Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA

The interaction of the homeodomain of the sunflower KNOX protein HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl radical footprinting and missing nucleoside experiments. Binding of HAKN1 to different oligonucleotides indicated that HAKN1 prefers the sequence TGACA (TGTCA), with changes within the GAC core more profoundly affecting the interaction. Footprinting and missing nucleoside experiments using hydroxyl radical cleavage of DNA showed that HAKN1 interacts with a 6-bp region of the strand carrying the GAC core, covering the core and nucleotides towards the 3' end. On the other strand, protection was observed along an 8-bp region, comprising two additional nucleotides complementary to those preceding the core. Changes in the residue present at position 50 produced proteins with different specificities. An I50S mutant showed a preference for TGACT, while the presence of lysine shifted the preference to TGACC, suggesting that residue 50 interacts with nucleotide(s) 3' to GAC. Mutation of Lys54-->Val produced a protein with reduced affinity and relaxed specificity, able to recognize the sequence TGAAA, while the conservative change of Arg55-->Lys completely abolished binding to DNA. Based on these results, we propose a model for the interaction of HAKN1 with DNA in which helix III of the homeodomain accommodates along the major groove with Arg55, Asn51, Lys54 and Ile50, establishing specific contacts with bases of the GACA sequence or their complements. This model can be extended to other KNOX proteins given the conservation of these amino acids in all members of the family.     

Peptidase activity of the Escherichia coli Hsp31 chaperone

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys(184), His(185), and Asp(213) catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His(85), His(122), and Glu(90) metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8-12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.     

Plasmalogens in rat liver chromatin: new molecules involved in cell proliferation

A minor component of chromatin, the phospholipid fraction, changes during cell cycle as result of the activation of intranuclear lipid metabolism enzymes including phosphatidylcholine-dependent phospholipase C activity. It is known that this enzyme may be activated by phosphatidylcholine plasmalogen (Plg). Until now, there has been little evidences for the presence of Plgs inside the nucleus. The aim of our study is to ascertain if they are present in the nucleus and are responsible of the activation of phosphatidylcholine-dependent phospholipase C during cell proliferation and apoptosis. Therefore, we have analysed the Plg composition of the whole homogenate, cytosol, nuclei and chromatin of hepatocytes. The phosphatidylcholine-dependent phospholipase C activity was assayed using both phosphatidylcholine and plasmalogenyl-phosphatidylcholine as substrates. Our results show, for the first time, that Plgs are present in chromatin and the plasmalogenyl-phosphatidylcholine stimulates the phosphatidylcholine-dependent phospholipase C activity more than phosphatidylcholine. Finally, in order to verify the possible role of these molecules during cell proliferation and apoptosis, we used liver of rats fed with ciprofibrate which stimulates hepatocytes proliferation during the treatment and, after withdrawal, apoptosis. After 3 days of ciprofibrate treatment, the chromatin plasmalogenyl-phosphatidylcholine increases as well as the phosphatidylcholine-dependent phospholipase C activity. After drug withdrawal, when the hepatocytes undergo to apoptosis, the plasmalogenyl-phosphatidylcholine content together with phosphatidylcholine-dependent phospholipase C activity decreases. Therefore, it can be concluded that plamalogens are present in the chromatin, and probably may have a function both in regulating phosphatidylcholine dependent phospholipase C and cell cycle.     

Catch-bond model derived from allostery explains force-activated bacterial adhesion

High shear enhances the adhesion of Escherichia coli bacteria binding to mannose coated surfaces via the adhesin FimH, raising the question as to whether FimH forms catch bonds that are stronger under tensile mechanical force. Here, we study the length of time that E. coli pause on mannosylated surfaces and report a double exponential decay in the duration of the pauses. This double exponential decay is unlike previous single molecule or whole cell data for other catch bonds, and indicates the existence of two distinct conformational states. We present a mathematical model, derived from the common notion of chemical allostery, which describes the lifetime of a catch bond in which mechanical force regulates the transitions between two conformational states that have different unbinding rates. The model explains these characteristics of the data: a double exponential decay, an increase in both the likelihood and lifetime of the high-binding state with shear stress, and a biphasic effect of force on detachment rates. The model parameters estimated from the data are consistent with the force-induced structural changes shown earlier in FimH. This strongly suggests that FimH forms allosteric catch bonds. The model advances our understanding of both catch bonds and the role of allostery in regulating protein activity.     

Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly

In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to be critical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contribution at this sixth ligand position to the kinetic and spectroscopic properties. The k(cat) value of N694G is approximately 230 times lower than that of WT sLO-1 at 25 degrees C, which can be partially explained by a lowered reduction potential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band (lambda(max) = 410 nm for N694G and lambda(max) = 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidation of N694G by the product than that of WT sLO-1 (k(2) = 606 s(-)(1) for N694G and k(2) = 349 s(-)(1) for WT sLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which is confirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circular dichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombic electron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependence suggest that the coordination environment of ferric N694G contains one hydroxide and one water. On the basis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likely be a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering the reaction rate of N694G more than would be expected from the lowered reduction potential alone.     

Local force and geometry sensing regulate cell functions

The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms.     

Co- and terpolyesters based on isosorbide and succinic acid for coating applications: synthesis and characterization

Co- and terpolyesters based on succinic acid and isosorbide in combination with other renewable monomers such as 2,3-butanediol, 1,3-propanediol, and citric acid were synthesized and characterized. Linear polyesters were obtained via melt polycondensation of nonactivated dicarboxylic acids with OH functional monomers. Polymer end functionality (i.e., hydroxyl or carboxylic acid) was controlled by adjusting the monomer stoichiometry. The glass transition temperatures of the resulting polyesters could be effectively adjusted by varying the polymer composition and molar mass. By adding polyfunctional monomers such as trimethylolpropane or citric acid, polyesters with enhanced functionality were obtained. These biobased polyesters displayed functionalities and Tg values in the appropriate range for (powder) coating applications. The polyesters were cross-linked using conventional curing agents. Coatings from branched polyesters--hydroxyl as well as acid functional--showed significantly improved mechanical and chemical resistance compared to those formulated from linear polymers. These renewable polyesters proved to be suitable materials for coating applications with respect to solvent resistance, impact resistance, and hardness.     

Endogenous mitochondrial oxidative stress in MnSOD-deficient mouse embryonic fibroblasts promotes mitochondrial DNA glycation

The accumulation of somatic mutations in mitochondrial DNA (mtDNA) induced by reactive oxygen species (ROS) is regarded as a major contributor to aging and age-related degenerative diseases. ROS have also been shown to facilitate the formation of certain advanced glycation end-products (AGEs) in proteins and DNA and N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) has been identified as a major DNA-bound AGE. Therefore, the influence of mitochondrial ROS on the glycation of mtDNA was investigated in primary embryonic fibroblasts derived from mutant mice (Sod2(-/+)) deficient in the mitochondrial antioxidant enzyme manganese superoxide dismutase. In Sod2(-/+) fibroblasts vs wild-type fibroblasts, the CEdG content of mtDNA was increased from 1.90 ± 1.39 to 17.14 ± 6.60 pg/μg DNA (p<0.001). On the other hand, the CEdG content of nuclear DNA did not differ between Sod2(+/+) and Sod2(-/+) cells. Similarly, cytosolic proteins did not show any difference in advanced glycation end-products or protein carbonyl contents between Sod2(+/+) and Sod2(-/+). Taken together, the data suggest that mitochondrial oxidative stress specifically promotes glycation of mtDNA and does not affect nuclear DNA or cytosolic proteins. Because DNA glycation can change DNA integrity and gene functions, glycation of mtDNA may play an important role in the decline of mitochondrial functions.