张炜
植物细胞程序性死亡,蛋白质工程。
个性化签名
- 姓名:张炜
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学术头衔:
博士生导师, 教育部“新世纪优秀人才支持计划”入选者
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学科领域:
生物化学
- 研究兴趣:植物细胞程序性死亡,蛋白质工程。
张炜,南京农业大学生命科学学院生物化学与分子生物学系教授,博士生导师。1970年10月生,陕西西安人。1992年毕业于兰州大学生物系,理学学士。1995年毕业于华东师范大学生物系,理学硕士。1998年毕业于复旦大学遗传学研究所,理学博士。1998年至2003年,美国加州大学洛杉矶分校(UCLA)、德克萨斯州立大学西南医学中心(UT Southwestern)博士后。2003年至今,南京农业大学生命科学学院教授。
目前研究方向:植物细胞程序性死亡,蛋白质工程。
承担科研项目:主持863计划课题1项;国家自然科学基金2项;江苏省基础研究计划创新人才项目1项;2005年教育部新世纪优秀人才计划资助;南京农业大学引进人才基金资助。
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411
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成果数
9
【期刊论文】New changes in the plasma-membrane-associated proteome of rice roots under salt stress
张炜, Yanwei Cheng, Yaocheng Qi, Qian Zhu, Xi Chen, Ning Wang, Xin Zhao, Haiyan Chen, Xiangju Cui, Langlai Xu and Wei Zhang
Proteomics 2009, 9, 3100-3114,-0001,():
-1年11月30日
To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma-membrane-associated proteins following treatment with 150mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt-sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two-phase partitioning, and plasma-membrane-associated proteins were separated by IEF/SDS-PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5-fold in response to salt stress. Of these proteins, nine were upregulated and nine were down-regulated. MS analysis indicated that most of these membraneassociated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine-rich-repeat type receptor-like protein kinase, OsRPK1, was identified as a salt-responding protein. Immuno-blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno-histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms.
Leucine-rich-repeat receptor-like protein kinase/, Oryza sativa/, Plasma membrane/, Proteomics/, Salt stress
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【期刊论文】Mitochondrial proteome during salt stress-induced programmed cell death in rice
张炜, Xi Chen, Ying Wang, Jianyou Li, Ailiang Jiang, Yanwei Cheng, Wei Zhang*
Plant Physiology and Biochemistry 47 (2009) 407-415,-0001,():
-1年11月30日
It has been shown that mitochondria play a pivotal role in plant programmed cell death (PCD). Previous study established a salt stress-induced PCD model in rice (Oryza sativa L. cv. WYJ 8th) root tip cells, demonstrated by DNA laddering, cytochrome c release, and TUNEL positive reaction. In this study, the role of mitochondria during the early phase of PCD (2h-PCD) was analyzed in rice roots. After 2h-PCD induction, the integrity of mitochondria decreased slightly, consistent with a small release of cytochrome c. 2h-PCD partially inhibited electron transport, resulting in oxidative burst in mitochondria. However, ATP production maintained constant. Mitochondria proteome were analyzed by two-dimensional IEF/SDS-PAGE before and after 2h-PCD induction, and eight PCD-related proteins were identified. Among them, four proteins were up-regulated after PCD induction, which included glycoside hydrolase, mitochondrial heat shock protein 70, 20S proteasome subunit, and Cu/Zn-superoxide dismutase, and four were down-regulated, namely ATP synthase beta subunit, cytochrome c oxidase subunit 6b, S-adenosylmethionine synthetase 2, and transcription initiation factor eIF-3 epsilon. These results suggested that ATP synthase may not be the major producer of ATP in mitochondria during the early stage of PCD in rice. Glycoside hydrolase may be involved in ETC impairment and ROS burst, and mitochondrial HSP70 is a potential candidate for PCD regulation. The possible roles of other proteins on PCD initiation were also discussed.
Oryza sativa, Programmed cell death, Proteomics, Mitochondria, Salt stress, Heat shock protein 70
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张炜, Ai-liang Jiang, Yanwei Cheng, Jianyou Li, Wei Zhang
Journal of Plant Physiology 165 (2008) 1134-1141,-0001,():
-1年11月30日
DNA laddering is one of the biochemical processes characteristic of programmed cell death (PCD) both in animals and plants. However, the mechanism of DNA laddering varies in different species, even in different tissues of one organism. In the present study, we used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonuclease activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20 kDa (OsCyt20) and a nuclear of 37 kDa (OsNuc37), were identified as PCD related. Our results indicated that OsCyt20 is a Ca2+/Mg2+-dependent nuclease, which is most active at neutral pH, and that OsNuc37 is Zn2+-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2h salt treatment) and exhibited the highest activity approximately 4 h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering. The different effects of both endonucleases on DNA degradation during salt-induced PCD are discussed.
