpublished data indicates an inhibitory effect of oleocanthal on amyloid beta peptide fibrillization, we reasoned that it might inhibit tau fibrillization as well. Herein, we demonstrate that oleocanthal abrogates fibrillization of tau by locking tau into the naturally unfolded state. Using PHF6 consisting of the amino acid residues VQIVYK, a hexapeptide within the third repeat of tau that is essential for fibrillization, we show that oleocanthal forms an adduct with the lysine via initial Schiff base formation. Structure and function studies demonstrate that the two aldehyde groups of oleocanthal are required for the inhibitory activity. These two aldehyde groups show certain specificity when titrated with free lysine and oleocanthal does not significantly affect the normal function of tau. These findings provide a potential scheme for the development of novel therapies for neurodegenerative tauopathies.