Proper phosphorylation of septin 12 regulates septin 4 and soluble adenylyl cyclase expression to induce sperm capacitation

Septin-based ring complexes maintain the sperm annulus. Defective annular structures are observed in the sperm of Sept12- and Sept4-null mice. In addition, sperm capacitation, a process required for proper fertilization, is inhibited in Sept4-null mice, implying that the sperm annulus might play a role in controlling sperm capacitation. Hence, we analyzed sperm capacitation of sperm obtained from SEPT12 Ser196 phosphomimetic (S196E), phosphorylation-deficient (S196A), and SEPT4-depleted mutant mice. Capacitation was reduced in the sperm of both the Sept12 S196E- and Sept12 S196A-knock-in mice. The protein levels of septins, namely, SEPT4 and SEPT12, were upregulated, and these proteins were concentrated in the sperm annulus during capacitation. Importantly, the expression of soluble adenylyl cyclase (sAC), a key enzyme that initiates capacitation, was upregulated, and sAC was recruited to the sperm annulus following capacitation stimulation. We further found that SEPT12, SEPT4, and sAC formed a complex and colocalized to the sperm annulus. Additionally, sAC expression was reduced and disappeared in the annulus of the SEPT12 S196E- and S196A-mutant mouse sperm. In the sperm of the SEPT4-knockout mice, sAC did not localize to the annulus. Thus, our data demonstrate that SEPT12 phosphorylation status and SEPT4 activity jointly regulate sAC protein levels and annular localization to induce sperm capacitation.     

At3g53630 encodes a GUN1-interacting protein under norflurazon treatment

Chloroplasts are semi-autonomous organelles, with more than 95% of their proteins encoded by the nuclear genome. The chloroplast-to-nucleus retrograde signals are critical for the nucleus to coordinate its gene expression for optimizing or repairing chloroplast functions in response to changing environments. In chloroplasts, the pentatricopeptide-repeat protein GENOMES UNCOUPLED 1 (GUN1) is a master switch that senses aberrant physiological states, such as the photooxidative stress induced by norflurazon (NF) treatment, and represses the expression of photosynthesis-associated nuclear genes (PhANGs). However, it is largely unknown how the retrograde signal is transmitted beyond GUN1. In this study, a protein GUN1-INTERACTING PROTEIN 1 (GIP1), encoded by At3g53630, was identified to interact with GUN1 by different approaches. We demonstrated that GIP1 has both cytosol and chloroplast localizations, and its abundance in chloroplasts is enhanced by NF treatment with the presence of GUN1. Our results suggest that GIP1 and GUN1 may function antagonistically in the retrograde signaling pathway.

     

Structure and mechanism of Helicobacter pylori fucosyltransferase. A basis for lipopolysaccharide variation and inhibitor design

Helicobacter pylori alpha1,3-fucosyltransferase (FucT) is involved in catalysis to produce the Lewis x trisaccharide, the major component of the bacteria's lipopolysaccharides, which has been suggested to mimic the surface sugars in gastric epithelium to escape host immune surveillance. We report here three x-ray crystal structures of FucT, including the FucT.GDP-fucose and FucT.GDP complexes. The protein structure is typical of the glycosyltransferase-B family despite little sequence homology. We identified a number of catalytically important residues, including Glu-95, which serves as the general base, and Glu-249, which stabilizes the developing oxonium ion during catalysis. The residues Arg-195, Tyr-246, Glu-249, and Lys-250 serve to interact with the donor substrate, GDP-fucose. Variations in the protein and ligand conformations, as well as a possible FucT dimer, were also observed. We propose a catalytic mechanism and a model of polysaccharide binding not only to explain the observed variations in H. pylori lipopolysaccharides, but also to facilitate the development of potent inhibitors.     

ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth

Receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is expressed during embryogenesis and by certain leukemias, but not by normal adult tissues. Here we show that the neoplastic cells of many human breast cancers express the ROR1 protein and high-level expression of ROR1 in breast adenocarcinoma was associated with aggressive disease. Silencing expression of ROR1 in human breast cancer cell lines found to express this protein impaired their growth in vitro and also in immune-deficient mice. We found that ROR1 could interact with casein kinase 1 epsilon (CK1ε) to activate phosphoinositide 3-kinase-mediated AKT phosphorylation and cAMP-response-element-binding protein (CREB), which was associated with enhanced tumor-cell growth. Wnt5a, a ligand of ROR1, could induce ROR1-dependent signaling and enhance cell growth. This study demonstrates that ROR1 is expressed in human breast cancers and has biological and clinical significance, indicating that it may be a potential target for breast cancer therapy.     

