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浙江大学学报（医学版） 2019年第1期48卷 50-57页

作者：杜东芬朱丽霞王云贵叶琇锦浙江大学医学院附属第一医院血液病高级病区浙江杭州310003 浙江省绍兴市中心血站浙江绍兴312000 浙江大学医学院附属第一医院血液科浙江大学血液病研究所浙江杭州310003

目的:分析急性髓系白血病(AML)患者肾母细胞瘤1(WT1)基因表达水平的差异,探讨WT1表达及其在核仁磷酸蛋白1(NPM1)或Fms样酪氨酸激酶3内部串联重复(FLT3-ITD)不同突变状态下与AML患者疗效和生存的相关性,评估其对于患者预后的预测价值。方... 详细信息

目的:分析急性髓系白血病(AML)患者肾母细胞瘤1(WT1)基因表达水平的差异,探讨WT1表达及其在核仁磷酸蛋白1(NPM1)或Fms样酪氨酸激酶3内部串联重复(FLT3-ITD)不同突变状态下与AML患者疗效和生存的相关性,评估其对于患者预后的预测价值。方法:以167例初发AML患者(除外M3亚型)为研究对象,根据初诊时WT1表达水平分为WT1高表达组和WT1低表达组,回顾性分析两组的临床及实验室检查资料,采用Kaplan-Meier法进行患者生存分析以明确WT1表达水平在预后评估中的价值,特别是在NPM1或FLT3-ITD不同突变状态AML患者中的预后评估价值。结果:WT1高表达组126例患者中治疗有效83例(65.9%),低表达组41例患者中治疗有效39例(95.1%),差异有统计学意义(P<0.01)。WT1高表达组2年总存活率低于WT1低表达组(46.1%和75.2%,P<0.05),2年无病存活率也低于WT1低表达组(43.5%和68.5%,P<0.05)。诱导化疗前后WT1表达量下降≥1 log患者总反应率和2年总存活率优于下降<1 log患者(均P<0.05),但两者2年无病存活率差异无统计学意义(P>0.05)。NPM1野生型患者中,WT1高表达组总反应率、2年总存活率较WT1低表达组低(均P<0.05);FLT3-ITD野生型患者中,WT1高表达组总反应率、2年总存活率和2年无病存活率均较WT1低表达组低(均P<0.05);而NPM1或FLT3-ITD突变患者中,不同WT1表达水平组疗效及生存分析结果差异无统计学意义(均P>0.05)。结论:初发AML患者中WT1基因过表达提示预后不良;诱导治疗后WT1表达量下降≥1 log的患者预后优于下降<1 log的患者;初诊WT1基因表达水平可以作为NPM1或FLT3-ITD野生型AML患者的预后判断指标。

关键词：白血病,髓样,急性/病理学基因,肾母细胞瘤磷蛋白类/遗传学核蛋白质类/遗传学蛋白酪氨酸激酶类/遗传学 fms样酪氨酸激酶3/遗传学突变预后

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A method for the large-scale cloning of nuclear proteins and nuclear targeting sequences on a functional basis

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ANALYTICAL BIOCHEMISTRY 2000年第2期284卷 231-239页

作者： Pichon, B Mercan, D Pouillon, V Christophe-Hobertus, C Christophe, D Free Univ Brussels Hop Erasme IRIBHN Fac Med B-1070 Brussels Belgium Free Univ Brussels Hop Erasme Serv Chim B-1070 Brussels Belgium

We describe here a selection strategy allowing the cloning of sequences that contain a functional nuclear targeting signal. Our method relies on the use of green fluorescent protein fusion proteins to identify nuclear targeting sequences. Transfected cells expressing nuclear protein fusions were isolated on the basis of their nuclear fluorescence using flow cytometry and the transfected DNAs were recovered after bacterial transformation with total DNA from pools of sorted cells. Starting from a cDNA expression library, in which only 1% of the expressed proteins were nuclear, we obtained a 70-fold enrichment in nuclear protein-encoding clones after a single round of selection. Among the 63 clones that have been partially sequenced to date, 25 (40%) corresponded to known nuclear proteins and 13 (20%) to previously uncharacterized sequences. Despite their ability to target the green fluorescent protein marker to the cell nucleus, about half of the cloned sequences did not encode canonical basic or bipartite nuclear localization signals. The method can thus be applied to the large-scale cloning of functional nuclear targeting sequences, which opens the way to a wide investigation of nuclear import mechanisms and to the identification of previously unknown nuclear proteins, (C) 2000 Academic Press.

