BBA - General Subjects (v.1860, #3)

Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis by Linda Celeste Montemiglio; Giacomo Parisi; Antonella Scaglione; Giuliano Sciara; Carmelinda Savino; Beatrice Vallone (465-475).
OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism.The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent.Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates.Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP.Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.Display Omitted
Keywords: Cyp107d1; P450; Epoxigenases; Oleandomycin; Clotrimazole and Ketoconazole; Crystal structure;

The North American wood frog, Rana sylvatica, endures whole body freezing while wintering on land and has developed multiple biochemical adaptations to elude cell/tissue damage and optimize its freeze tolerance. Blood flow is halted in the frozen state, imparting both ischemic and oxidative stress on cells. A potential build-up of H2O2 may occur due to increased superoxide dismutase activity previously discovered. The effect of freezing on catalase (CAT), which catalyzes the breakdown of H2O2 into molecular oxygen and water, was investigated as a result.The present study investigated the purification and kinetic profile of CAT in relation to the phosphorylation state of CAT from the skeletal muscle of control and frozen R. sylvatica.Catalase from skeletal muscle of frozen wood frogs showed a significantly higher Vmax (1.48 fold) and significantly lower Km for H2O2 (0.64 fold) in comparison to CAT from control frogs (5 °C acclimated). CAT from frozen frogs also showed higher overall phosphorylation (1.73 fold) and significantly higher levels of phosphoserine (1.60 fold) and phosphotyrosine (1.27 fold) compared to control animals. Phosphorylation via protein kinase A or the AMP-activated protein kinase significantly decreased the Km for H2O2 of CAT, whereas protein phosphatase 2B or 2C action significantly increased the Km.The physiological consequence of freeze-induced CAT phosphorylation appears to improve CAT function to alleviate H2O2 build-up in freezing frogs.Augmented CAT activity via reversible phosphorylation may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.
Keywords: Rana sylvatica; Freeze tolerance; Ischemia; Oxidative stress; Antioxidant; Reversible protein phosphorylation;

Towards a characterization of the structural determinants of specificity in the macrocyclizing thioesterase for deoxyerythronolide B biosynthesis by Panos Argyropoulos; Fabien Bergeret; Christophe Pardin; Janice M. Reimer; Atahualpa Pinto; Christopher N. Boddy; T. Martin Schmeing (486-497).
Type I polyketide synthases (PKSs) are giant multidomain proteins that synthesize many therapeutics and other natural products. The synthesis proceeds by a thiotemplate mechanism whereby intermediates are covalently attached to the PKS. The release of the final polyketide is catalyzed by the terminal thioesterase (TE) domain through hydrolysis, transesterification, or macrocyclization. The PKS 6-deoxyerythronolide B synthase (DEBS) produces the 14-membered macrolide core of the clinically important antibiotic erythromycin. The TE domain of DEBS (DEBS TE) has well-established, empirically-defined specificities for hydrolysis or macrocyclization of native and modified substrates. We present efforts towards understanding the structural basis for the specificity of the thioesterase reaction in DEBS TE using a set of novel diphenyl alkylphosphonates, which mimic substrates that are specifically cyclized or hydrolyzed by DEBS TE. We have determined structures of a new construct of DEBS TE alone at 1.7 Å, and DEBS TE bound with a simple allylphosphonate at 2.1 Å resolution. Other, more complex diphenyl alkylphosphonates inhibit DEBS TE, but we were unable to visualize these faithful cyclization analogs in complex with DEBS TE. This work represents a first step towards using DEBS TE complexed with sophisticated substrate analogs to decipher the specificity determinants in this important reaction.Display Omitted
Keywords: Thioesterase; Polyketide synthase; Non-hydrolyzable acyl-enzyme intermediates; Inhibition; High-resolution structure;

