李俊发
①学习、记忆和神经可塑性方面;②蛋白质化学修饰方面;③脑低氧预适应细胞分子机制方面。
个性化签名
- 姓名:李俊发
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学术头衔:
博士生导师
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学科领域:
神经生物学
- 研究兴趣:①学习、记忆和神经可塑性方面;②蛋白质化学修饰方面;③脑低氧预适应细胞分子机制方面。
李俊发,1962年10月出生,男,汉族,籍贯北京。1986年8月毕业于首都医科大学,获医学学士学位;1992年6月毕业于中国协和医科大学,获医学硕士学位;1996年2月至2001年2月,在美国国立卫生研究院(NIH)完成5年博士后培训。现任首都医科大学分子生物学实验测试室主任;神经生物学系副主任、副研究员。兼任中国生理学学会理事,神经科学专业委员会和科普工作委员会委员;中国神经科学学会分子神经生物学专业委员会委员;中国生物化学和分子生物学学会、美国生物化学和分子生物学学会(ASBMB)等学术组织会员;【生理学通讯】编委、【基础医学与临床】常务编委。获奖情况:曾获“美籍华人葛克全基金奖”(1992.6);“北京市高等学校优秀青年骨干教师”(1992.8);“全国青年优秀生理学学术论文奖”(1993.7);“北京市卫生系统优秀青年教师”(1993.9)等。研究领域及成果:①在学习、记忆和神经可塑性方面,发现脑特异性蛋白-神经颗粒素(Neurogranin, Ng) 基因敲除小鼠的学习、记忆受损,并证明这一受损程度与细胞内的Ng表达水平有关(J. Neurosci., 2004;Proc.Natl.Acad.Sci. U.S.A., 2000);②在蛋白质化学修饰方面,尤其是蛋白质的谷胱甘肽化 (S-glutathiolation) 修饰,发现Ng的氧化(J.Biol.Chem.,1999)及谷胱甘肽化(Biochemistry,2000)修饰可能参与了Ng对钙调蛋白(CaM)的细胞调节机制;并首次发现报道了一种重要化学物质-Glutathione disulfide S-oxide(GS(O)SG)存在于神经细胞内,并通过离体和在体实验证明,这一物质能有效地对蛋白质的-SH基团进行高效的谷胱甘肽化修饰(J.Biol.Chem.,2001)。蛋白质的谷胱甘肽化修饰参与体内蛋白功能的调节在国际上是一个刚刚引起人们注意的研究领域,鉴于目前人们尚不知何种机制参与了体内蛋白质谷胱甘肽化修饰,这一发现将具有重要的科学意义。因为根据GS(O)SG这一化学物质的存在及其可能的产生机制,它很可能在正常生理状态下参与一些蛋白功能的调节,并可能与病理生理状态下的氧化应激、衰老等过程中一些重要蛋白功能的改变有关;③脑低氧预适应细胞分子机制方面,目前作为项目负责人,在国家自然科学基金、北京市自然科学基金、教育部“优秀青年教师资助计划”等多项科研基金的资助下,正以蛋白激酶C(PKCs)、丝裂原激活的蛋白激酶(MAPKs) 及其下游作用底物等为靶蛋白,探讨蛋白质的化学修饰及其细胞信号转导机制在脑低氧预适发生发展过程中的作用,以进一步丰富人们对脑低氧预适应细胞分子机制的认识,并为临床防治缺血/低氧性脑损伤提供新的可能措施。
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李俊发, 李洪燕, 李俊发△, 吕国蔚
生理科学进展,2003,34(2):111~115,-0001,():
-1年11月30日
神经颗粒素(Neurogranin,Ng)是一种新发现的由78个氨基酸组成的脑特异性蛋白,主要分布于人类或动物的大脑皮层、海马和嗅球等脑区的神经突触后。作为Calpacitin蛋白家族中的一员,Ng是蛋白激酶C的天然作用底物及钙调蛋白(CaM)的储库。在生理状态下,Ng与CaM结合形成复合体,而在蛋白激酶C或氧化剂的作用下,Ng可被磷酸化、氧化及谷胱甘肽化等化学修饰,降低其与CaM的亲和力,从而参与对CaM及CaM2激活的蛋白酶,如CaM2依赖性NO合酶、CaM2依赖性蛋白激酶II(CaMKII)及CaM2依赖性腺苷酸环化酶的调节。同时,由于CaM2依赖性蛋白酶大多参与长时程增强(LTP)和长时程抑制(LTD)的诱导,并且Ng的基因表达和蛋白质合成与神经元的突触形成、分化同步,因此,Ng可能在学习、记忆、神经系统发育(可塑性)等生理性变化中具有重要作用。此外,一些研究表明,Ng还可能参与甲状腺机能减退、睡眠剥夺、衰老及脑低氧预适应等病理生理学变化所造成的神经系统功能的改变。
神经颗粒素, 钙调蛋白, CaM2依赖性蛋白酶II, CaM2依赖性NO合酶, CaM2依赖性腺苷酸环化酶
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【期刊论文】Glutathiolation of Proteins by Glutathione Disulfide S-Oxide Derived from S-Nitrosoglutathione
李俊发, Junfa Li, Freesia L. Huang, and Kuo-Ping Huang ‡
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (5): 3098-3105,-0001,():
-1年11月30日
S-Nitrosoglutathione (GSNO) undergoes spontaneous degradation that generates several nitrogen-containing compounds and oxidized glutathione derivatives. We identified glutathione sulfonic acid, glutathione disulfide S-oxide (GS (O) SG), glutathione disulfide S-dioxide, and GSSG as the major decomposition products of GSNO. Each of these compounds and GSNO were tested for their efficacies to modify rat brain neurogranin/RC3 (Ng) and neuromodulin/GAP-43 (Nm). Among them, GS (O) SG was found to be the most potent in causing glutathiolation