Crystallization of the terminal oxygenase component of biphenyl dioxygenase derived from Rhodococcus sp. strain RHA1

The terminal oxygenase component of the biphenyl dioxygenase (BphA1A2 complex) was over-expressed with a novel over expression system in recombinant Rhodococcus strain and purified. The purified enzyme has been crystallized by the hanging drop vapor diffusion method and subjected to X-ray diffraction analysis. The crystals belong to the tetragonal system in the space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to better than 2.2A resolution.     

Three mitogen-activated protein kinases inhibit insulin signaling by different mechanisms in 3T3-L1 adipocytes

TNFalpha, which activates three different MAPKs [ERK, p38, and jun amino terminal kinase (JNK)], also induces insulin resistance. To better understand the respective roles of these three MAPK pathways in insulin signaling and their contribution to insulin resistance, constitutively active MAPK/ERK kinase (MEK)1, MAPK kinase (MKK6), and MKK7 mutants were overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated transfection procedure. The MEK1 mutant, which activates ERK, markedly down-regulated expression of the insulin receptor (IR) and its major substrates, IRS-1 and IRS-2, mRNA and protein, and in turn reduced tyrosine phosphorylation of IR as well as IRS-1 and IRS-2 and their associated phosphatidyl inositol 3-kinase (PI3K) activity. The MKK6 mutant, which activates p38, moderately inhibited IRS-1 and IRS-2 expressions and IRS-1-associated PI3K activity without exerting a significant effect on the IR. Finally, the MKK7 mutant, which activates JNK, reduced tyrosine phosphorylation of IRS-1 and IRS-2 and IRS-associated PI3K activity without affecting expression of the IR, IRS-1, or IRS-2. In the context of our earlier report showing down-regulation of glucose transporter 4 by MEK1-ERK and MKK6/3-p38, the present findings suggest that chronic activation of ERK, p38, or JNK can induce insulin resistance by affecting glucose transporter expression and insulin signaling, though via distinctly different mechanisms. The contribution of ERK is, however, the strongest.     

Identification of a second member of the ponticulin gene family and its differential expression pattern

We have identified a homologue (ponB) of the ponticulin gene (ponA), an F-actin binding protein, in the expressed sequence tag library generated to mRNA isolated from fusion-competent cells of Dictyostelium discoideum. PonB is predicted to have many of the same characteristics as ponticulin. Both proteins are predicted to possess a cleaved signal peptide, a glycosyl anchor, an amphipathic beta-strand structure and six conserved cysteines. Because of the sequence similarity and predicted conserved structures, this gene constitutes the second member of a ponticulin gene family. Unlike ponticulin, ponB is not expressed in axenically grown cells or during the asexual reproductive phase of D. discoideum. PonB is expressed by cells grown on bacterial lawns and by cells induced to be fusion-competent, i.e., gametes. The expression of ponB correlates with the appearance of a new F-actin binding activity in cell lysates of bacterially grown ponA(-) cells. By immunofluorescence microscopy, ponB appears to be localized to vesicles and to the plasma membrane of bacterially grown cells. Because ponticulin is the major high-affinity link between the plasma membrane and the cytoskeleton, the ponticulin gene family is likely to be part of the redundant system of proteins involved in connecting the cytoskeleton to the plasma membrane.     

Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein

Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.     

Importance of local structures of second and third repeat fragments of microtubule-binding domain for tau filament formation

To investigate the importance of the seventh residue of the second and third repeat fragments (R2 and R3 peptides) of the microtubule-binding domain (MBD) for tau filamentous assembly, the residues Lys and Pro were substituted (R2-K7P and R3-P7K). The filament formations of the R2 and R3 peptides were almost lost due to their substitutions despite their overall conformational similarities. The NOE analyses showed the importance of the conformational flexibility for the R2 peptide and the coupled extended and helical conformations for the R3 peptide in their limited N-terminal regions around their seventh residues. The result shows that the filament formation of MBD is initiated from a short fragment region containing the minimal conformational or functional motif.     

Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces

The interaction of apolipoprotein E (apoE) with cell-surface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136-150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic alpha-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a two-step process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the N-terminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the alpha-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cell-surface heparan sulfate proteoglycan localizes apoE-enriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation.     

Binding of two flaviolin substrate molecules, oxidative coupling, and crystal structure of Streptomyces coelicolor A3(2) cytochrome P450 158A2

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.