Identification of FANCA as a protein interacting with centromere-associated protein E

This study sought to isolate and identify proteins that interact with centromere-associated protein E （CENP- E）, provide new clues for exploring the function of CENP-E in cell cycle control and the pathogenesis of tumor. Yeast two-hybrid screen and regular molecular biologic techniques were undertaken to screen human HeLa cDNA library with the kinetochore binding domain of CENP-E. The bait from the C-terminus of CENP-E was created by subcloning methods to find out optimal candidate proteins that interact with the kinetochore binding domain of CENP-E. Eight novel CENP-E interacting proteins including Homo sapiens Fanconi anemia complementation group A （FANCA） were obtained. In yeast two-hybrid assay, the N-terminal 260 amino acids of FANCA were found to be necessary and sufficient for the interaction with the C-terminus of CENP-E. The interaction was confirmed by in vitro glutathione S-transferase pull-down assay and in vivo coimmunoprecipitation assay. Our finding of the interaction of CENP-E with FANCA demonstrates that CENP-E and FANCA may play important roles in the functional regulation of the mitotic checkpoint signal pathway.     

Modeling of protein refolding from inclusion bodies

Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies （IBs）, which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble pro- teins following dilution refoiding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical proper- ties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high effi- cacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight （R2= 0.98, P = 0.000, F-test）. Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refold- ing developed here could be used to predict the refold- ing yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refoiding from IBs according to their intrinsic properties.     

Different effect of glutamine on macrophage tumor necrosis factor-alpha release and heat shock protein 72 expression in vitro and in vivo

Macrophage plays a vital role in sepsis. However, the modulatory effect of glutamine （Gin） on macrophage/ monocyte-mediate cytokines release is still controversial. Thus, we investigated the effect of Gin on macro- phage tumor necrosis factor （TNF）-α release and heat shock protein （HSP） 72 expression in vivo and in vitro. Data from our study indicated that the increase of HSP72 expression was significant at 8 mM of Gin 4 h after lipopolysaccharide （LPS） stimulation and became independent of Gin concentrations at 24 h, whereas TNF-α release was dose-and time-dependent on Gin. Heat stress （HS） induced more HSP72 and less TNF-α production compared with the non-HS group. However, the production of TNF-α in cells pretreated with HS was increased with increasing concentrations of Gin. Treatment with various concentrations of Gin for 1 h and then 0.5mM Gin for 4h led to an increase in HSP72 expression, but not in TNF-α production. In sepsis model mice, Gin treatment led to a significantly lower intracellular TNF-α level and an increase in HSP72 expression in mouse peritoneal macrophages. Our results demonstrate that Gin directly increases TNF-α release of LPS-stimulated RAW264.7 macro- phages in a dose-dependent manner, and also decreases mouse peritoneal macrophages TNF-α release in the sepsis model. Taken together, our data suggest that there may be more additional pathways by which Gin modulates cytokine production besides HSP72 expression in macrophage during sepsis.     

低氧运动后不同恢复环境腓肠肌热休克蛋白70表达变化

目的：实验旨在观察急性间歇低氧（氧含量12．7％）跑台运动后不同恢复环境下大鼠腓肠肌热休克蛋白HSP70表达的时程变化。方法：雄性sD大鼠进行低氧环境下的急性间歇跑台运动,用Western blot方法检测低氧运动后即刻、低氧运动后低氧恢复及常氧氧恢复1d,2d,7d的大鼠腓肠肌HSP70蛋白表达水平。结果：急性间歇低氧运动后即刻HSP70蛋白表达水平开始升高。常氧恢复第1dHSP70蛋白表达水平显著高于常氧对照组,P〈0．05。第2d、第7d呈现先降低后升高的趋势;低氧恢复中HSP70蛋白表达水平均显著高于常氧对照组,P〈0．05。结论：急性间歇低氧运动后能诱导HSP70蛋白表达水平升高;低氧恢复过程中HSP70蛋白高水平表达维持的时间要比常氧恢复长。     

Shoot-Specific Down-Regulation of Protein Farnesyltransferase （α-Subunit） for Yield Protection against Drought in Canola