of both proteins; four glutathiones were incorporated into the four Cys residues of Ng, and two were incorporated into the two Cys residues of Nm. Ng and Nm are two in vivo substrates of protein kinase C; their phosphorylations by protein kinase C attenuate the binding affinities of both proteins for calmodulin. When compared with their respective unmodified forms, the glutathiolated Ng was a poorer substrate and glutathiolated Nm a better substrate for protein kinase C. Glutathiolation of these two proteins caused no change in their binding affinities for calmodulin. Treatment of [35S] cysteine-labeled rat brain slices with xanthine/xanthine oxidase or a combination of xanthine/xanthine oxidase with sodium nitroprusside resulted in an increase in cellular level of GS (O) SG. These treatments, as well as those by other oxidants, all resulted in an increase in thiolation of proteins; among them, thiolation of Ng was positively identified by immunoprecipitation. These results show that GS (O) SG is one of the most potent glutathiolating agents generated upon oxidative stress.
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【期刊论文】N-Methyl-D-aspartate Induces Neurogranin/RC3 Oxidation in Rat Brain Slices*
李俊发, Junfa Li, Jhang Ho Pak, Freesia L. Huang, and Kuo-Ping Huang ‡
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (3): 1294-1300,-0001,():
-1年11月30日
Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca21. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter, N-methyl-D-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3-5 min and declined to base line in 30min. The response was dose-dependent (EC50 ;100mm) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitor NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. Ng was oxidized by NO donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione, and H2O2 at concentrations less than 0.5mm. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca21 and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.
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李俊发, Kuo-Ping Huang, *, ‡ Freesia L. Huang, ‡ Junfa Li, ‡ Peter Schuck, § and Peter McPhie|
Biochemistry 2000, 39, 7291-7299,-0001,():
-1年11月30日
Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca2+ concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the R-helical content of this protein. Mutation of the four Cys residues [Cys (-)-NG] to Gly and Ser did not affect the R-helical content either. Interaction of CaM with the reduced (red), glutathiolated (GS)-, or Cys(-)-NG in the Ca2+-free solution resulted in an increase in the R-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO4-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys (-)-NG > red-NG, GS-NG > ox-NG > PO4-NG. Unlike Cys (-)-, red-, and GS-NG, neither ox-nor PO4-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.