关键词：碱基序列克隆分子 DNA引物 DNA 互补分子序列数据核定位信号/遗传学核蛋白质类/遗传学质粒皂苷类/化学动物人类

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Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第41期274卷 29211-29219页

作者： Serpe, M Joshi, A Kosman, DJ SUNY Buffalo Dept Biochem Sch Med & Biomed Sci Buffalo NY 14214 USA

The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of yeast high affinity copper uptake. A positive transcription factor, Mac1p binds via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1, encoding a copper permease and metal reductase, respectively. Mac1p-dependent transcriptional. activation is negatively regulated by copper. We have mapped the domains in Mac1p responsible for its nuclear localization and for the protein-protein interactions that underlie its transcriptional activity. Immunofluorescence studies indicate that Mac1p contains two nuclear localization signals, one each in the N- and C-terminal halves of the protein. Yeast one-hybrid analysis demonstrates that the copper-dependent transcriptional activity in Mac1p resides primarily in a cysteine-rich element encompassing residues 264-279. Two-hybrid analysis indicates that a copper-independent Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted helix in the C-terminal region of the protein encompassing residues 388-406. Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro. When produced in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support transcriptional activation in vivo consistent with an essential Mac1p dimerization in transcriptional activation. Lastly, the one- and two-hybrid data indicate that an intramolecular interaction between the DNA-binding and transactivation domains negatively modulates Mac1p activity.

关键词：结合部位阳离子转运蛋白质类铜/代谢铜/药理学 DNA结合蛋白质类/化学 DNA结合蛋白质类/遗传学二聚作用荧光抗体技术真菌蛋白质类/化学真菌蛋白质类/遗传学膜蛋白质类/遗传学核定位信号核蛋白质类/化学核蛋白质类/遗传学点突变启动区遗传蛋白质结构二级酿酒酵母菌/代谢酿酒酵母蛋白质类构效关系反式激活因子类/化学反式激活因子类/遗传学转录因子/化学转录因子/遗传学

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Binding of p53 to the KIX domain of CREB binding protein - A potential link to human T-cell leukemia virus, type I-associated leukemogenesis

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第37期274卷 26321-26328页

作者： Van Orden, K Giebler, HA Lemasson, I Gonzales, M Nyborg, JK Colorado State Univ Dept Biochem & Mol Biol Ft Collins CO 80523 USA

The pleiotropic cellular coactivator CREB binding protein (CBP) plays a critical role in supporting p53-dependent tumor suppressor functions. p53 has been shown to directly interact with a carboxyl-terminal region of CBP for recruitment of the coactivator to p53-responsive genes. In this report, we identify the KM domain as a new p53 contact point on CBP. We show that both recombinant and endogenous forms of p53 specifically interact with KM. We demonstrate that the activation domain of p53 participates in KM binding and provide evidence showing that this interaction is critical for p53 transactivation function. The human T-cell leukemia virus, type-I-encoded oncoprotein Tax is a well established repressor of p53 transcription function. Like p53, Tax also binds to KIX. The finding that both transcription factors bind to a common region of CBP suggests that coactivator competition may account for the observed repression. We demonstrate reciprocal repression between Tax and p53 in transient transfection assays, supporting the idea of intracellular coactivator competition. We biochemically confirm coactivator competition by directly showing that both transcription factors bind to KM in a mutually exclusive fashion. These data provide molecular evidence for the observed intracellular competition and suggest that Tax inhibits p53 function by abrogating a novel p53-KIX interaction. Thus, Tax competition for the p53-KIX complex may be a pivotal event in the human T-cell leukemia virus, type I transformation pathway.

关键词：结合竞争性 CREB结合蛋白质克隆分子基因产物 tax/代谢人类嗜T淋巴细胞病毒1型/生理学 Jurkat细胞白血病 T细胞/病毒学核蛋白质类/遗传学核蛋白质类/代谢蛋白质结合重组融合蛋白质类/遗传学重组融合蛋白质类/代谢反式激活因子类/遗传学反式激活因子类/代谢肿瘤抑制蛋白质p53/代谢人类

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E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control

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MOLECULAR CELL 2000年第3期6卷 729-735页

作者： Gaubatz, S Lindeman, GJ Ishida, S Jakoi, L Nevins, JR Livingston, DM Rempel, RE Dana Farber Canc Inst Boston MA 02115 USA Harvard Univ Sch Med Boston MA 02115 USA Duke Univ Med Ctr Howard Hughes Med Inst Dept Genet Durham NC 27710 USA

E2F transcription factors are major regulators of cell proliferation. The diversity of the E2F family suggests that individual members perform distinct functions in cell cycle control. E2F4 and E2F5 constitute a defined subset of the family. Until now, there has been little understanding of their individual biochemical and biological functions. Here, we report that simultaneous inactivation of E2F4 and E2F5 in mice results in neonatal lethality, suggesting that they perform overlapping functions during mouse development. Embryonic fibroblasts isolated from these mice proliferated normally and reentered from Go with normal kinetics compared to wild-type cells. However, they failed to arrest in G1 in response to p16(INK4a). Thus, E2F4 and E2F5 are dispensable for cell cycle progression but necessary for pocket protein-mediated G1 arrest of cycling cells.