Homodimeric hexaprenyl pyrophosphate synthase from the thermoacidophilic crenarchaeon Sulfolobus solfataricus displays asymmetric subunit structures

Hexaprenyl pyrophosphate synthase (HexPPs) from Sulfolobus solfataricus catalyzes the synthesis of trans-C(30)-hexaprenyl pyrophosphate (HexPP) by reacting two isopentenyl pyrophosphate molecules with one geranylgeranyl pyrophosphate. The crystal structure of the homodimeric C(30)-HexPPs resembles those of other trans-prenyltransferases, including farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase (OPPs). In both subunits, 10 core helices are arranged about a central active site cavity. Leu164 in the middle of the cavity controls the product chain length. Two protein conformers are observed in the S. solfataricus HexPPs structure, and the major difference between them occurs in the flexible region of residues 84 to 100. Several helices (alphaI, alphaJ, alphaK, and part of alphaH) and the associated loops have high-temperature factors in one monomer, which may be related to the domain motion that controls the entrance to the active site. Different side chain conformations of Trp136 in two HexPPs subunits result in weaker hydrophobic interactions at the dimer interface, in contrast to the symmetric pi-pi stacking interactions of aromatic side chains found in FPPs and OPPs. Finally, the three-conformer switched model may explain the catalytic process for HexPPs.     

Regulation of TCRβ allelic exclusion by gene segment proximity and accessibility

Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR β-chain gene triggers feedback inhibition of V(β)-to-DJ(β) recombination in double-positive (DP) thymocytes, which correlates with reduced V(β) chromatin accessibility and a locus conformational change that separates V(β) from DJ(β) gene segments. We previously generated a Tcrb allele that maintained V(β) accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced V(β) accessibility and increased distance between V(β) and DJ(β) gene segments both enforce feedback inhibition in DP thymocytes.     

Sucrose metabolism in grape (Vitis vinifera L.) branches under low temperature during overwintering covered with soil

The effect of low temperature on sugar content and activities of key enzymes related to sucrose metabolism in grape (Vitis vinifera L.) branches during overwintering covered with soil was investigated. We measured the contents of soluble sugar and the activities of sucrose-phosphate synthase (SPS), sucrose synthase (SS), acid invertase (AI) and neutral invertase (NI) of three grape varieties with different freezing tolerance, Beta, Vidal and Merlot, in October, 2011, January, 2012 and March, 2012. The result showed that: total soluble sugar had the significant negative correlation, ?0.988, with temperature during overwintering covered with soil. The content of hexose was about twofold content of sucrose in January, while sucrose increased and the hexose decreased to a very low level in March, the ratios between hexose and sucrose declined to 0.26, 0.15 and 0.18. Sucrose was more important than hexose in protecting grape branches from cold injury under low temperature, but non-freezing. The accumulation of sucrose was mostly due to the elevation of the SPS activity, whereas the increase of hexose was due to the enhanced AI activity. Three grape varieties responded to low temperature positively as reflected by the variations of physiological and biochemical characteristics, such as superoxide dismutase, catalase and proline. Besides, by the principal components analysis and combined with cultivation practices, among twelve characteristics, the sugar metabolism mainly contributed to the difference of the cold resistance. The results indicated that sucrose metabolism regulation played an important role during overwintering covered with soil, and it was the key factor to explain the difference of cold resistance.     

一株邻苯二甲酸二丁酯降解菌的筛选及其降解特性

【目的】从自然环境中筛选邻苯二甲酸二丁酯(Dibutyl phthalate,DBP)降解能力较强的微生物,并研究其降解特性和代谢途径。【方法】从杭州市河道污水出口的淤泥中筛选到DBP降解菌ZJUTW,对其进行形态、生理生化特征、16SrRNA基因序列分析,考察该菌株对DBP的降解特性,并用GC-MS分析降解中间产物。【结果】该菌株经鉴定为Arthrobacter sp.,降解DBP的最适温度和最适pH值分别为30°C和7.0-8.0,可降解多种邻苯二甲酸酯类化合物;当DBP浓度为800 mg/L时,半衰期为10.47 h;菌株的休止细胞(OD_(600)=1.2)可在20 h内将1 200 mg/L的DBP完全降解。利用GC-MS进行中间产物分析,该菌株可通过酯交换方式起始DBP的降解。【结论】Arthrobacter sp.ZJUTW对DBP有较强的降解能力和较高的耐受性,具有潜在的应用前景。     

Roles of carbohydrate supply and ethylene, polyamines in maize kernel set

Glucose appears to have an antagonistic relationship with ethylene and ethylene and polyamines appear to play antagonistic roles in the abortion of seeds and fruits. Moreover, ethylene, spermidine, and spermine share a common biosynthetic precursor. The synchronous changes of them and the relationships with kernel set are currently unclear. Here, we stimulated maize (Zea mays L.) apical kernel set and studied their changes at 4, 8, 12, and 16 d after pollination (DAP). The status of the apical kernels changed from abortion to set, showing a pattern similar to that of the middle kernels, with slow decrease in glucose and rapid decline in ethylene production, and a sharp increase in spermidine and spermine after four DAP. Synchronous changes in ethylene and spermidine were also observed. However, the ethylene production decreased slowly in the aborted apical kernels, the glucose and polyamines concentrations were lower. Ethephon application did not block the change from abortion to set for the setting apical kernels. These data indicate that the developmental change may be accompanied by an inhibition of adequate glucose to ethylene synthesis and subsequent promotion of spermidine and spermine synthesis, and adequate carbohydrate supply may play a key role in the developmental process.