DNA laddering, Endonuclease, Oryza sativa, Programmed cell death, Salt stress
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【期刊论文】水稻LRR型类受体蛋白激酶胞外区的原核表达及多克隆抗体制备*
张炜, 程彦伟**, 李亮**, 沈嵘, 齐耀程, 刘晓宇, 王宁, 张炜***
生物化学与生物物理进展,2008,35(9):1077~1083,-0001,():
-1年11月30日
前期研究表明,水稻根尖细胞质膜类受体蛋白激酶OsRLK的表达受盐胁迫诱导。为了进一步研究该激酶的生理功能,通过反转录PCR得到OsRLK胞外区cDNA片段,将其亚克隆至pET29a原核表达载体并在大肠杆菌中实现了高表达,表达量约为细胞总蛋白的30%。重组蛋白经SDS-PAGE分离,染色切胶收集后,作为抗原免疫新西兰家兔,分离抗血清,经纯化得到1:20000效价的多克隆抗体。Western blot结果显示,该抗体能特异识别在原核表达系统内表达的抗原,以及水稻根尖细胞质膜组分中的LRR型类受体蛋白激酶,并且在蛋白质水平证实该激酶为盐胁迫响应蛋白。
LRR型类受体蛋白激酶,, 水稻,, 原核表达,, 多克隆抗体
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张炜, Xi Chen, Wei Zhang *, Yajing Xie, Wei Lu, Rongxian Zhang
Plant Science 173 (2007) 397-407,-0001,():
-1年11月30日
A novel rice mutant (Oryza sativa L. var. Zhenhui 249Y) was identified as a low chlorophyll b mutant with a chlorophyll a/b ratio of 4.7. Chloroplast ultrastructure showed decreased grana lamellae and slightly swelling thylakoid in the mutant. Chlorophyll fluorescence assay demonstrated that the mutant noticeably reduced PSII thermostability. However, heat-induced PsbO release from thylakoid membrane indicated an increased thermo-resistance of PSII structure in the mutant. In order to explain these phenomena, two different 2D electrophoresis approaches have been used to compare the thylakoid proteome between the mutant and the wild type: two-dimensional blue native-SDS-PAGE and isoelectric focusing-SDS-PAGE, which was followed by MS identification. Totally, 52 proteins were identified. The results indicated that the LHC-IIb in the mutant was about 40% less than that in wild type. However, both LHC-Ia and LHC-Ib decreased more greatly in this mutant, only 20% remaining. IEF–SDS-PAGE showed that H+-transporting ATP synthase, fructose-bisphosphate aldolase, PSII assembly/stability factor HCF136 increased in the mutant, while PsaE and 8.7 kDa Fe-S protein, decreased. The results suggest that in our mutant, maintenance of the water-oxidizing system is not the major reason of LHC-II stabilization effect on PSII complexes. In addition, the reduction of Chl b affected LHC-I assembly more severely than LHC-II.
Blue native–SDS-PAGE, IEF–SDS-PAGE, Chlorophyll b-less mutant, PSII stability, Oryza sativa
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【期刊论文】Proteomic Analysis of Integral Plasma Membrane Proteins
张炜, Yingxin Zhao, Wei Zhang, Yoonjung Kho, and Yingming Zhao*
Anal. Chem. 2004, 76, 1817-1823,-0001,():
-1年11月30日
Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotindirected affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membraneassociated cytosolic proteins by harsh washes with highsalt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation as almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 íg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.
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【期刊论文】Site-Directed Sulfhydryl Labeling of Helix IX in the Lactose Permease of Escherichia coli†
张炜, Wei Zhang, Yonglin Hu, and H. Ronald Kaback*
Biochemistry 2003, 42, 4904-4908,-0001,():
-1年11月30日
Site-directed sulfhydryl modification of transmembrane helix IX in the lactose permease of Escherichia coli was studied in right-side-out membrane vesicles with the thiol-specific reagents N-[14C]-ethylmaleimide (NEM) and methanethiosulfonate ethylsulfonate (MTSES) which are permeant and impermeant, respectively. Out of 20 mutants with a single Cys residue at each position in the helix, only five mutants label with NEM. (i) Cys residues at positions 291, 308, and 310 label at 25℃, and binding of substrate has no effect. (ii) Cys residues at positions 295 and 298 label only in the presence of substrate. NEM labeling at 0℃ indicates that alkylation of Cys residues at positions 295 and 308 is dependent on the thermal motion of the protein. In contrast, temperature has little effect on labeling of Cys residues at positions 291, 298, and 310. Interestingly, pretreatment with MTSES blocks NEM labeling of all the mutants. The findings demonstrate that the face of helix IX on which Arg302 is located is involved in ligand-induced conformational changes and accessible to water from the periplasmic surface of the membrane. Since Arg302 facilitates deprotonation of Glu325 (helix X) during turnover [Sahin-Toth, M., and Kaback, H. R. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6068-6073], the findings are consistent with the idea that this face of helix IX may comprise part of the H+ translocation pathway.
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张炜, Wei Zhang, Lan Guan and H. Ronald Kaback*
J. Mol. Biol. (2002) 315, 53-62,-0001,():
-1年11月30日
By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N7/C5 split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-speci®c cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 AÊ), N,N0-ophenylenedimaleimide (6 AÊ) and N,N0-p-phenylenedimaleimide (10 AÊ). Mutant Asp240→Cys (helix VII)/Lys319→Cys (helix X) cross-links most ef®ciently with the 3 AÊ reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired. Furthermore, cross-linking the two positions inactivates the protein. Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices. Thus, helices VII and X are in close proximity in the middle of the membrane. In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases. In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices. The results are consistent with a recently proposed mechanism for lactose/H+ symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H+ from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X).
bioenergetics, transport, membrane proteins, helix packing, author thiol cross-linking
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【期刊论文】Effect of the Lipid Phase Transition on the Lactose Permease from Escherichia coli†
张炜, Wei Zhang and H. Ronald Kaback*
Biochemistry 2000, 39, 14538-14542,-0001,():
-1年11月30日
The temperature dependence of lactose active transport, efflux down a concentration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose permease and mutant Glu325 f Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve H+ symport, Arrhenius plots with wild-ype permease exhibit a discontinuity at 18-19℃ with a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve H+ symport, the change in activation energy is much less pronounced (2-3-fold) than that observed for active transport or efflux. Strikingly, mutant Glu325f Ala, which catalyzes equilibrium exchange as well as wild-type permease but is defective in all translocation reactions that involve net H+ translocation, exhibits no change whatsoever in activation energy. The findings are consistent with the conclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and H+ translocation rather than the conformational change (s) responsible for translocation across the membrane.
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