Thermodynamic interactions of a cis and trans benzanilide with Escherichia coli bacterial membranes

The activity of a membrane interactive cis and trans benzanilide against Escherichia coli membrane mimics was investigated using Langmuir monolayers. It was found that in the presence of E. coli lipid mix monolayers, cis-benzanilide induced maximal surface pressure changes of 1?mN?m(-1), whereas a reduced interaction was observed with trans-benzanilide. Compression isotherm analysis of these monolayers showed ?G (mix)?相似文献   

A cis and trans adenine-dependent hairpin ribozyme against Tpl-2 target

Ribozymes are catalytic RNAs that possess the property of cutting an RNA target via site-specific cleavage after sequence-specific recognition. Ribozymes can moreover cleave multiple substrate molecules. An increasing number of studies show that ribozymes are particularly well adapted tools against cancer, silencing or down-regulating gene expression at the RNA level. We have constructed an adenine-dependent hairpin ribozyme that cleaves the sequence at nucleotides A(225)(downward arrow)G(226) relative to the start codon of translation of the Tpl-2 kinase mRNA; this serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in breast cancer. An adenine-dependent hairpin ribozyme 1 (ADHR1) was previously isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure. Switch on/switch off ribozymes are particularly useful since high amounts of stable ribozyme can be produced in the absence of adenine and the ribozyme specifically cleaves its target in the presence of adenine. The ADHR1 target sequence was replaced by a sequence derived from the Tpl-2 kinase mRNA. The resulting Tpl-2 ribozyme is active in cis cleavage: kinetic studies have been performed as a function of Mg2+ concentration, adenine concentration, as well as at different pH and with various cofactors. Finally, the Tpl-2 ribozyme was shown to cleave its target in trans successfully. These findings demonstrate that a potential therapeutic ribozyme can be produced by simple sequence modification.     

Structural mechanism governing cis and trans isomeric states and an intramolecular switch for cis/trans isomerization of a non-proline peptide bond observed in crystal structures of scorpion toxins

Non-proline cis peptide bonds have been observed in numerous protein crystal structures even though the energetic barrier to this conformation is significant and no non-prolyl-cis/trans-isomerase has been identified to date. While some external factors, such as metal binding or co-factor interaction, have been identified that appear to induce cis/trans isomerization of non-proline peptide bonds, the intrinsic structural basis for their existence and the mechanism governing cis/trans isomerization in proteins remains poorly understood. Here, we report the crystal structure of a newly isolated neurotoxin, the scorpion alpha-like toxin Buthus martensii Karsch (BmK) M7, at 1.4A resolution. BmK M7 crystallizes as a dimer in which the identical non-proline peptide bond between residues 9 and 10 exists either in the cis conformation or as a mixture of cis and trans conformations in either monomer. We also determined the crystal structures of several mutants of BmK M1, a representative scorpion alpha-like toxin that contains an identical non-proline cis peptide bond as that observed in BmK M7, in which residues within or neighboring the cis peptide bond were altered. Substitution of an aspartic acid residue for lysine at residue 8 in the BmK M1 (K8D) mutant converted the cis form of the non-proline peptide bond 9-10 into the trans form, revealing an intramolecular switch for cis-to-trans isomerization. Cis/trans interconversion of the switch residue at position 8 appears to be sequence-dependent as the peptide bond between residues 9 and 10 retains its wild-type cis conformation in the BmK M1 (K8Q) mutant structure. The structural interconversion of the isomeric states of the BmK M1 non-proline cis peptide bond may relate to the conversion of the scorpion alpha-toxins subgroups.     

