The ICK/KRP family of cyclin‐dependent kinase (CDK) inhibitors modulates the activity of plant CDKs through protein binding. Previous work has shown that changing the levels of ICK/KRP proteins by overexpression or downregulation affects cell proliferation and plant growth, and also that the ubiquitin proteasome system is involved in degradation of ICK/KRPs. We show in this study that the region encompassing amino acids 21 to 40 is critical for ICK1 levels in both Arabidopsis and yeast. To determine how degradation of ICK1 is controlled, we analyzed the accumulation of hemagglutinin (HA) epitope‐tagged ICK1 proteins in yeast mutants defective for two ubiquitin E3 ligases. The highest level of HA‐ICK1 protein was observed when both the N‐terminal 1–40 sequence was removed and the SCF (SKP1–Cullin1–F‐box complex) function disrupted, suggesting the involvement of both SCF‐dependent and SCF‐independent mechanisms in the degradation of ICK1 in yeast. A short motif consisting of residues 21–30 is sufficient to render green fluorescent protein (GFP) unstable in plants and had a similar effect in plants regardless of whether it was fused to the N‐terminus or C‐terminus of GFP. Furthermore, results from a yeast ubiquitin receptor mutant rpn10Δ indicate that protein ubiquitination is not critical in the degradation of GFP‐ICK11–40 in yeast. These results thus identify a protein‐destabilizing sequence motif that does not contain a typical ubiquitination residue, suggesting that it probably functions through an SCF‐independent mechanism. 相似文献
Our previous studies have indicated that TGF-beta1 exerts its effect on the expression of A-type potassium channels (I(A)) in rat vascular myofibroblasts by activation of protein kinase C during the phenotypic transformation of vascular fibroblasts to myofibroblasts. In the present study, patch-clamp whole-cell recording and transwell-migration assays were used to examine the effects of TGF-beta1- and phorbol 12-myristate 13-acetate (PMA)-induced expression of I(A) channels on myofibroblast migration and its modulation by the protein kinase A (PKA) pathway. Our results reveal that incubation of fibroblasts with TGF-beta1 or PMA up-regulates the expression of I(A) channels and increases myofibroblast migration. Blocking I(A) channel expression by 4-aminopyridine (4-AP) significantly inhibits TGF-beta1- and PMA-induced myofibroblast migration. Incubation of fibroblasts with forskolin does not result in increased expression of I(A) channels but does cause a slight increase in fibroblast migration at higher concentrations. In addition, forskolin increases the TGF-beta1- and PMA-induced myofibroblast migration but inhibits TGF-beta1- and PMA-induced the expression of I(A) channels. Whole-cell current recordings showed that forskolin augments the delayed rectifier outward K(+) (I(K)) current amplitude of fibroblasts, but not the I(A) of myofibroblasts. Our results also indicate that TGF-beta1- and PMA-induced expression of I(A) channels might be related to increase TGF-beta1- or PMA-induced myofibroblast migration. Promoting fibroblast and myofibroblast migration via the PKA pathway does not seem to involve the expression of I(A) channels, but the modulation of I(K) and I(A) channels might be implicated. 相似文献
Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium. 相似文献
Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27(-/-) mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27(-/-) mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27(-/-) mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27(-/-)and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27(-/-) mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1alpha were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27(-/-) mice. Remarkably, Fsp27(-/-) MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity. 相似文献
Membrane-bound proteins represent the single most important class of drug targets. This article discusses the issues surrounding fabrication of membrane-protein microarrays by conventional robotic pin printing techniques. Ligand binding selectivity and specificity to G protein-coupled receptor (GPCR) microarrays are presented. The potential applications of these arrays for drug screening are discussed. 相似文献
Optically pure methyl (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepared with 100 % theoretical yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid ((S)-2-CMA). In the present work, we proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidation system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to the native MR was obtained. Kinetic analysis indicated that the enhanced catalytic efficiency mainly arose from the elevated kcat. Further insight into the source of improved catalytic activity was gained by molecular simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate binding sites. In addition, the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.