The hormone-like polypeptide, fibroblast growth factor 21 (FGF21), is a major modulator of lipid and glucose metabolism and an exploratory treatment strategy for obesity related metabolic disorders. The costs of recombinant FGF21 and mode of delivery by injection are important constraints to its wide therapeutic use. The stimulation of endogenous FGF21 production through diet is being explored as an alternative approach. To that end, we examined the mechanism(s) by which serum manipulation and lipoic acid (a dietary activator of FGF21) induce FGF21 in human hepatocellular carcinoma HepG2 cells. Serum withdrawal markedly induced FGF21 mRNA levels (88 fold) and FGF21 secreted in the media (19 fold). Lipoic acid induced FGF21 mRNA 7 fold above DMSO-treated control cells and FGF21 secretion 3 fold. These effects were several-fold greater than those of PPARα agonist, Wy14643, which failed to induce FGF21 above and beyond the induction seen with serum withdrawal. The use of transcription inhibitor, actinomycin D, revealed that de novo mRNA synthesis drives FGF21 secretion in response to serum starvation. Four previously unrecognized loci in FGF21 promoter were nucleosome depleted and enriched in acetylated histone H3 revealing their role as transcriptional enhancers and putative transcription factor binding sites. FGF21 did not accumulate to a significant degree in induced HepG2 cells, which secreted FGF21 time dependently in media. We conclude that lipoic acid cell signaling connects with the transcriptional upregulation of FGF21 and it may prove to be a safe and affordable means to stimulate FGF21 production.
Keywords: FAIRE assay; Nucleosome; Histone H3; Dihydrolipoic acid; PPARα; Fucosyltransferase 1;

CREB-binding protein (CBP) is a promiscuous transcriptional co-regulator. In insects, CBP has been studied in the fly Drosophila melanogaster, where it is known as Nejire. Studies in D. melanogaster have revealed that Nejire is involved in the regulation of many pathways during embryo development, especially in anterior/posterior polarity, through Hedgehog and Wingless signaling, and in dorsal/ventral patterning, through TGF-ß signaling. Regarding post-embryonic development, Nejire influences histone acetyl transferase activity on the ecdysone signaling pathway.Functional genomics studies using RNAi have shown that CBP contributes to the regulation of feeding and ecdysis during the pre-metamorphic nymphal instar of the cockroach Blattella germanica and is involved in TGF-ß, ecdysone, and MEKRE93 pathways, contributing to the activation of Kr-h1 and E93 expression. In D. melanogaster, Nejire's involvement in the ecdysone pathway in pre-metamorphic stages is conserved, whereas the TGF-ß pathway has only been described in the embryo. CBP role in ecdysis pathway and in the activation of Kr-h1 and E93 expression is described here for the first time.Studies in D. melanogaster may have been suggestive that CBP functions in insects are concentrated in the embryo. Results obtained in B. germanica indicate, however, that CBP have diverse and important functions in post-embryonic development and metamorphosis, especially regarding endocrine signaling.Further research into a higher diversity of models will probably reveal that the multiple post-embryonic roles of CBP observed in B. germanica are general in insects.
Keywords: CBP; Metamorphosis; TGF-beta pathway; Ecdysone; MEKRE93; Juvenile hormone; Kr-h1; E93;

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions by Milica M. Grozdanovic; Milena Čavić; Andrijana Nešić; Uroš Andjelković; Peyman Akbari; Joost J. Smit; Marija Gavrović-Jankulović (516-526).
The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease — actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs).Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry.Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 μg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 μg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin.Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice.In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.Display Omitted
Keywords: Actinidin; Cysteine protease; Intestinal permeability; Occludin; Tight junctions;

Sequential protein expression and selective labeling for in-cell NMR in human cells by Enrico Luchinat; Erica Secci; Francesca Cencetti; Paola Bruni (527-533).
In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein–protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells.We established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR.SOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by 1H–15N heteronuclear NMR.We showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein–protein interactions by NMR in human cells.Display Omitted
Keywords: In-cell NMR; Nuclear magnetic resonance; Protein–protein interactions; Isotope labeling; Mammalian cells;

Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA, the soluble component of the ZnuABC transporter by Andrea Ilari; Luca Pescatori; Roberto Di Santo; Andrea Battistoni; Serena Ammendola; Mattia Falconi; Francesca Berlutti; Piera Valenti; Emilia Chiancone (534-541).
Under conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake.Antibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15 Å resolution.Two di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound.Two molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)-binding drugs against Salmonella.The data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria.Display Omitted
Keywords: ZnuA; ZnuABC zinc transporter; Salmonella enterica; X-ray structure; Lead compounds;

