Biomedical subjects

Ying-Jie Lu

Publications and source records attributed to Ying-Jie Lu.

12 recordsLinked to original sources

PubMed2006

Acyl-phosphates initiate membrane phospholipid synthesis in Gram-positive pathogens.

It is not known how Gram-positive bacterial pathogens carry out glycerol-3-phosphate (G3P) acylation, which is the first step in the formation of phosphatidic acid, the key intermediate in membrane phospholipid synthesis. In Escherichia coli, acylation of the 1-position of G3P is carried out by PlsB; however, the majority of bacteria lack a plsB gene and in others it is not essential. We describe a two-step pathway that utilizes a new fatty acid intermediate for the initiation of phospholipid formation. First, PlsX produces a unique activated fatty acid by catalyzing the synthesis of fatty acyl-phosphate from acyl-acyl carrier protein, and then PlsY transfers the fatty acid from acyl-phosphate to the 1-position of G3P. The PlsX/Y pathway defines the most widely distributed pathway for the initiation of phospholipid formation in bacteria and represents a new target for the development of antibacterial therapeutics.

Bacterial Proteins↗

PubMed2006

Transcriptional regulation of fatty acid biosynthesis in Streptococcus pneumoniae.

The transcriptional regulation of membrane fatty acid composition in the human pathogen Streptococcus pneumoniae is distinct from the systems utilized in the model organisms Escherichia coli and Bacillus subtilis. The genes encoding the components of type II fatty acid biosynthesis cluster at a single location within the S. pneumoniae genome, and the second gene in this cluster (SPR0376) encodes a transcription factor (FabT) that belongs to the MarR superfamily. Derivatives of S. pneumoniae strain D39 were constructed that lacked functional FabT. This strain had significantly elevated levels of saturated fatty acids and longer chain lengths than the control strain, was unable to grow at pH 5.5 and had increased sensitivity to detergent. Eliminating FabT function increased the expression levels of all of fab genes with the notable exception of fabM. FabT was purified and bound to the DNA palindrome located within the promoter regions of the fabT and fabK genes within the cluster. The analysis of cells with increased expression of individual genes leads to a model where the physical properties of the S. pneumoniae membrane is controlled primarily by the activity of FabK, the enoyl reductase, which diverts intermediates to saturated fatty acid formation, in contrast to E. coli where FabB, an elongation condensing enzyme, pulls the pathway in the direction of unsaturated acid synthesis.

Bacterial Proteins↗

PubMed2005

Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity.

Anaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of beta-hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction in vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489-34495). We carried out a series of domain swapping and mutagenesis experiments coupled with in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the beta3 and beta4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of beta-hydroxyacyl-ACP dehydratases is determined by the properties of the beta-sheets that dictate the orientation of the central alpha-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site.

Amino Acid Sequence↗

PubMed2005

Specific interaction between Smad1 and CHIP: a surface plasmon resonance study.

The TGF-beta superfamily signaling pathway regulates many important biological processes, including cell growth, differentiation and embryonic pattern formation. Smad1, a member of this signaling pathway that functions downstream of serine/threonine kinase receptors, has ability to interact with carboxyl terminus of Hsc70-interacting protein (CHIP), which is an E3 ubiquitin ligase in other cases. It has been reported that Smurf1, a member of the Hect family E3 ubiquitin ligases, can target Smad1 to 26S proteasome for degradation. In this paper, we studied the interaction of Smad1 and CHIP by combination of surface plasmon resonance and supported monolayer approach. The specific binding of Smad1 to CHIP indicates that the degradation of Smad1 may also be mediated by CHIP, and CHIP may play an essential role in the TGF-beta signaling pathway.

DNA-Binding Proteins↗

PubMed2004

The reductase steps of the type II fatty acid synthase as antimicrobial targets.

The increasing of multidrug resistance of clinically important pathogens calls for the development of novel antibiotics with unexploited cellular targets. FA biosynthesis in bacteria is catalyzed by a group of highly conserved proteins known as the type II FA synthase (FAS II) system. Bacterial FAS II organization is distinct from its mammalian counterpart; thus the FAS II pathway offers several unique steps for selective inhibition by antibacterial agents. Some known antibiotics that target the FAS II system include triclosan, isoniazid, and thiolactomycin. Recent years have seen remarkable progress in the understanding of the genetics, biochemistry, and regulation of the FAS II system with the availability of the complete genome sequence for many bacteria. Crystal structures of the FAS II pathway enzymes have been determined for not only the Escherichia coli model system but also other gram-negative and gram-positive pathogens. The protein structures have greatly facilitated structure-based design of novel inhibitors and the improvement of existing antibacterial agents. This review discusses new developments in the discovery of inhibitors that specifically target the two reductase steps of the FAS II system, beta-ketoacyl-acyl carrier potein (ACP) reductase and enoyl-ACP reductase.