关键词：等位基因载体蛋白质类/遗传学细胞存活/遗传学周期素依赖激酶抑制剂p16 周期素依赖激酶抑制剂p21 细胞周期蛋白类/遗传学 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/代谢 E2F4转录因子 E2F5转录因子成纤维细胞/细胞学 G1期/生理学基因表达调控发育期基因 ras/生理学基因型杂合子纯合子小鼠近交BALBC 小鼠近交C57BL 小鼠基因敲除核蛋白质类/遗传学核蛋白质类/代谢表型磷蛋白类/遗传学磷蛋白类/代谢磷酰化蛋白质类视网膜母细胞瘤蛋白质/遗传学视网膜母细胞瘤蛋白质/代谢视网膜母细胞瘤样蛋白质p107 视网膜母细胞瘤样蛋白质p130 转录因子/遗传学转录因子/代谢转录遗传/生理学转染动物小鼠

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Isolation of a 40-kDa Huntingtin-associated protein

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第5期276卷 3188-3194页

作者： Peters, MF Ross, CA Johns Hopkins Univ Sch Med Dept Psychiat Mol Neurobiol LabDiv Neurobiol Baltimore MD 21205 USA Johns Hopkins Univ Sch Med Dept Neurosci Baltimore MD 21205 USA Johns Hopkins Univ Sch Med Program Cellular Mol Med Baltimore MD 21205 USA

Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, the function of normal huntingtin and the mechanism by which expanded huntingtin causes selective neurotoxicity remain unknown. Clues may come from the identification of huntingtin-associated proteins (HAPs). Here, we show that huntingtin copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is encoded by the open reading frame factor VIII-associated gene A (F8A) located within intron 22 of the factor VIII gene. In transfected cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is cytoplasmic in the presence of huntingtin but is actively targeted to the nucleus in the absence of huntingtin. These data indicate that HAP40 is likely to contribute to the function of normal huntingtin and is a candidate for involvement in the aberrant nuclear localization of mutant huntingtin found in degenerating neurons in Huntington's disease.

关键词：氨基酸序列载体蛋白质类/遗传学载体蛋白质类/免疫学载体蛋白质类/分离和提纯疾病模型动物杭廷顿病/代谢分子序列数据分子量神经组织蛋白质类/遗传学神经组织蛋白质类/免疫学神经组织蛋白质类/分离和提纯核蛋白质类/遗传学核蛋白质类/免疫学核蛋白质类/分离和提纯动物人类小鼠大鼠

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CCAAT enhancer-binding protein β and GATA-4 binding regions within the promoter of the steroidogenic acute regulatory protein (StAR) gene are required for transcription in rat ovarian cells

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第25期274卷 17987-17996页

作者： Silverman, E Eimerl, S Orly, J Hebrew Univ Jerusalem Alexander Silberman Inst Life Sci Dept Biol Chem IL-91904 Jerusalem Israel

Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the -1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10-40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (-81/-72) for CCAAT enhancer-binding protein beta (C/EBP beta) and a consensus motif for GATA-4 binding (-61/-66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBP beta were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental -96/+6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBP beta and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter.

关键词：碱基序列结合部位 CCAAT增强子结合蛋白类氯霉素O-乙酰转移酶/遗传学 DNA结合蛋白质类/遗传学卵泡刺激素/药理学 fushitarazu转录因子类 GATA4转录因子基因表达调控/遗传学基因报告粒层细胞/代谢同源盒结构域蛋白质类分子序列数据诱变定点核蛋白质类/遗传学卵巢/代谢磷蛋白类/遗传学启动区遗传 RNA 信使/代谢大鼠 Sprague-Dawley 受体胞质和核 Sp1转录因子/遗传学类固醇生成因子1 转录因子/遗传学转录遗传动物女(雌)性大鼠

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A redox mechanism controls differential DNA binding activities of hypoxia-inducible factor (HIF) 1α and the HIF-like factor

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第7期275卷 4618-4627页

作者： Lando, D Pongratz, I Poellingers, L Whitelaw, ML Univ Adelaide Dept Biochem Adelaide SA 5005 Australia Karolinska Inst Med Nobel Inst Dept Cell & Mol Biol S-17177 Stockholm Sweden