A novel fibrinolytic metalloproteinase, barnettlysin-I from Bothrops barnetti (barnett´s pitviper) snake venom with anti-platelet properties by Eladio Flores Sanchez; Michael Richardson; Luiza Helena Gremski; Silvio Sanches Veiga; Armando Yarleque; Stephan Niland; Augusto Martins Lima; Maria Inácia Estevao-Costa; Johannes Andreas Eble (542-556).
Viperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential.Bar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function.Bar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC50  = 1.3 and 3.2 μM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2β1-integrin. This explains why it additionally inhibits collagen-induced platelet activation.A non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function.This study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect.Display Omitted
Keywords: Direct acting fibrinolytic enzyme; Metalloproteinase; Antithrombotic; Snake venoms; Integrins; von willebrand factor;

A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes by Vanessa W. Petit; Jean-Luc Rolland; Alain Blond; Chantal Cazevieille; Chakib Djediat; Jean Peduzzi; Christophe Goulard; Evelyne Bachère; Joëlle Dupont; Delphine Destoumieux-Garzón; Sylvie Rebuffat (557-568).
Hemocyanins are respiratory proteins with multiple functions. In diverse crustaceans hemocyanins can release histidine-rich antimicrobial peptides in response to microbial challenge. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of hemocyanins.The three-dimensional structure of the antifungal peptide PvHCt from Litopenaeus vannamei was determined by NMR. Its mechanism of action against the shrimp pathogen Fusarium oxysporum was investigated using immunochemistry, fluorescence and transmission electron microscopy.PvHCt folded into an amphipathic α-helix in membrane-mimicking media and displayed a random conformation in aqueous environment. In contact with F. oxysporum, PvHCt bound massively to the surface of fungal hyphae without being imported into the cytoplasm. At minimal inhibitory concentrations, PvHCt made the fungal membrane permeable to SYTOX-green and fluorescent dextran beads of 4 kDa. Higher size beads could not enter the cytoplasm. Therefore, PvHCt likely creates local damages to the fungal membrane. While the fungal cell wall appeared preserved, gradual degeneration of the cytoplasm most often resulting in cell lysis was observed in fungal spores and hyphae. In the remaining fungal cells, PvHCt induced a protective response by the formation of daughter hyphae.The massive accumulation of PvHCt at the surface of fungal hyphae and subsequent insertion into the plasma membrane disrupt its integrity as a permeability barrier, leading to disruption of internal homeostasis and fungal death.The histidine-rich antimicrobial peptide PvHCt derived from shrimp hemocyanin is a strictly antifungal peptide, which adopts an amphipathic α-helical structure, and selectively binds to and permeabilizes fungal cells.Display Omitted
Keywords: Antimicrobial peptide; Amphipathic helix; Fungi; Membrane bilayer; Nuclear magnetic resonance (NMR); Fluorescence microscopy;

Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2 + to the second metal binding site by Lisa M. Oppegard; Heidi A. Schwanz; Tyrell R. Towle; Robert J. Kerns; Hiroshi Hiasa (569-575).
Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase–DNA covalent complex as a topoisomerase–fluoroquinolone–DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction.We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg2 +-, Mn2 +-, or Ca2 +-supported DNA cleavage activity of Escherichia coli Topo IV.In the absence of any drug, 20–30 mM Mg2 + was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn2 + or Ca2 + was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg2 + concentrations where Topo IV alone could not efficiently cleave DNA.At low Mg2 + concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg2 + binding to metal binding site B through the structural distortion in DNA. As Mg2 + concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg2 + at site B or inhibition the binding of Mg2 + to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg2 + binding.
Keywords: Divalent metal ion; DNA gyrase; Fluoroquinolone; Quinazolinedione; Topoisomerase IV;

Analysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of this formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms.To overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlo sampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined.The framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions.Our results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation.This framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.Graphical abstractDisplay Omitted
Keywords: Enzyme kinetics; Control analysis; Rate-limiting steps; Sequential Monte Carlo; Bayesian inference;

