Additional Binding Sites Research Articles

Syntrophin binds directly to multiple spectrin-like repeats in dystrophin and mediates binding of nNOS to repeats 16-17.

Mutation of the gene encoding dystrophin leads to Duchenne and Becker muscular dystrophy (DMD and BMD). Currently, dystrophin is thought to function primarily as a structural protein, connecting the muscle cell actin cytoskeleton to the extra-cellular matrix. In addition to this structural role, dystrophin also plays an important role as a scaffold that organizes an array of signaling proteins including sodium, potassium, and calcium channels, kinases, and nitric oxide synthase (nNOS). Many of these signaling proteins are linked to dystrophin via syntrophin, an adapter protein that is known to bind directly to two sites in the carboxyl terminal region of dystrophin. A search of the dystrophin sequence revealed three additional potential syntrophin binding sites (SBSs) within the spectrin-like repeat (SLR) region of dystrophin. Binding assays revealed that the site at SLR 17 bound specifically to the α isoform of syntrophin while the site at SLR 22 bound specifically to the β-syntrophins. The SLR 17 α-SBS contained the core sequence known to be required for nNOS-dystrophin interaction. In vitro and in vivo assays indicate that α-syntrophin facilitates the nNOS-dystrophin interaction at this site rather than nNOS binding directly to dystrophin as previously reported. The identification of multiple SBSs within the SLR region of dystrophin demonstrates that this region functions as a signaling scaffold. The signaling role of the SLR region of dystrophin will need to be considered for effective gene replacement or exon skipping based DMD/BMD therapies.

The Transcriptional Regulator TFB-RF1 Activates Transcription of a Putative ABC Transporter in Pyrococcus furiosus.

Transcription factor B recruiting factor 1 (TFB-RF1; PF1088) is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in Pyrococcus furiosus. The protein is located in an operon structure together with the hypothetical gene pf1089 and western blot as well as end-point RT-PCR experiments revealed an extremely low expression rate of both proteins. Furthermore, conditions to induce the expression of the operon are not known. By introducing an additional copy of tfb-RF1 using a Pyrococcus shuttle vector we could circumvent the lacking expression of both proteins under standard growth conditions as indicated by western blot as well as end-point RT-PCR experiments. A ChIP-seq experiment revealed an additional binding site of TFB-RF1 in the upstream region of the pf1011/1012 operon, beside the expected target of the pf1089/tfb-RF1 region. This operon codes for a putative ABC transporter which is most-related to a multidrug export system and in vitro analysis using gel shift assays, DNase I footprinting and in vitro transcription confirmed the activator function of TFB-RF1 on the corresponding promoter. These findings are also in agreement with in vivo data, as RT-qPCR experiments also indicate transcriptional activation of both operons. Taken together, the overexpression strategy of tfb-RF1 enabled the identification of an additional operon of the TFB-RF1 regulon which indicates a transport-related function and provides a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network in P. furiosus.

Distinct functions for the flexible loops of zinc specific solute binding proteins from Paracoccus denitrificans

In bacteria, ATP binding cassette (ABC) transporters of cluster A‐I exist for the acquisition of the essential trace element zinc from limiting environments. These transporters are crucial for bacterial survival, and the gene knockout strains often demonstrate a pronounced growth defect in minimal media and attenuated virulence in the case of pathogens. Thus such systems are attractive targets for the development of novel antibiotics. Zinc binding with high affinity and specificity is mediated by the solute binding protein (SBP) component of ABC transporters. Zinc‐specific SBPs are characterized by the presence of a flexible loop near zinc binding pocket of the protein. P. denitrificans encodes two zinc ABC transporter operons, znuABC and aztABCD. The SBPs for these systems (AztC and ZnuA) are distinct in the length and sequence of flexible loop regions. The function of the flexible loop in each protein was investigated using fluorescence binding assays and structural methods for WT and loop mutant proteins. The flexible loop of AztC has no impact on zinc binding affinity, stoichiometry or protein structure, yet it is essential for zinc transfer from the metallochaperone AztD. Site specific mutagenesis was employed to evaluate the roles of specific residues in this process and the results showed that three His residues in the loop temporarily coordinate zinc and then convey it to the high‐affinity binding site. Consistent with this, mutation of any of these residues to Ala abrogated zinc transfer from AztD. AztD does not transfer zinc to ZnuA, despite the fact that ZnuA also contains a flexible loop rich in His residues. The ZnuA flexible loop contains 15 His, 11 Glu and 3 Asp residues, providing multiple additional zinc binding sites. In fact, P. denitrificans ZnuA binds up to 6 zinc ions, the most for any zinc SBP studied to date. The results demonstrate distinct roles for zinc SBP flexible loops depending on loop structure and context, potentially allowing for acquisition of zinc from different environments.Support or Funding InformationNational Institute of General Medical Science of the National Institutes of Health under award number 1SC2GM111170‐01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Surface engineering of a chromium metal-organic framework with bifunctional ionic liquids for selective CO2 adsorption: Synergistic effect between multiple active sites