Anti-Bacterial Agents↗

PubMed2004

Product diversity and regulation of type II fatty acid synthases.

Fatty acid biosynthesis is catalyzed in most bacteria by a group of highly conserved proteins known as the type II fatty acid synthase (FAS II) system. FAS II has been extensively studied in the Escherichia coli model system, and the recent explosion of bioinformatic information has accelerated the investigation of the pathway in other organisms, mostly important human pathogens. All FAS II systems possess a basic set of enzymes for the initiation and elongation of acyl chains. This review focuses on the variations on this basic theme that give rise to the diversity of products produced by the pathway. These include multiple mechanisms to generate unsaturated fatty acids and the accessory components required for branched-chain fatty acid synthesis in Gram-positive bacteria. Most of the known mechanisms that regulate product distribution of the pathway arise from the fundamental biochemical properties of the expressed enzymes. However, newly identified transcriptional factors in bacterial fatty acid biosynthetic pathways are a fertile field for new investigation into the genetic control of the FAS II system. Much more work is needed to define the role of these factors and the mechanisms that regulate their DNA binding capability, but there appear to be fundamental differences in how the expression of the pathway genes is controlled in Gram-negative and in Gram-positive bacteria.

Acetyltransferases↗

PubMed2003

Phosphotransferase-mediated transport of the osmolyte 2-O-alpha-mannosyl-D-glycerate in Escherichia coli occurs by the product of the mngA (hrsA) gene and is regulated by the mngR (farR) gene product acting as repressor.

2-O-alpha-mannosyl-D-glycerate (MGs) has been recognized as an osmolyte in hyperthermophilic but not mesophilic prokaryotes. We report that MG is taken up and utilized as sole carbon source by Escherichia coli K12, strainMC4100. Uptake is mediated by the P-enolpyruvate-dependent phosphotransferase system with the MG-inducible HrsA (now called MngA) protein as its specific EIIABC complex. The apparent Km of MG uptake in induced cells was 10 microm, and the Vmax was 0.65 nmol/min/10(9) cells. Inverted membrane vesicles harboring plasmid-encoded MngA phosphorylated MG in a P-enolpyruvate-dependent manner. A deletion mutant in mngA was devoid of MG transport but is complemented by a plasmid harboring mngA. Uptake of MG in MC4100 also caused induction of a regulon specifying the uptake and the metabolism of galactarate and glucarate controlled by the CdaR activator. The ybgG gene (now called mngB) the gene immediately downstream of mngA encodes a protein with alpha-mannosidase activity. farR, the gene upstream of mngA (now called mngR) had previously been characterized as a fatty acyl-responsive regulator; however, deletion of mngR resulted in the up-regulation of only two genes, mngA and mngB. The mngR deletion caused constitutive MG transport that became MG-inducible after transformation with plasmid expressed mngR. Thus, MngR is the regulator (repressor) of the MG transport/metabolism system. Thus, the mngR mngA mngB gene cluster encodes an MG utilizing system.

Amino Acid Sequence↗

PubMed2003

Two-dimensional crystallization of a small heat shock protein HSP16.3 on lipid layer.

As a member of small heat shock proteins, HSP16.3 was identified as the major membrane-bound protein of Mycobacterium tuberculosis during stationary phase. Previous studies revealed that HSP16.3 was in a nonameric form in solution. Here, two-dimensional crystal of HSP16.3 molecules on lipid monolayer was obtained for the first time. The crystal exhibited p422 symmetry with lattice parameters a=b=90A, gamma=90 degrees. The projection map of untilted crystals showed that the basic unit of the crystal was a rod-like structure with two high-density regions. The three-dimensional map at 2.2 nm resolution revealed a rod-like structure with a dimension of 56A x 32A x 25A, similar to the dimeric forms of M. jannaschii HSP16.5 and wheat HSP16.9. Cross-linking experiments confirmed that HSP16.3 nonamers dissociated into dimers upon interaction with the positively charged lipid layer. Surface plasmon resonance measurements revealed that both electrostatic and hydrophobic forces involved in the formation of the 2D crystal on the lipid monolayer. These results provide a basis for further investigation on the unique dimeric structure of HSP16.3 and its functions in vivo.