Syk negatively regulates TLR4-mediated IFNβ and IL-10 production and promotes inflammatory responses in dendritic cells by Hui Yin; Huaxin Zhou; Yi Kang; Xiaoju Zhang; Xiaoxian Duan; Ridab Alnabhan; Shuang Liang; David A. Scott; Richard J. Lamont; Jia Shang; Huizhi Wang (588-598).
While Syk has been shown to associate with TLR4, the immune consequences of Syk–TLR interactions and related molecular mechanisms are unclear.Gain- and loss-of-function approaches were utilized to determine the regulatory function of Syk and elucidate the related molecular mechanisms in TLR4-mediated inflammatory responses. Cytokine production was measured by ELISA and phosphorylation of signaling molecules determined by Western blotting.Syk deficiency in murine dendritic cells resulted in the enhancement of LPS-induced IFNβ and IL-10 but suppression of pro-inflammatory cytokines (TNFα, IL-6). Deficiency of Syk enhanced the activity of PI3K and elevated the phosphorylation of PI3K and Akt, which in turn, lead to the phospho-inactivation of the downstream, central gatekeeper of the innate response, GSK3β. Inhibition of PI3K or Akt abrogated the ability of Syk deficiency to enhance IFNβ and IL-10 in Syk deficient cells, confirmed by the overexpression of Akt (Myr–Akt) or constitutively active GSK3β (GSK3 S9A). Moreover, neither inhibition of PI3K–Akt signaling nor neutralization of de novo synthesized IFNβ could rescue TNFα and IL-6 production in LPS-stimulated Syk deficient cells. Syk deficiency resulted in decreased phosphorylation of IKKβ and the NF-κB p65 subunit, further suggesting a divergent influence of Syk on pro- and anti-inflammatory TLR responses.Syk negatively regulates TLR4-mediated production of IFNβ and IL-10 and promotes inflammatory responses in dendritic cells through divergent regulation of downstream PI3K–Akt and NF-κB signaling pathways.Syk may represent a novel target for manipulating the direction or intensity of the innate response, depending on clinical necessity.
Keywords: Syk; Toll-like receptor 4; Interferon-β; IL-10; PI3K; GSK3β;

Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate by Longguang Jiang; Lisbeth Moreau Andersen; Peter A. Andreasen; Liqing Chen; Mingdong Huang (599-606).
Serine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A).By taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W3A peptide at four different pH values by X-ray crystallography.In the structures obtained at low pH (pH 4.6 and 5.5), the cyclic peptide upain-1-W3A was found to be intact and remained in the active site of uPA. At 7.4, the scissile bond of the peptide was found cleaved, showing that the peptide became a uPA substrate. At pH 9.0, the C-terminal part of the substrate was no longer visible, and only the P1 residue occupying the S1 pocket was identified.The analysis of these structures provides explanations why the upain-1-W3A is a slow substrate. In addition, we clearly identified the cleaved fragments of the peptide at both sides of the scissile bond in the active site of the enzyme, showing a slow release of the cleaved peptide.This work indicates that the quick release of the cleaved P′ fragment after the first step of hydrolysis may not always be needed for the second hydrolysis.
Keywords: Serine proteases; uPA; Enzyme catalysis; Cyclic peptidyl inhibitor/substrate; Crystal structure;

Replication of the HIV-1 virus requires reverse transcription of the viral RNA genome, a process that is specifically initiated by human tRNA3 Lys packaged within the infectious virion. The primary binding site for the tRNA involves the 3′ 18 nucleotides with an additional interaction between an adenine rich loop (A-loop) in the template and the anticodon stem–loop region of the tRNA3 Lys. The loop of the tRNA primer contains two hypermodified base residues and a pseudouridine that are required for a proper binding and activity. Here, we investigate the influence on the structure, dynamics and binding stability of the three modified residues (mnm5s2U34, t6A37 and Ψ39) using extensive molecular dynamics and Quantum Theory of Atoms in Molecules (QTAIM) analysis. Consistent with experiment, the results suggest that the three modified residues are required for faithful binding. Residues mnm5s2U34 and Ψ39 have a major influence in stabilizing the anticodon loop whereas mnm5s2U34 and t6A37 appear to stabilize the formation of the complex of tRNA3 Lys with the HIV-1 A-loop.Display Omitted
Keywords: RNA dynamics; HIV genome; HIV transcription; tRNA; Anti-codon loop; Molecular dynamics; Quantum mechanics; QTAIM;

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells by Vanessa Lopes-Rodrigues; Alessio Di Luca; Diana Sousa; Hugo Seca; Paula Meleady; Michael Henry; Raquel T. Lima; Robert O'Connor; M. Helena Vasconcelos (618-627).
Multidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.Two pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.We found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.The determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.This work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.
Keywords: Multidrug resistance; Tumour cells; Extracellular vesicles;