Targeting CO2 capture application, a new strategy for building multiple adsorption sites in metal-organic framework MIL-101(Cr) was constructed through the incorporation of diethylenetriamine-based ionic liquid (DETA-Ac) via a post-synthetic modification approach. The DETA-Ac, with multi-amine-tethered cation and acetate anion, could not only provide additional binding sites, but also enhance the affinity of framework surfaces toward CO2. Simultaneously, the high surface area and large cage size of MIL-101(Cr) ensured the better dispersion of IL, thus exposing more active sites for CO2 adsorption. In addition, enough free space was still retained after functionalization, which facilitated CO2 transport and allowed the Cr(III) sites deep within the pores to be accessed. The multiple adsorption sites originating from IL and MOF were found to synergistically affect the CO2 capture performance of the composite. The adsorption capacity and selectivity of DETA-Ac@MIL-101(Cr) for CO2 were significantly improved. The higher isosteric heats of adsorption (Qst) evidenced the stronger interaction between the composite and CO2 molecules. Moreover, a possible two-step mechanism was proposed to reveal the manner in which CO2 bound to the IL-incorporated frameworks. Despite the relatively high initial Qst value, the DETA-Ac@MIL-101(Cr) could be easily regenerated with almost no drop in CO2 uptake during six cycles.

Insights into structural features of HDAC1 and its selectivity inhibition elucidated by Molecular dynamic simulation and Molecular Docking

Histone deacetylases (HDACs) are a family of proteins whose main function is the removal of acetyl groups from lysine residues located on histone and non-histone substrates, which regulates gene transcription and other activities in cells. HDAC1 dysfunction has been implicated in cancer development and progression; thus, its inhibition has emerged as a new therapeutic strategy. Two additional metal binding sites (Site 1 and Site 2) in HDACs have been described that are primarily occupied by potassium ions, suggesting a possible structural role that affects HDAC activity. In this work, we explored the structural role of potassium ions in Site 1 and Site 2 and how they affect the interactions of compounds with high affinities for HDAC1 (AC1OCG0B, Chlamydocin, Dacinostat and Quisinostat) and SAHA (a pan-inhibitor) using molecular docking and molecular dynamics (MD) simulations in concert with a Molecular-Mechanics-Generalized-Born-Surface-Area (MMGBSA) approach. Four models were generated: one with a potassium ion (K+) in both sites (HDAC1k), a second with K+ only at site 1 (HDAC1ks1), a third with K+ only at site 2 (HDAC1ks2) and a fourth with no K+ (HDAC1wk). We found that the presence or absence of K+ not only impacted the structural flexibility of HDAC1, but also its molecular recognition, consistent with experimental findings. These results could therefore be useful for further structure-based drug design studies addressing new HDAC1 inhibitors.

The enzymatic processing of α-dystroglycan by MMP-2 is controlled by two anchoring sites distinct from the active site.

Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and β-dystroglycan (β -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of β -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483–628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621–628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613–651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613–651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.

Discovery of C-1 modified oseltamivir derivatives as potent influenza neuraminidase inhibitors

Inspired by our initial discovery about a series of neuraminidase (NA) inhibitors targeting the 150-cavity, in present study, we designed, synthesized, and biologically tested a panel of novel oseltamivir derivatives with C-1 modification, targeting the 430-cavity, an additional binding site which widely and stably existed in both group-1 and group-2 NAs. Some of the synthesized compounds displayed robust anti-influenza potencies against H5N1 and H5N6 viruses. Among them, compound 8b exerted the greatest inhibition, with IC50 values of 0.088 and 0.097 μM and EC50 values of 4.26 and 1.31 μM against H5N1 and H5N6 strains, respectively, which are similar to those of oseltamivir carboxylate (OSC). And its potency against mutant H5N1-H274Y NA was just 7-fold weaker than OSC. Molecular modeling revealed the elongated group at C-1 position being projected toward the 430-cavity. Notably, although compound 8b was not sensitive toward H5N1 strain relative to OSC in the embryonated egg model, it displayed greater anti-influenza virus effect against H5N6 strain than OSC at the concentration of 10 mmol/L. Overall, this work provided unique insights in the discovery of potent inhibitors against both group-1 and group-2 NAs.