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李俊发, Chenchen Niu, Junfa Li*, Xiuyu Cui, Song Han, Penyu Zu, Hua Li, Qunyuan Xu*
,-0001,():
-1年11月30日
Previous studies have shown that the level of total conventional protein kinase C (cPKC) membrane translocation (activation) was increased in brain of hypoxic preconditioned mice. In order to find out which isoform of cPKC may participate in the development of cerebral hypoxic preconditioning (HPC), we used Western bolt and immunohistochemistry to observe the effects of repetitive hypoxic exposure (H1-H6, n=6 for each group) on the level of cPKC isoform-specific protein expression and its membrane translocation in cortex and hippocampus of mice. We found that levels of cPKC βII and γ membrane translocation were increased significantly (p<0.05 versus normoxic H0 group, n=6) in response to repetitive hypoxic exposure (H1-H4) at early phase of hypoxic preconditioning, but no significant changes of cPKC α and βI membrane translocation while cPKC α, βI, βII and γ protein expression were found both in hippocampus and cortex. In addition, an extensive subcellular redistribution of cPKC βII and γ was detected by immunohistochemistry staining in cortex after repetitive hypoxic exposures (H3). However, a significant decrease in the expression of cPKC γ protein (p<0.05 versus H0 group) was found only in cortex of delayed hypoxic preconditioned mice (H5-6). These results suggested that the activation of cPKC βII and γ may be involved in the early phase of cerebral hypoxic preconditioning and the changes in cPKC γ protein expression may participate in the development of late phase of cerebral hypoxic preconditioning, as well as selective vulnerability to hypoxia both in cortex and hippocampus.
cPKC isoforms, Hypoxic preconditioning, Membrane translocation, Protein expression, Brain
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李俊发, Kuo-Ping Huang, * Freesia L. Huang, * Tino Jager, Junfa Li, Klaus G. Reymann, and Detlef Balschun
The Journal of Neuroscience, November 2004, 24 (47): 10660-10669,-0001,():
-1年11月30日
Neurogranin (Ng) is a brain-specific, postsynaptically located protein kinase C (PKC) substrate, highly expressed in the cortex, hippocampus, striatum, and amygdala. This protein is a Ca2-sensitive calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. To investigate the role of Ng in neural function, a strain of Ng knockout mouse (KO) was generated. Previously we reported (Pak, J. H., Huang, F. L., Li, J., Balschun, D., Reymann, K. G., Chiang, C., Westphal, H., and Huang, K. -P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11232- 11237) that these KO mice displayed no obvious neuroanatomical abnormality, but exhibited deficits in learning and memory and activation of Ca2/CaM-dependent protein kinase II. In this report, we analyzed several downstream phosphorylation targets in phorbol 12-myristate 13-acetate-and forskolin-treated hippocampal slices from wild type (WT) and KO mice. Phorbol 12-myristate 13-acetate caused phosphorylation of Ng in WT mice and promoted the translocation of PKC from the cytosolic to the particulate fractions of both the WT and KO mice, albeit to a lesser extent in the latter. Phosphorylation of downstream targets, including mitogenactivated protein kinases, 90-kDa ribosomal S6 kinase, and the cAMP response element binding protein (CREB) was significantly attenuated in KO mice. Stimulation of hippocampal slices with forskolin also caused greater stimulation of protein kinase A (PKA) in the WT as compared with those of the KO mice. Again, phosphorylation of the downstream targets of PKA was attenuated in the KO mice. These results suggest that Ng plays a pivotal role in regulating both PKC-and PKA-mediated signaling pathways, and that the deficits in learning and memory of spatial tasks detected in the KO mice may be the result of defects in the signaling pathways leading to the phosphorylation of CREB.