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the HIF-like factor (HLF) are two highly related basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) homology transcription factors that undergo dramatically increased function at low oxygen levels. Despite strong similarities in their activation mechanisms (e.g, they both undergo rapid hypoxia-induced protein stabilization, bind identical target DNA sequences, and induce synthetic reporter genes to similar degrees), they are both essential for embryo survival via distinct functions during vascularization (HIF-1 alpha) or catecholamine production (HLF). It is currently unknown how such specificity of action is achieved. We report here that DNA binding by HLF, but not by HIF-1 alpha, is dependent upon reducing redox conditions. In vitro DNA binding and mammalian two-hybrid assays showed that a unique cysteine in the DNA-binding basic region of HLF is a target for the reducing activity of redox factor Ref-1. Although the N-terminal DNA-binding domain of HIF-1 alpha can function in the absence of Ref-1, we found that the C-terminal region containing the transactivation domain requires Ref-1 for fall activity. Our data reveal that the hypoxia-inducible factors are subject to complex redox control mechanisms that can target discrete regions of the proteins and are the first to establish a discriminating control mechanism for differential regulation of RIF-la and HLF activity.

关键词：氨基酸序列碳氧裂合酶类/遗传学半胱氨酸/遗传学半胱氨酸/代谢 DNA/代谢 DNA-(无嘌呤或无嘧啶位点)裂合酶 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/代谢基因表达调控 HeLa细胞螺旋-环-螺旋构型缺氧诱导因子1 缺氧诱导因子1 α亚基分子序列数据诱变定点核蛋白质类/遗传学核蛋白质类/代谢寡核苷酸类反义/代谢氧化还原重组蛋白质类/代谢序列同源性氨基酸丝氨酸/遗传学丝氨酸/代谢转录因子/遗传学转录因子/代谢人类

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Nuclear export of proteins in plants:: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana

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PLANT JOURNAL 1999年第6期20卷 695-705页

作者： Haasen, D Köhler, C Neuhaus, G Merkle, T Univ Freiburg Inst Biol 2 D-79104 Freiburg Germany

Transport across the nuclear envelope is mediated by transport receptors from the Importin beta family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-rich nuclear export signals (NESs). AtXPO1 shares 42-50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L(176)A, L(179)A, V(181)A). In BY-2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.

关键词：氨基酸序列拟南芥属/遗传学拟南芥属/代谢生物转运主动载体蛋白质类/遗传学载体蛋白质类/代谢克隆分子 DNA引物/遗传学 DNA 互补/遗传学 DNA 互补/分离和提纯 DNA 植物/遗传学 DNA 植物/分离和提纯基因表达基因植物核胞浆转运蛋白类分子序列数据核蛋白质类/遗传学核蛋白质类/代谢植物蛋白质类/遗传学植物蛋白质类/代谢植物基因修饰植物有毒 RNA 信使/遗传学 RNA 信使/代谢 RNA 植物/遗传学 RNA 植物/代谢受体胞质和核重组融合蛋白质类/遗传学重组融合蛋白质类/代谢序列同源性氨基酸组织分布烟草/遗传学烟草/代谢双杂交系统技术动物人类

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Requirements for cell cycle arrest by p16^INK4a

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MOLECULAR CELL 2000年第3期6卷 737-742页

作者： Bruce, JL Hurford, RK Classon, M Koh, J Dyson, N Massachusetts Gen Hosp Ctr Canc Oncol Mol Lab Charlestown MA 02129 USA

Analysis of tumor-derived mutations has led to the suggestion that p16(INK4a), cyclin D1, cdk4, and the retinoblastoma protein (pRB) are components of a regulatory pathway that is inactivated in most tumor cells. Cell cycle arrest induced by p16(INK4a), inhibitor of cyclin D-dependent kinases, requires pRB, and it has been proposed that this G1 arrest is mediated by pRB-E2F repressor complexes. By comparing the properties of primary mouse embryonic fibroblasts specifically lacking pRB-family members, we find that PRE is insufficient for a p16(INK4a)-induced arrest. In addition to pRB, a second function provided by either p107 or p130, two pRB-related proteins, is required for p16(INK4a) to block DNA synthesis. We infer that p16(INK4a)-induced arrest is not mediated exclusively by pRB, but depends on the nonredundant functions of at least two pRB-family members.

关键词：印迹法蛋白质载体蛋白质类/分析载体蛋白质类/遗传学细胞培养的细胞周期蛋白依赖激酶4 周期素依赖激酶抑制剂p16 细胞周期蛋白质依赖激酶类/分析胎儿/细胞学成纤维细胞/化学成纤维细胞/细胞学 G1期/生理学基因表达调控肿瘤/生理学小鼠基因敲除核蛋白质类/遗传学磷蛋白类/遗传学蛋白质类原癌基因蛋白质类视网膜母细胞瘤蛋白质/遗传学视网膜母细胞瘤样蛋白质p107 视网膜母细胞瘤样蛋白质p130 S期/生理学动物小鼠

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