Identification and characterization of a 14 kDa human protein as a novel parvulin-like peptidyl prolyl cis/trans isomerase

A second member of the parvulin family of peptidyl-prolyl cis/trans isomerases was identified in a human lung cDNA library. The gene encoded a protein named hPar14 that has 131 amino acid residues and a molecular mass of 13676 Da. Sequence comparison showed 34.5% identity to E. coli Par10 and 34% identity to human Pin1 (hPar18) within a C-terminal region of 87 or 120 amino acid residues, respectively. In comparison to the E. coli Par10, hPar14 possesses a N-terminal extension of 41 amino acid residues. This extension does not contain a polyproline II helix-binding motif typical of the known eukaryotic parvulins. The hPar14 does not accelerate the cis to trans interconversion of oligopeptides with side chain-phosphorylated Ser(Thr)-Pro moieties as hPin1 did. In contrast, it showed preference of an arginine residue adjacent N-terminal to proline. Northern blot analysis revealed expression of the gene within various human tissues like heart, placenta, liver, kidney and pancreas.     

Influence of glycation on cis/trans isomerization and tautomerization in novel morphiceptin-related Amadori compounds

The influence of N-glycation of the N-terminus on amide bond stereochemistry and tautomeric distribution has been explored via the synthesis and NMR analysis of novel N-(1-deoxy-D-fructos-1-yl) derivatives (Amadori compounds) of the exogenous, milk derived, opioid tetrapeptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH2). NMR analysis of the protected Amadori compounds revealed the presence of four configurational isomers in DMSO solution arising from cis/trans isomerization about Tyr1-Pro2 and Phe3-Pro4 peptide bonds. Comparison of the data obtained for protected Amadori compounds with those obtained for morphiceptin showed that equilibrium fraction of all-trans isomers in N-glycated peptide derivatives was smaller than in the parent peptide compound. Spectroscopic investigation of unprotected morphiceptin-related Amadori compound revealed the presence of multiple conformers in solution due to cis/trans isomerization of the peptide backbone and tautomerization of the sugar moiety. The equilibrium composition in DMSO is markedly shifted towards furanose forms, amounting to two-thirds of the mixture. The estimated equilibrium of the tautomeric forms in water solution revealed the -pyranose form as the major tautomer (66%).     

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans.

Highly efficient cap-independent translation initiation at the 5'-proximal AUG is facilitated by the 3' translation enhancer sequence (3'TE) located near the 3' end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3'TE in regulating viral translation was examined. The 3'TE is required for translation and thus replication of the genomic RNA that lacks a 5' cap (Allen et al., 1999, Virology253:139-144). Here we show that the 3'TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3'TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5' extremity of the sequence required in the 3' untranslated region (UTR) for cap-independent translation in vivo coincides with the 5' end of sgRNA2. Thus, sgRNA2 has the 3'TE in its 5' UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3'TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5' UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.     

Analogs of oxytocin containing a pseudopeptide Leu-Gly bond of cis and trans configuration

Analogs of deamino-oxytocin wherein the Leu-Gly peptide bond has been replaced by a tetrazole moiety or by a double bond of trans configuration were synthesized and their biological activities evaluated. Trans double bond was found to be the most appropriate substitution for the amide bond (uterotonic activity 24% of the deamino-oxytocin). In the case of all three analogs low but prolonged galactogogic activity was found and the ratio of uterotonic in vitro and in vivo activity was surprisingly high (ranging from 4.5 to 20).     

Synaptotagmin 13: structure and expression of a novel synaptotagmin

Synaptotagmins represent a family of putative vesicular trafficking proteins. With synaptotagmin 13, we have now identified a novel synaptotagmin, making this one of the largest families of trafficking proteins. Similar to synaptotagmins 3, 4, 6, 7, 9, and 11, synaptotagmin 13 is expressed at highest levels in brain but is also detectable at lower levels in non-neuronal tissues. Synaptotagmin 13 is composed of the canonical domains of synaptotagmins that include an N-terminal transmembrane region and two C-terminal cytoplasmic C2-domains (C2A- and C2B-domain) and a connecting sequence between the transmembrane region and the C2-domains. Different from most other synaptotagmins, however, synaptotagmin 13 does not have an N-terminal sequence preceding the transmembrane region, and features an unusually long connecting sequence that is proline-rich. Furthermore, the C2-domains of synaptotagmin are degenerate and lack almost all of the residues involved in Ca2+ binding, suggesting that synaptotagmin 13 is not a Ca2+-binding protein unlike most other synaptotagmins. Our data demonstrate that synaptotagmins represent a larger and more complex gene family than previously envisioned.     