Designed Enzymes: Creating a more Efficient Nitric Oxide Dioxygenase

We have designed an artificial nitric oxide dioxygenase (NOD) which, when paired with ferredoxin NADPH+ oxidoreductase (FNR) as an electron donor, transforms nitric oxide into nitrate ions at rates comparable to that of natural NOD enzymes. Nitric oxide has been implicated in several neurodegenerative disorders, and unwanted NOD activity has been identified as a major problem in artificial blood substitutes. We have characterized the binding affinities and the kinetics of each step along the reaction pathway and competing off-pathways. While capable of acting as a NOD, HFHF has several deficiencies: poor selectivity in gaseous binding and a slow rate of electron transfer to the porphyrin cofactor from FNR. As we found electron transfer to the porphyrin cofactor to be rate-limiting, we created an improved NOD by covalently linking the artificial hemoglobin directly to an electron donor. Building upon the original design, we combined a natural and a designed protein to create a chimera in which the diflavin domain of cytochrome P-450 BM3 is connected to a variant of our designed enzyme that contains an additional porphyrin binding site intended to relay electrons from the diflavin domain to the site of NOD activity. This single chain protein contains one FMN, one FAD, and two porphyrin cofactors. The chimera reacts with NADPH, taking in its two electrons at the FAD cofactor, breaking them into single electrons at the FMN cofactor, and then transferring them into the artificial porphyrin domain. This construct increases the electron transfer rate to the porphyrin by more than an order of magnitude.

Stabilizing vitamin D3 using the molten globule state of α-lactalbumin

α-Lactalbumin (α-LA) is the second most abundant bovine whey protein. It has been intensively studied because of its readiness to populate the molten globular (MG) state, a partially folded state with native levels of secondary structure but loss of tertiary structure. The MG state of α-LA exposes a significant number of hydrophobic patches that could be used to bind and stabilize small hydrophobic molecules such as vitamin D3 (vitD). Accordingly, we tested the ability of α-LA to stabilize vitD in a pH interval from 7.4 to 2; over this pH interval, α-LA transitions from the folded state to the MG state. The MG state stabilized vitD better than the folded state and was superior to the major bovine whey protein β-lactoglobulin (β-LG), which is known to stabilize vitD. At pH 7.4, β-LG and α-LA stabilized vitD to the same extent. Tryptophan fluorescence quenching measurements indicated that α-LA has one binding site at pH 7.4 but acquires an additional binding site when the pH is lowered to pH 2 to 4. Stability measurements of the vitD in the α-LA-vitD complex at different temperatures suggest that UHT processing would lead to little loss of vitD. This study demonstrates the potential of α-LA as a component in vitD fortification, particularly for low pH applications.

Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibitors

A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of MDM2. To select for the anticipated modifications we employed molecular docking. Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma U2OS cells) by Annexin V staining. The target activity was estimated using high-content imaging system Operetta. Valine and phenylglycine ester derivatives were identified as potentially active MDM2-p53 inhibitors.

BAK1 is involved in AtRALF1-induced inhibition of root cell expansion.

The rapid alkalinization factor (RALF) peptide negatively regulates cell expansion, and an antagonistic relationship has been demonstrated between AtRALF1, a root-specific RALF isoform in Arabidopsis, and brassinosteroids (BRs). An evaluation of the response of BR signaling mutants to AtRALF1 revealed that BRI1-associated receptor kinase1 (bak1) mutants are insensitive to AtRALF1 root growth inhibition activity. BAK1 was essential for the induction of AtRALF1-responsive genes but showed no effect on the mobilization of Ca2+ and alkalinization responses. Homozygous plants accumulating AtRALF1 and lacking the BAK1 gene did not exhibit the characteristic semi-dwarf phenotype of AtRALF1-overexpressors. Biochemical evidence indicates that AtRALF1 and BAK1 physically interact with a Kd of 4.6 μM and acridinium-labeled AtRALF1 was used to demonstrate that part of the specific binding of AtRALF1 to intact seedlings and to a microsomal fraction derived from the roots of Arabidopsis plants is BAK1-dependent. Moreover, AtRALF1 induces an increase in BAK1 phosphorylation, suggesting that the binding of AtRALF1 to BAK1 is functional.These findings show that BAK1 contains an additional AtRALF1 binding site, indicating that this protein may be part of a AtRALF1-containing complex as a co-receptor, and it is required for the negative regulation of cell expansion.