Canola （Brassica napus L.） is one of the most important oilseed crops in the world and its seed yield and quality are significantly affected by drought stress. As an innate and adaptive response to water deficit, land plants avoid potential damage by rapid biosynthesis of the phytohormone abscisic acid （ABA）, which triggers stomatal closure to reduce transpirational water loss. The ABA-mediated stomatal response is a dosage-dependent process; thus, one genetic engineering approach for achieving drought avoidance could be to sensitize the guard cell＇s responsiveness to this hormone. Recent genetic studies have pinpointed protein farnesyltransferase as a key negative regulator controlling ABA sensitivity in the guard cells. We have previously shown that down-regulation of the gene encoding Arabidopsis β-subunit of farnesyltransferase （ERA1） enhances the plant＇s sensitivity to ABA and drought tolerance. Although the β-subunit of famesyltransferase （AtFTA） is also implicated in ABA sensing, the effectiveness of using such a gene target for improving drought tolerance in a crop plant has not been validated. Here, we report the identification and characterization of the promoter of Arabidopsis hydroxypyruvate reductase （AtHPR1）, which expresses specifically in the shoot and not in non-photosynthetic tissues such as root. The promoter region of AtHPR1 contains the core motif of the well characterized dehydration-responsive cis-acting element and we have confirmed thatAtHPR1 expression is inducible by drought stress. Conditional and specific down-regulation of FTA in canola using the AtHPR1 promoter driving an RNAi construct resulted in yield protection against drought stress in the field. Using this molecular strategy, we have made significant progress in engineering drought tolerance in this important crop species.     

Plant Cell Wall Proteomics： Mass Spectrometry Data,a Trove for Research on Protein Structure/Function Relationships

Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organelles. In particular, mass spectrometry （MS） analysis of gel-separated proteins produces data not only for protein identification, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics ofArabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins （H/PRPs） is arduous, different types of MS spectra were exploited to identify and characterize an H/PRP. Maturation events in a couple of cell wall proteins （CWPs） were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.     

Protein engineering of a fibroblast growth factor-1 fusion protein with cell adhesive activity

Fibroblast growth factor-1 （FGF1） is one of the most potent angiogenic growth factors, and also plays an important role in regulating cellular functions including cell proliferation, motility, differentiation, survival, and tissue regeneration processes. Here we described a novel fusion protein that was designed by combining the cell adhesion sequence from fibronectin with FGF1. The F1-Fn fusion protein functions as a minimized protein that directs integrin-dependent cell adhesion and stimulates cellular responses including cell proliferation and differentiation. Moreover, our results indicate that Fn-mediated signaling synergizes with signals from FGF1 in promoting cellular adhesion, proliferation, and differentiation in MG63 cells.     

Protein-Repairing Methionine Sulfoxide Reductases in Photosynthetic Organisms： Gene Organization,Reduction Mechanisms,and Physiological Roles

Methionine oxidation to methionine sulfoxide （MetSO） is reversed by two types of methionine sulfoxide reducrases （MSRs）, A and B, specific to the S- and R-diastereomers of MetSO, respectively. MSR genes are found in most organisms from bacteria to human. In the current review, we first compare the organization of the MSR gene families in photosynthetic organisms from cyanobacteria to higher plants. The analysis reveals that MSRs constitute complex families in higher plants, bryophytes, and algae compared to cyanobacteria and all non-photosynthetic organisms. We also perform a classification, based on gene number and structure, position of redox-active cysteines and predicted sub-cellular localization. The various catalytic mechanisms and potential physiological electron donors involved in the regeneration of MSR activity are then de- scribed. Data available from higher plants reveal that MSRs fulfill an essential physiological function during environmental constraints through a role in protein repair and in protection against oxidative damage. Taking into consideration the ex- pression patterns of MSR genes in plants and the known roles of these genes in non-photosynthetic cells, other functions of MSRs are discussed during specific developmental stages and ageing in photosynthetic organisms.     

