NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H₂CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε–Cε–Nζ of Lys and Hδ–Cδ–Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H₂CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H₂CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.     

Growth Promotion and Salt-Tolerance Improvement of Gerbera jamesonii by Root Colonization of Piriformospora indica

Journal of Plant Growth Regulation - Interaction of plants with beneficial microbes is one of the strategies for increasing growth and resistance under environmental stresses. In this study, the...     

Integrated small RNA profiling and degradome analysis of Anthurium andraeanum cultivars with different-colored spathes

MicroRNAs (miRNAs) are known to play vital roles in coloration of leaves, flowers, and fruits in plants. However, their functions in spathe coloration are poorly known. Anthurium andraeanum is a popular ornamental plant with various spathe colors. In this study, small RNA and degradome libraries from three A. andraeanum cultivars with different-colored spathes were constructed and sequenced. Illumina sequencing resulted in 94 conserved miRNAs, and 34 novel miRNAs in total were then identified based on precursor sequences and hairpin structures. Differential expression analysis showed that 52, 51, and 49 miRNAs were differentially expressed in comparisons of orange- versus white-colored spathe, purple- versus white-colored spathe, and purple- versus orange-colored spathe, respectively. The expression patterns of miRNAs and their corresponding targets involved in spathe coloration were further analyzed, and displayed that miR156b and miR529 were highly abundant in the spathes with higher anthocyanin content. These two miRNAs co-targeted a gene encoding SPL17, which may function as a negative regulator in anthocyanin accumulation. In addition, miR408 was also abundantly expressed in purple- and orange-colored spathes, and its typical targets were also identified. This comprehensive integrated analysis provides insight into the miRNA-mediated genetic regulation in spathe coloration of A. andraeanum.

     

Dynamic molecular structure of DPPC-DLPC-cholesterol ternary lipid system by spin-label electron spin resonance

The hydrated ternary lamellar lipid mixture of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) has been studied by electron spin resonance (ESR) to reveal the dynamic structure on a molecular level of the different phases that exist and coexist over virtually the full range of composition. The spectra for more than 100 different compositions at room temperature were analyzed by nonlinear least-squares fitting to provide the rotational diffusion rates and order parameters of the end-chain labeled phospholipid 16-PC. The ESR spectra exhibit substantial variation as a function of composition, even though the respective phases generally differ rather modestly from each other. The Lalpha and Lbeta phases are clearly distinguished, with the former exhibiting substantially lower ordering and greater motional rates, whereas the well-defined Lo phase exhibits the greatest ordering and relatively fast motional rates. Typically, smaller variations occur within a given phase. The ESR spectral analysis also yields phase boundaries and coexistence regions which are found to be consistent with previous results from fluorescence methods, although new features are found. Phase coexistence regions were in some cases confirmed by observing the existence of isosbestic points in the absorption mode ESR spectra from the phases. The dynamic structural properties of the DPPC-rich Lbeta and DLPC-rich Lalpha phases, within their two-phase coexistence region do not change with composition along a tie-line, but the ratio of the two phases follows the lever rule in accordance with thermodynamic principles. The analysis shows that 16-PC spin-label partitions nearly equally between the Lalpha and Lbeta phases, making it a useful probe for studying such coexisting phases. Extensive study of two-phase coexistence regions requires the determination of tie-lines, which were approximated in this study. However, a method is suggested to accurately determine the tie-lines by ESR.     

Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling

The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339-342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser(296)/Ser(323) domain, which was not affected by substituting Ala for Ser at Ser(359)/Ser(364). The DEF and Ser(296)/Ser(323) sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser(359)/Ser(364) sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser(296) MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser(296) MKP-1 bound to SCF(Skp2) ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCF(Skp2) cooperation, thereby sustaining ERK activation.     

ESR Study of Interfacial Hydration Layers of Polypeptides in Water-Filled Nanochannels and in Vitrified Bulk Solvents

There is considerable evidence for the essential role of surface water in protein function and structure. However, it is unclear to what extent the hydration water and protein are coupled and interact with each other. Here, we show by ESR experiments (cw, DEER, ESEEM, and ESE techniques) with spin-labeling and nanoconfinement techniques that the vitrified hydration layers can be evidently recognized in the ESR spectra, providing nanoscale understanding for the biological interfacial water. Two peptides of different secondary structures and lengths are studied in vitrified bulk solvents and in water-filled nanochannels of different pore diameter (6.1∼7.6 nm). The existence of surface hydration and bulk shells are demonstrated. Water in the immediate vicinity of the nitroxide label (within the van der Waals contacts, ∼0.35 nm) at the water-peptide interface is verified to be non-crystalline at 50 K, and the water accessibility changes little with the nanochannel dimension. Nevertheless, this water accessibility for the nanochannel cases is only half the value for the bulk solvent, even though the peptide structures remain largely the same as those immersed in the bulk solvents. On the other hand, the hydration density in the range of ∼2 nm from the nitroxide spin increases substantially with decreasing pore size, as the density for the largest pore size (7.6 nm) is comparable to that for the bulk solvent. The results demonstrate that while the peptides are confined but structurally unaltered in the nanochannels, their surrounding water exhibits density heterogeneity along the peptide surface normal. The causes and implications, especially those involving the interactions between the first hydration water and peptides, of these observations are discussed. Spin-label ESR techniques are proven useful for studying the structure and influences of interfacial hydration.     

显性失活缺氧诱导因子-1通过下调VEGF表达促进宫颈癌细胞凋亡

将表达野生型缺氧诱导因子-1α (hypoxia inducible factor-1 α, HIF-1α)的重组质粒pcDNA3.1-full length HIF-1α,表达抑制型HIF-1α的重组质粒pcDNA3.1-dominant negative HIF-1α和空质粒pcDNA3.1稳定转染人宫颈癌SiHa细胞,研究HIF-1α对人宫颈癌SiHa细胞生物学行为的影响．采用免疫细胞化学法和Western 印迹检测HIF-1α与VEGF蛋白的表达;CoCl2化学缺氧法处理细胞,采用原位缺口末端标记(TUNEL)法检测细胞凋亡情况．结果显示,显性失活HIF-1α能下调VEGF蛋白的表达,促进细胞缺氧条件下的凋亡,这提示HIF-1α可能在宫颈癌的发生发展中起作用,利用显性失活HIF-1α转染抑制HIF-1α可望成为宫颈癌治疗基因治疗的又一新途径．     

ERK1/2 achieves sustained activation by stimulating MAPK phosphatase-1 degradation via the ubiquitin-proteasome pathway

Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogen-activated protein kinase phosphatase 1 (MKP-1) protein levels in time- and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb(II)-treated cells, MKP-1 is polyubiquitinated, and proteasome inhibitors markedly alleviate the ubiquitination and degradation of MKP-1. Inhibiting the Pb(II)-induced ERK1/2 activation by PD98059 greatly suppresses MKP-1 ubiquitination and degradation. It is remarkable that constitutive activation of MKK1/2 triggers endogenous MKP-1 ubiquitination and degradation in various mammalian cell lines. Furthermore, expression of functional MKP-1 decreases ERK1/2 activation and the c-Fos protein level and enhances cytotoxicity under Pb(II) exposure. Taken together, these results demonstrate that activated ERK1/2 can trigger MKP-1 degradation via the ubiquitin-proteasome pathway, thus facilitating long-term activation of ERK1/2 against cytotoxicity.