PtII, PdII and AuIII complexes with a thiosemicarbazone derived from diacethylmonooxime: Structural analysis, trypanocidal activity, cytotoxicity and first insight into the antiparasitic mechanism of action

New complexes of composition [MX(HL1)] (M = PtII, PdII, X = Cl− or I−) and [MX(L1)] (M = AuIII, X = Cl−; M = PtII, PdII, X = PPh3) have been synthesized using a potentially tridentate thiosemicarbazone (H2L1) containing an additional oxime binding site. Among other analytical methods, all the seven complexes have been structurally characterized by single crystal X-ray diffractometry. Interesting structural features such as the influence of the halide ligands on hydrogen bonds and the formation of supramolecular structures for the phosphine derivatives are discussed. The in vitro trypanocidal activity of the free ligand H2L1 and its derivatives against both extracellular trypomastigote and intracellular amastigote (IC50try/ama) forms of Trypanosoma cruzi (Tulahuen Lac-Z strain) and the cytotoxicity was assessed on LLC-MK2 cell line. The results showed that complexation of the thiosemicarbazone ligand H2L1 to PtII, PdII and AuIII metal centers enhances the in vitro trypanocidal activity and that the cytotoxicity is dependent on both the metal center and coligands. Within the studied series, the AuIII complex showed the greatest potential, being not the most active but the most selective compound with a similar selectivity index to that of the standard drug benznidazole. In order to get a preliminary insight into the mechanism of action of these compounds, in vitro experiments of fluorescence quenching and enzymatic activity were performed using the AuIII complex and Trypanosoma cruzi Old Yellow Enzyme (TcOYE) which indicated that the gold derivative was capable of abstracting the hydride from the prosthetic FMN group of the enzyme. Additionally, molecular docking studies followed by semiempirical simulations showed that the [AuCl(L1)] binds to the binary complex TcOYE/FMN, almost parallel to the FMN prosthetic group, in a close distance that an electron/proton transfer might occur among them.

Genetic polymorphisms and haplotypes of the DJ-1 gene promoter associated with the susceptibility to male infertility.

In this study, we evaluate the relationship between genetic polymorphisms of the DJ-1 gene, g.-6_+10del, and g.168_185del with male infertility susceptibility. Four hundred and twenty-two male infertile patients and 285 fertile male controls were recruited. Genotyping was performed by polymerase chain reaction. In silico analysis was performed by EPD, ElemeNT, SNPnexus, and PROMO to predict the potential functions of rs901561484 and rs373653682 polymorphisms. The Del (D) allele carriers of DJ-1 g.-6_+10del polymorphism were significantly associated with the risk of male infertility in total infertile, asthenozoospermia, and oligoasthenozoospermia patients. Moreover, the Del (D) allele of DJ-1 g.-6_+10del polymorphism significantly increased in total male infertile, asthenozoospermia, and oligoasthenozoospermia groups. In addition, the frequencies of different genotypes and the Del allele and Dup allele carriers of DJ-1 g.168_185del gene polymorphisms were associated with male infertility in total infertile and four different sub-group patients. Furthermore, haplotype analysis of DJ-1 g.-6_+10del and g.168_185del polymorphisms revealed that the D-Dup and I-Del haplotype frequencies significantly increased the risk of male infertility, while I-Ins haplotypes were associated with a decreased risk of male infertility in total and sub-group patients. The in silico analysis showed that the presence of Ins and/or Dup alleles of the DJ-1 g.-6_+10del and g.168_185del polymorphisms could provide additional binding sites of more nuclear factors and probably affect transcriptional activity. Our study presents evidence of a strong association between functional polymorphisms of the DJ-1 promoter, g.-6_+10del, and g.168_185del with the risk of male infertility.

PnpM, a LysR-Type Transcriptional Regulator Activates the Hydroquinone Pathway in para-Nitrophenol Degradation in Pseudomonas sp. Strain WBC-3.