A Myosin XI Tail Domain Homologous to the Yeast Myosin Vacuole-Binding Domain Interacts with Plastids and Stromules in Nicotiana benthamiana

The actin cytoskeleton plays a role in mobility of many different organelles in plant cells, including chloroplasts, mitochondria, Golgi, and peroxisomes. While progress has been made in identifying the myosin motors involved in trafficking of various plant organelles, not all of the cargoes mobilized by different members of the myosin XI family have yet been identified. The involvement of myosins in chloroplast positioning and mitochondrial movement was demonstrated by expression of a virus-induced gene silencing （VIGS） construct in tobacco. When VIGS with two different conserved sequences from a myosin Xl motor was performed in plants with either GFP-labeled plastids or mitochondria, chloroplast positioning in the dark was abnormal, and mitochondrial movement ceased. Because these and prior obser- vations have implicated a role for myosins and the actin cytoskeleton in plastid and stromule movement, we searched for myosin tail domains that could associate with plastids and stromules. While a yellow fluorescent protein （YFP） fusion with the entire tail region of myosin XI-F was usually found only in the cytoplasm, we observed that an Arabidopsis or Nicotiana benthamiana YFP：：myosin XI-F tail domain homologous to the yeast myo2p vacuole-binding domain associated with plastids and stromules after transient expression in N. benthamiana. Taken together, these observations implicate myosin motor proteins in dynamics of plastids and stromules.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

Induction of Protection against Paraquat-induced Oxidative Damage by Abscisic Acid in Maize Leaves is Mediated through Mitogen-activated Protein Kinase

Mitogen-activated protein kinase （MAPK） cascade has been shown to be important components in stress signal transduction pathway. In the present study, protection of maize seedlings （Zea mays L.） against paraquat-generated oxidative toxicity by abscisic acid （ABA）, its association with MAPK and ZmMPK5, a candidate for MAPK were investigated. Treatment of maize leaves with exogenous ABA led to significant decreases in the content of malondialdehyde, the percentage of ion leakage and the level of protein oxidation （in terms of carbonyl groups） under paraquat （PQ） stress. However, such decreases were blocked by the pretreatment with two MAPK kinase inhibitors PD98059 and U0126. The damage caused by PQ was further aggravated by inhibitors. Two inhibitors also suppressed the total activities of the antioxidant enzymes superoxide dismutase （SOD, EC 1.15.1.1）, catalase （CAT, EC 1.11.1.6）, ascorbate peroxidase （APX, EC 1.11.1.tl）, and glutathione reductase （GR, EC 1.6.4.2）. Besides, treatment with PQ stimulated the activation of a 46 kDa MAPK, which was identified as ZmMPK5 by in-gel kinase assay with immunoprecipitation. These results reveal that ABA-induced protection against PQ-generated oxidative damage is mediated through MAPK cascade in maize leaves, in which ZmMPK5, a candidate for MAPK, is demonstrated to be involved.     

RanBPM is an acetylcholinesterase-interacting protein that translocates into the nucleus during apoptosis

Acetylcholinesterase （ACHE） expression may be induced during apoptosis in various cell types. Here, we used the C-terminal of AChE to screen the human fetal brain library and found that it interacted with Ran-binding protein in the microtubule-organizing center （RanBPM）. This interaction was further confirmed by coimmunoprecipitation analysis. In HEK293T cells, RanBPM and AChE were heterogeneously expressed in the cisplatin-untreated cytoplasmic extracts and in the cisplatin-treated cytoplasmic or nuclear extracts. Our previous studies performed using morphologic methods have shown that AChE translocates from the cytoplasm to the nucleus during apoptosis. Taken together, these results suggest that RanBPM is an AChE-interacting protein that is translocated from the cytoplasm into the nucleus during apoptosis, similar to the translocation observed in case of ACHE.     

The structure-function relationship of MSI7, a matrix protein from pearl oyster Pinctada fucata

We previously identified a matrix protein, MSI7, from pearl oyster Pinctada fucata. According to the structural analysis, the DGD site in the N-terminal of MSI7 is crucial for its role in the shell formation. In this study, we expressed a series of recombinant MSI7 proteins, including the wild-type and several mutants directed at the DGD site, using an Escherichia coli expression system to reveal the structure-function relationship of MSI7. Furthermore, in vitro crystallization, crystallization speed assay, and circular dichroism spectrometry were carried out. Results indicated that wild-type MSI7 could induce the nucleation of aragonite and inhibit the crystallization of calcite. However, none of the mutants could induce the nucleation of aragonite, but all of them could inhibit the crystallization of calcite to some extent. And all the proteins accelerated the crystallization process. Taken together, the results indicated that MSI7 could contribute to aragonite crystallization by inducing the nucleation of aragonite and inhibiting the crystallization of calcite, which agrees with our prediction about its role in the nacreous layer formation of the shell. The DGD site was critical for the induction of the nucleation of aragonite.