neurogranin, calmodulin, water maze, learning, LTP, calcium signaling
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李俊发, Junfang Wu, Junfa Li, Kuo-Ping Huang, and Freesia L. Huang‡
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (22): 19498-19505,-0001,():
-1年11月30日
Neurogranin (Ng) is a brain-specific, postsynaptically located protein kinase C (PKC) substrate, highly expressed in the cortex, hippocampus, striatum, and amygdala. This protein is a Ca2-sensitive calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. To investigate the role of Ng in neural function, a strain of Ng knockout mouse (KO) was generated. Previously we reported (Pak, J. H., Huang, F. L., Li, J., Balschun, D., Reymann, K. G., Chiang, C., Westphal, H., and Huang, K. -P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11232- 11237) that these KO mice displayed no obvious neuroanatomical abnormality, but exhibited deficits in learning and memory and activation of Ca2/CaM-dependent protein kinase II. In this report, we analyzed several downstream phosphorylation targets in phorbol 12-myristate 13-acetate-and forskolin-treated hippocampal slices from wild type (WT) and KO mice. Phorbol 12-myristate 13-acetate caused phosphorylation of Ng in WT mice and promoted the translocation of PKC from the cytosolic to the particulate fractions of both the WT and KO mice, albeit to a lesser extent in the latter. Phosphorylation of downstream targets, including mitogenactivated protein kinases, 90-kDa ribosomal S6 kinase, and the cAMP response element binding protein (CREB) was significantly attenuated in KO mice. Stimulation of hippocampal slices with forskolin also caused greater stimulation of protein kinase A (PKA) in the WT as compared with those of the KO mice. Again, phosphorylation of the downstream targets of PKA was attenuated in the KO mice. These results suggest that Ng plays a pivotal role in regulating both PKC-and PKA-mediated signaling pathways, and that the deficits in learning and memory of spatial tasks detected in the KO mice may be the result of defects in the signaling pathways leading to the phosphorylation of CREB.
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李俊发, Jhang Ho Pak*†‡, Freesia L. Huang*†, Junfa Li†, Detlef Balschun§, Klaus G. Reymann¶, Chin Chiangi**, Heiner Westphal**, and Kuo-Ping Huang†, ††
PNAS October 10, 2000 vol. 97 no.21,-0001,():
-1年11月30日
NeurograninyRC3 is a neural-specific Ca21-sensitive calmodulin CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. Here we show that deletion of the Ng gene in mice did not result in obvious developmental or neuroanatomical abnormalities but caused an impairment of spatial learning and changes in hippocampal short- and long-term plasticity (paired-pulse depression, synaptic fatigue, long-term potentiation induction). These deficits were accompanied by a decreased basal level of the activated Ca21yCaM-dependent kinase II (CaMKII) ('60% of wild type). Furthermore, hippocampal slices of the mutant mice displayed a reduced ability to generate activated CaMKII after stimulation of protein phosphorylation and oxidation by treatments with okadaic acid and sodium nitroprusside, respectively. These results indicate a central role of Ng in the regulation of CaMKII activity with decisive influences on synaptic plasticity and spatial learning.
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【期刊论文】The FBP Interacting Repressor Targets TFIIH to Inhibit Activated Transcription
李俊发, Juhong Liu, * Liusheng He, *# Irene Collins, *# Hui Ge, † Daniel Libutti, * Junfa Li, ‡ Jean-Marc Egly, § and David Levens*ш
Molecular Cell, Vol.5, 331-341, February, 2000, Copyright,-0001,():
-1年11月30日
FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes, pos- sesses potent transcription activation and repression domains, and is necessary for c-myc expression. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocked activator-dependent, but not basal, transcription through TFIIH. Recruited through FBP' s nucleic acid-binding domain, FIR formed a ternary complex with FBP and FUSE. FIR repressed a c-myc reporter via the FUSE. The amino terminus of FIR con-tained an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targeted only TFIIH' s p89/XPB helicase, required at several stages in tran-scription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription.
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【期刊论文】重复性低氧对小鼠脑内cPKCα和γ膜转位及蛋白表达量的影响
李俊发, 牛晨晨, 李俊发*, 徐群渊
基础医学与临床,2005,25(1):30~34,-0001,():
-1年11月30日
探讨cPKCs特定亚型在脑低氧预适应形成过程中的作用,借助已建立的小鼠整体低氧预适应模型,应用SDS-PAGE和Western bolt等生化技术,并结合Gel Doc成像系统,半定量检测脑组织内cPKCα和γ的膜转位水平和蛋白表达量。结果发现,随低氧暴露次数(低氧2-4次)增加,在小鼠海马和皮层组织内cPKCγ膜转位水平增高显著(p<0.05,n=6);然而,cPKCα的膜转位水平和cPKCα及γ的蛋白表达量的变化均不显著。提示,cPKCγ的激活可能参与了脑低氧预适应的形成过程。
脑低氧预适应, 经典型蛋白激酶C, 膜转位, 蛋白表达量, 脑组织。
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