Amines↗

PubMed2003

Change in pH-dependent membrane insertion characteristics of trichosanthin caused by deletion of its last seven C-terminal amino acid residues.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) that can selectively kill some types of cells at low concentration (0.1-1 nM). The pH-dependent membrane insertion ability of TCS makes it possible that the internalized toxin avoids degradation in lysosomes and further undergoes transportation into the cytosol by some still unidentified mechanism. Here, we show that deletion of C-terminal residues affects interactions of modified TCS (C7-TCS) with lipids and reduces its pH-dependent membrane insertion ability. Fluorescence measurements indicate that at low pH C7-TCS undergoes profound conformational changes that causes exposure of a hydrophobic region and leads to oligomerization of the C7-TCS molecules. The results suggest that the membrane insertion of TCS at low pH might be important for translocation of TCS into the cytosol, which is important for exertion of the RIP activity of TCS. Deletion of the last seven C-terminal residues of TCS would reduce both its RIP activity in vitro and cytotoxicity in vivo, with the degree of decrease being more significant for the cytotoxicity in vivo.

Amino Acid Sequence↗

PubMed2002

Inositol hexakisphosphate (InsP6) can weaken the Ca(2+)-dependent membrane binding of C2AB domain of synaptotagmin I.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. In the present work, two fragments of the large cytoplasmic domain of synaptotagmin I, C2A and C2AB, were compared by combining surface plasmon resonance with circular dichroism and fluorescence techniques. C2AB and C2A had almost identical membrane binding constants, indicating that C2A is the predominate domain to bind to negatively charged phospholipids. After reacting with inositol hexakisphosphate (InsP6) a conformational change of C2AB was detected in the presence of liposome. The InsP6 binding notably weakened the Ca(2+)-dependent C2AB-membrane interaction, which suggests that InsP6 may act as a modulator of neurotransmitter release by altering the state of synaptotagmin-phospholipid interaction.

Animals↗

PubMed2002

The effects of protein hydrophobic exposure on the slope of the line in the deltapi vs pi(i) plot.

The deltapi vs pi(i) curve from monolayer surface pressure detection is a powerful method to characterize the membrane insertion. The deltapi vs pi(i) curve is fixed by two parameters, the pi(c) and the slope. The intersected point (pi(c)) of deltapi vs pi(i) curve with abscissa is generally used as a quantitative measure of the membrane insertion ability of a protein. In the current work we demonstrate a correlation between the variation in the slope of the deltapi vs pi(i) curve with protein hydrophobic exposure by performing monolayer experiments on three different proteins, human apolipoprotein H, trichosanthin, and mutant trichosanthin. The value of /slope/ increases gradually following the degree of hydrophobic exposure. These findings suggest that the two parameters, the pi(c) and the slope, will complement each other to interpret the lipid/protein interaction involved in membrane insertion.

Cell Membrane↗

PubMed1996

Deoxygenation of Polynuclear Metal-Oxo Anions: Synthesis, Structure, and Reactivity of the Condensed Polyoxoanion [(C(4)H(9))(4)N](4)(NbW(5)O(18))(2)O.

The deoxygenation of the mixed-metal polyoxoanion [(C(4)H(9))(4)N](3)NbW(5)O(19) with benzoyl chloride in dichloromethane forms quantitatively the condensed polyoxanion [(C(4)H(9))(4)N](4)(NbW(5)O(18))(2)O, in which two polyoxoanion fragments are linked together by a Nb-O-Nb oxo bridge. The product is characterized by a strong IR band at 692 cm(-)(1) assigned to a Nb-O-Nb stretch and a broad single (93)Nb NMR resonance at 975 ppm. Partial hydrolysis of [(C(4)H(9))(4)N](4)(NbW(5)O(18))(2)O to NbW(5)O(19)O(3)(-) in wet acetonitrile was observed by IR and (17)O NMR spectroscopy. The reaction of [(C(4)H(9))(4)N](4)(NbW(5)O(18))(2)O with a variety of alcohols and phenol forms alkoxide-derivatized polyoxoanions [(C(4)H(9))(4)N](2)Nb(OR)W(5)O(18) (R = methyl, ethyl, isopropyl, cholesteryl, phenyl). The similarity of the IR spectra of these deriviatives suggests that functionalization occurs at the terminal NbO oxygen. A crystallographic study of [(C(4)H(9))(4)N](4)(NbW(5)O(18))(2)O revealed a crystallographically imposed linear Nb-O-Nb oxo bridge (Nb-O(bridge) = 1.887(3) Å) and a structure in which the terminal tungsten-oxo bonds on the adjoining polyoxoanion fragments are eclipsed. Crystal data: orthorhombic, Cmca; Z = 4, a = 15.817(2) Å, b = 17.870(2) Å, c = 35.058(2) Å; V = 9928.0(10) Å(3); R = 5.52%.

Journal Article↗