A LysR-type transcriptional regulator (LTTR), PnpR, has previously been shown to activate the transcription of operons pnpA, pnpB, and pnpCDEFG for para-nitrophenol (PNP) degradation in Pseudomonas sp. strain WBC-3. Further preliminary evidence suggested the possible presence of an LTTR additional binding site in the promoter region of pnpCDEFG. In this study, an additional LTTR PnpM, which shows 44% homology to PnpR, was determined to activate the expression of pnpCDEFG. Interestingly, a pnpM-deleted WBC-3 strain was unable to grow on PNP but accumulating hydroquinone (HQ), which is the catabolic product from PNP degradation by PnpAB and the substrate for PnpCD. Through electrophoretic mobility shift assays (EMSAs) and promoter activity detection, only PnpR was involved in the activation of pnpA and pnpB, but both PnpR and PnpM were involved in the activation of pnpCDEFG. DNase I footprinting analysis suggested that PnpR and PnpM shared the same DNA-binding regions of 27 bp in the pnpCDEFG promoter. In the presence of PNP, the protection region increased to 39 bp by PnpR and to 38 bp by PnpM. Our data suggested that both PnpR and PnpM were involved in activating pnpCDEFG expression, in which PNP rather than the substrate hydroquinone for PnpCD is the inducer. Thus, during the PNP catabolism in Pseudomonas sp. strain WBC-3, pnpA and pnpB operons for the initial two reactions were controlled by PnpR, while the third operon (pnpCDEFG) for HQ degradation was activated by PnpM and PnpR. This study builds upon our previous findings and shows that two LTTRs PnpR and PnpM are involved in the transcriptional activation of these three catabolic operons. Specifically, our identification that an LTTR, PnpM, regulates pnpCDEFG expression provides new insights in an intriguing regulation system of PNP catabolism that is controlled by two regulators.

Characterization of Protein-Protein Interfaces in Large Complexes by Solid-State NMR Solvent Paramagnetic Relaxation Enhancements.

Solid-state NMR is becoming a viable alternative for obtaining information about structures and dynamics of large biomolecular complexes, including ones that are not accessible to other high-resolution biophysical techniques. In this context, methods for probing protein–protein interfaces at atomic resolution are highly desirable. Solvent paramagnetic relaxation enhancements (sPREs) proved to be a powerful method for probing protein–protein interfaces in large complexes in solution but have not been employed toward this goal in the solid state. We demonstrate that 1H and 15N relaxation-based sPREs provide a powerful tool for characterizing intermolecular interactions in large assemblies in the solid state. We present approaches for measuring sPREs in practically the entire range of magic angle spinning frequencies used for biomolecular studies and discuss their benefits and limitations. We validate the approach on crystalline GB1, with our experimental results in good agreement with theoretical predictions. Finally, we use sPREs to characterize protein–protein interfaces in the GB1 complex with immunoglobulin G (IgG). Our results suggest the potential existence of an additional binding site and provide new insights into GB1:IgG complex structure that amend and revise the current model available from studies with IgG fragments. We demonstrate sPREs as a practical, widely applicable, robust, and very sensitive technique for determining intermolecular interaction interfaces in large biomolecular complexes in the solid state.

Facilitating Proton Transport in Nafion-Based Membranes at Low Humidity by Incorporating Multifunctional Graphene Oxide Nanosheets.

Nafion, as a state-of-the-art solid electrolyte for proton exchange membrane fuel cells (PEMFCs), suffers from drastic decline in proton conductivity with decreasing humidity, which significantly restricts the efficient and stable operation of the fuel cell system. In this study, the proton conductivity of Nafion at low relative humidity (RH) was remarkably enhanced by incorporating multifunctional graphene oxide (GO) nanosheets as multifunctional fillers. Through surface-initiated atom transfer radical polymerization of sulfopropyl methacrylate (SPM) and poly(ethylene glycol) methyl ether methacrylate, the copolymer-grafted GO was synthesized and incorporated into the Nafion matrix, generating efficient paths at the Nafion-GO interface for proton conduction. The Lewis basic oxygen atoms of ethylene oxide (EO) units and sulfonated acid groups of SPM monomers served as additional proton binding and release sites to facilitate the proton hopping through the membrane. Meanwhile, the hygroscopic EO units enhanced the water retention property of the composite membrane, conferring a dramatic increase in proton conductivity under low humidity. With 1 wt % filler loading, the composite membrane displayed the highest proton conductivity of 2.98 × 10-2 S cm-1 at 80 °C and 40% RH, which was 10 times higher than that of recast Nafion. Meanwhile, the Nafion composite exhibited a 135.5% increase in peak power density at 60 °C and 50% RH, indicating its great application potential in PEMFCs.

