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991.
992.
The Escherichia coli mispair-binding protein MutS forms dimers and tetramers in vitro, although the functional form in vivo is under debate. Here we demonstrate that the MutS tetramer is extended in solution using small angle x-ray scattering and the crystal structure of the C-terminal 34 amino acids of MutS containing the tetramer-forming domain fused to maltose-binding protein (MBP). Wild-type C-terminal MBP fusions formed tetramers and could bind MutS and MutS-MutL-DNA complexes. In contrast, D835R and R840E mutations predicted to disrupt tetrameric interactions only allowed dimerization of MBP. A chromosomal MutS truncation mutation eliminating the dimerization/tetramerization domain eliminated mismatch repair, whereas the tetramer-disrupting MutS D835R and R840E mutations only modestly affected MutS function. These results demonstrate that dimerization but not tetramerization of the MutS C terminus is essential for mismatch repair. 相似文献
993.
Isenberg JS Jia Y Fukuyama J Switzer CH Wink DA Roberts DD 《The Journal of biological chemistry》2007,282(21):15404-15415
Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47. 相似文献
994.
Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes. 相似文献
995.
The protein C (PC) pathway plays an important role in coagulation and inflammation. Many components of the PC pathway have been identified in epidermal keratinocytes, including endothelial protein C receptor (EPCR), which is the specific receptor for PC/activated PC (APC), but the core member of this pathway, PC, and its function in keratinocytes has not been defined. In this study, we reveal that PC is strongly expressed by human keratinocytes at both gene and protein levels. When endogenous PC was blocked by siRNA the proliferation of keratinocytes was significantly decreased. This inhibitory effect was restored by the addition of recombinant APC. PC siRNA treatment also increased cell apoptosis by 3-fold and inhibited cell migration by more than 20%. When keratinocytes were pretreated with RCR252, an EPCR-blocking antibody, or PD153035, an epidermal growth factor receptor (EGFR) inhibitor, cell proliferation was hindered by more than 30%. These inhibitors also completely abolished recombinant APC (10 mug/ml)-stimulated proliferation. Blocking PC expression or inhibiting its binding to EPCR/EGFR decreased the phosphorylation of ERK1/2 but increased p38 activation. Furthermore, inhibition of ERK decreased cell proliferation by approximately 30% and completely abolished the stimulatory effect of APC on proliferation. Taken together, these results indicate that keratinocyte-derived PC promotes cell survival, growth, and migration in an autocrine manner via EPCR, EGFR, and activation of ERK1/2. Our results highlight a novel role for the PC pathway in normal skin physiology and wound healing. 相似文献
996.
Millard CJ Ellis IR Pickford AR Schor AM Schor SL Campbell ID 《The Journal of biological chemistry》2007,282(49):35530-35535
The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN. 相似文献
997.
Janus kinase 3 (Jak3) is a non-receptor tyrosine kinase known to be expressed in hematopoietic cells. Studies of whole organ homogenates show that Jak3 is also expressed in the intestines of both human and mice. However, neither its expression nor its function has been defined in intestinal epithelial enterocytes. The present studies demonstrate that functional Jak3 is expressed in human intestinal enterocytes HT-29 Cl-19A and Caco-2 and plays an essential role in the intestinal epithelial wound repair process in response to interleukin 2 (IL-2). Exogenous IL-2 enhanced the wound repair of intestinal enterocytes in a dose-dependent manner. Activation by IL-2 led to rapid tyrosine phosphorylation and redistribution of Jak3. IL-2-stimulated redistribution of Jak3 was inhibited by the Jak3-specific inhibitor WHI-P131. IL-2 also induced Jak3-dependent redistribution of the actin cytoskeleton in migrating cells. In these cells Jak3 interacted with the intestinal and renal epithelial cell-specific cytoskeletal protein villin in an IL-2-dependent manner. Inhibition of Jak3 activation resulted in loss of tyrosine phosphorylation of villin and a significant decrease in wound repair of the intestinal epithelial cells. Previously, we had shown that tyrosine phosphorylation of villin is important for cytoskeletal remodeling and cell migration. The present study demonstrates a novel pathway in intestinal enterocytes in which IL-2 enhances intestinal wound repair through mechanisms involving Jak3 and its interactions with villin. 相似文献
998.
PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex 总被引:4,自引:0,他引:4
Fonseca BD Smith EM Lee VH MacKintosh C Proud CG 《The Journal of biological chemistry》2007,282(34):24514-24524
Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1. 相似文献
999.
Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester 总被引:2,自引:0,他引:2
This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry. 相似文献
1000.
Unnithan VB Baynard T Potter CR Barker P Heffernan KS Kelly E Yates G Fernhall B 《Obesity (Silver Spring, Md.)》2007,15(11):2673-2682
Objective: Obesity has been proposed to negatively impact cardiac function in overweight (OW) individuals. The relationship between diastolic dysfunction and oxygen uptake (V?o 2) kinetics is equivocal. This exploratory investigation evaluated the relationship between resting left ventricular function and V?o 2 kinetics during cycle ergometry in OW and non‐overweight (NO) children and adolescents. Research Methods and Procedures: Fourteen OW (>85 percentile for BMI for age and gender) children, 10 boys and 4 girls (age, 11.7 ± 1.9 years; body mass, 80.6 ± 45.5 kg) and 10 NO children (4 boys, 6 girls) volunteered to participate in the study (age, 12.5 ± 2.1 years; body mass, 45.8 ± 13.8 kg). Resting cardiovascular structure and function were assessed using spectral Doppler echocardiography. All subjects underwent two sub‐maximal exercise stages on a cycle ergometer (3 minutes unloaded and 5 minutes at 50 W, both at a cadence of 50 rpm). Respiratory data were measured on a breath‐by‐breath basis at both workloads and the mean response time (MRT) was calculated. Results: Analysis of the MRT data demonstrated that there were no significant differences between OW and NO (OW, 52.6 ± 11.7 seconds vs. NO, 45.6 ± 7.4 seconds). Significant correlations (p < 0.05) were obtained between MRT V?o 2 and echocardiographic‐derived mitral valve inflow pressure half‐time (r = 0.55) and between MRT V?o 2, and mitral valve inflow deceleration time (r = 0.55). Discussion: The evidence from this research suggests a possible link between left ventricular diastolic function at rest and oxygen uptake kinetics during sub‐maximal exercise in OW and NO children and adolescents. 相似文献