Sequence-specific cleavage of hepatitis X RNA in cis and trans by novel monotarget and multitarget hammerhead motif-containing ribozymes

We constructed two monoribozymes and a diribozyme against the conserved region of the X RNA of hepatitis B virus (HBV). All the ribozymes (Rzs) possessed sequence-specific cleavage activities under standard and simulated physiologic conditions. Specific cleavage was also obtained when the same Rzs were placed in cis configuration with respect to X gene in multiple combinations. Rz-expressing cells were able to specifically interfere with the functional expression of X RNA and protein production in a liver-specific cell line HepG2. Potential applications of these novel Rzs are discussed.     

Topological mapping of complement component C9 by recombinant DNA techniques suggests a novel mechanism for its insertion into target membranes.

cDNA molecules coding for mouse and trout C9 have been isolated and the derived amino acid sequences compared with that of human C9. Regions of high homology between the closely related species (mouse and human) correlate with putative domains in the protein structure supporting a model of C9 having five globular domains. Comparison between the more distant species (trout and human) suggests regions of particular importance to C9 structure and function. In addition the three related sequences allow the secondary structure to be predicted with more confidence and we have tested the prediction by mapping surface features of the protein. Reported here is a recombinant DNA approach to fine mapping of antibody epitopes. Two of the putative domains of C9 are connected by a stretch of about 40 amino acid residues in which features characteristic of individual conformational forms of C9 are concentrated. We suggest that this region might act as a hinge allowing the rearrangement of globular domains necessary for membrane insertion. In the membrane inserting domain one highly conserved sequence has the potential to form an amphipathic alpha-helix once it is buried in the lipid bilayer. These features suggest a novel mechanism for the irreversible, post-translational insertion of C9 into target membranes.     

A novel mechanism of interaction between alpha-synuclein and biological membranes

Conformational abnormalities and aggregation of alpha-synuclein (alpha-syn) have been linked to the pathogenesis of Parkinson's (PD) and related diseases. It has been shown that alpha-syn can stably bind artificial phospholipid vesicles through alpha-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of alpha-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within alpha-syn, we demonstrated that the membrane interaction of alpha-syn in cells is also mediated by alpha-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of alpha-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of alpha-syn similar to the interaction with biological membranes. These results suggest that in cells, alpha-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of alpha-syn itself and by the cytoplasmic context.     

Biosynthesis,isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.     

Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism

The molecular mode of action of the lipopeptide SF with zwitterionic and negatively charged model membranes has been investigated with solid-state NMR, light scattering, and electron microscopy. It has been found that this acidic lipopeptide (negatively charged) induces a strong destabilization of negatively charged micrometer-scale liposomes, leading to the formation of small unilamellar vesicles of a few 10s of nanometers. This transformation is detected for very low doses of SF (Ri = 200) and is complete for Ri = 50. The phenomenon has been observed for several membrane mixtures containing phosphatidylglycerol or phosphatidylserine. The vesicularization is not observed when the lipid negative charges are neutralized and a cholesterol-like effect is then evidenced, i.e., increase of gel membrane dynamics and decrease of fluid membrane microfluidity. The mechanism for small vesicle formation thus appears to be linked to severe changes in membrane curvature and could be described by a two-step action: 1), peptide insertion into membranes because of favorable van der Waals forces between the rather rigid cyclic and lipophilic part of SF and lipid chains and 2), electrostatic repulsion between like charges borne by lipid headgroups and the negatively charged SF amino acids. This might provide the basis for a novel mode of action of negatively charged lipopeptides.