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a “non-specific” fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

Biochemical Characterization of WbkC, an N-Formyltransferase from Brucella melitensis.

It has become increasingly apparent within the last several years that unusual N-formylated sugars are often found on the O-antigens of such Gram negative pathogenic organisms as Francisella tularensis, Campylobacter jejuni, and Providencia alcalifaciens, among others. Indeed, in some species of Brucella, for example, the O-antigen contains 1,2-linked 4-formamido-4,6-dideoxy-α-d-mannosyl groups. These sugars, often referred to as N-formylperosamine, are synthesized in pathways initiating with GDP-mannose. One of the enzymes required for the production of N-formylperosamine, namely, WbkC, was first identified in 2000 and was suggested to function as an N-formyltransferase. Its biochemical activity was never experimentally verified, however. Here we describe a combined structural and functional investigation of WbkC from Brucella melitensis. Four high resolution X-ray structures of WbkC were determined in various complexes to address its active site architecture. Unexpectedly, the quaternary structure of WbkC was shown to be different from that previously observed for other sugar N-formyltransferases. Additionally, the structures revealed a second binding site for a GDP molecule distinct from that required for GDP-perosamine positioning. In keeping with this additional binding site, kinetic data with the wild type enzyme revealed complex patterns. Removal of GDP binding by mutating Phe 142 to an alanine residue resulted in an enzyme variant displaying normal Michaelis-Menten kinetics. These data suggest that this nucleotide binding pocket plays a role in enzyme regulation. Finally, by using an alternative substrate, we demonstrate that WbkC can be utilized to produce a trideoxysugar not found in nature.

Estimation of the optimal dosing regimen of escitalopram in dogs: A dose occupancy study with [11C]DASB.

Although the favourable characteristics of escitalopram as being the most selective serotonin reuptake inhibitor and having an increased therapeutic efficacy via binding on an additional allosteric binding site of the serotonin transporter, its dosing regimen has not yet been optimized for its use in dogs. This study aimed to estimate the optimal dosing frequency and the required dose for achieving 80% occupancy of the serotonin transporters in the basal ganglia. The dosing frequency was investigated by determining the elimination half-life after a four day oral pre-treatment period with 0.83 mg/kg escitalopram (3 administrations/day) and a subsequent i.v. injection 0.83 mg/kg. Blood samples were taken up to 12 hours after i.v. injection and the concentration of escitalopram in plasma was analysed via LC-MSMS. The dose-occupancy relationship was then determined by performing two PET scans in five adult beagles: a baseline PET scan and a second scan after steady state conditions were achieved following oral treatment with a specific dose of escitalopram ranging from 0.5 to 2.5 mg/kg/day. As the elimination half-life was determined to be 6.7 hours a dosing frequency of three administrations a day was proposed for the second part of the study. Further it was opted for a treatment period of four days, which well exceeded the minimum period to achieve steady state conditions. The optimal dosing regimen to achieve 80% occupancy in the basal ganglia and elicit a therapeutic effect, was calculated to be 1.85 mg/kg/day, divided over three administrations. Under several circumstances, such as insufficient response to other SSRIs, concurrent drug intake or in research studies focused on SERT, the use of escitalopram can be preferred over the use of the already for veterinary use registered fluoxetine, however, in case of long-term treatment with escitalopram, regularly cardiac screening is recommended.

Identification of iron-chelating peptides from Pacific cod skin gelatin and the possible binding mode

Pacific cod skin gelatin was hydrolyzed under optimized conditions (trypsin, 240min) to generate iron-chelating peptides. Gelatin tryptic hydrolysates were purified by immobilized metal affinity chromatography and reversed phase high performance liquid chromatography. Three novel iron-chelating peptides identified by LC-HRMS/MS were GPAGPHGPPGKDGR, AGPHGPPGKDGR and AGPAGPAGAR, which exhibited high affinity to ferrous ions. The iron-peptide complexation, investigated by ESI-MS and FTIR spectroscopy, showed that the three peptides bound with iron mainly at the ratio of 1:1. Among the groups of the three peptides, the amino and carboxylate terminal groups and peptide bond from peptide backbone, as well as the amino and imine from arginine side chain were involved in the complexation. Moreover, several amino acid side chain groups of GPAGPHGPPGKDGR and AGPHGPPGKDGR, including amino (Lys), imine (His) and carboxylate (Asp), supplied additional iron binding sites. This study suggests a potential application of gelatin-derived peptides as novel carriers to combat iron deficiency.