CN1771039A - sgk基因家族用于诊断和治疗白内障和青光眼的用途 - Google Patents
sgk基因家族用于诊断和治疗白内障和青光眼的用途 Download PDFInfo
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Abstract
本发明涉及hsgk1蛋白或hsgk3蛋白的功能抑制剂或者hsgk1基因或hsgk3基因的负转录调节剂在制备治疗和/或预防白内障、青光眼或糖尿病性神经病变的药物中的用途。本发明的另一方面涉及包含AccNo.NM_005627的hsgk1序列或其片段、或者包含Acc No.AF169035的hsgk3序列或其片段的单链或双链核酸用于诊断形成白内障、青光眼和/或糖尿病性神经病变的倾向的用途,以及诊断形成白内障、青光眼和/或糖尿病性神经病变倾向的试剂盒,其中包含上述核酸。本发明还涉及从许多测试物质中鉴定和表征治疗活性物质的各种筛选方法,所述治疗活性物质用于治疗和/或预防选自白内障、青光眼或糖尿病性神经病变的至少一种疾病。
Description
本发明涉及hsgk1蛋白或hsgk3蛋白的功能抑制剂或者hsgk1基因或hsgk3基因的负转录调节剂用于制备治疗和/或预防白内障、青光眼或糖尿病性神经病变的药物的用途。
在另一个方面,本发明涉及包含根据Acc No.NM_005627的hsgk1序列或其片段,或者包含根据登记号AF169035的hsgk3序列或其片段的单链或双链核酸用于诊断发展白内障、青光眼和/或糖尿病性神经病变的倾向的用途,以及诊断发展白内障、青光眼和/或糖尿病性神经病变的倾向的试剂盒,该试剂盒包括上述核酸。
此外,本发明涉及从许多测试物质中鉴定和表征治疗活性物质的不同筛选方法,该治疗活性物质用于治疗和/或预防选自白内障、青光眼和糖尿病性神经病变中的至少一种疾病。
血清和糖皮质激素诱导型激酶hsgk1最初是以糖皮质激素敏感性基因形式克隆的[Webster et al.Characterization of sgk,anovel member of the serine/threonine protein kinase gene familywhich is transcriptionally induced by glucocorticoids and serum.Mol Cell Biol 1993;13:2031-2040]。
后来的调查揭示hsgk1处于很多刺激物的影响之下[Lang F,Cohen P Regulation and physiological roles of serum-andglucocorticoid-induced protein kinase isoforms.Science STKE.2001 Nov 13;2001(108):RE17],例如,尤其是盐皮质激素的影响[Chenet al.Epithelial sodium channel regulated by aldosterone-induced protein sgk.Proc Natl Acad Sci USA 1999;96:2514-2519,Náray-Fejes-Tóth et al.sgk is an aldosterone-induced kinasein the renal collecting duct.Effects on epithelial Na+ channels.J Biol Chem 1999;274:16973-16978;Shigaev et al.Regulation ofsgk by aldosterone and its effects on the epithelial Na(+)channel.Am J Physiol 2000;278:F613-F619;Brenan FE,Fuller PJ.Rapid upregulation of serum and glucocorticoid-regulatedkinase(sgk)gene expression by corticosteroids in vivo.MolCell Endocrinol.2000;30;166:129-36;Cowling RT,Birnboim HC.Expression of serum-and glucocorticoid-regulated kinase(sgk)mRNA is up-regulated by GM-CSF and other proinflammatorymediators in human granulocytes.J Leukoc Biol.2000;67:240-248]。
胰岛素样生长因子IGF1、胰岛素和氧化应力通过磷酸肌醇-3-激酶(PI3激酶)和磷酸肌醇依赖性激酶PDK1,经由信号级联来刺激hsgk1[Park et al.Serum and glucocorticoid-inducible kinase(SGK)is a target of the PI 3-kinase-stimulatdsignaling pathway,EMBOJ 1999;18:3024-3033;Kobayashi et al.Characterization of thestructure and regulation of two novel isoforms of serum-andglucocorticoid-induced protein kinase.Biochem.J.1999;344:189-197]。PDK1对hsgk1的活化包括在422位丝氨酸处的磷酸化。这个丝氨酸向天冬氨酸的突变(S422DSGK1)产生了具有组成型活性的激酶[Kobayashi T,Cohen P:Activation of serum-andglucocorticoid-regulated protein kinase by agonists thatactivate phosphatidylinositide 3-kinase is mediated by3-phosphoinositide-dependent protein kinase-1(PDK1)and PDK2.Biochem J.1999;339:319-328]。
早期的研究显示,hsgk1是肾上皮Na+通道的有力刺激物[De laRosa et al.The serum and glucocorticoid kinase sgk increasesthe abundance of epithelial sodium channels in the plasmamembrane of Xenopus oocytes.J Biol Chem 1999;274:37834-37839;Bhmer et al.The Shrinkage-activated Na+ Conductance of RatHepatocytes and its Possible Correlation to rENaC.Cell PhysBiochem.2000;10:187-194;Lang et al.Deranged transcriptionalregulation of cell volume sensitive kinase hSGK in diabeticnephropathy.Proc Natl Acad Sci USA 2000;97:8157-8162]。由于在不表达上皮Na+通道ENaC的很多组织中发现了hsgk1,因此hsgk1的作用应该不只限于调节Na+通道[Klingel et al.Expression of thecell volume regulated kinase h-sgk in pancreatic tissue.Am JPhysiol(Gastroint.Liver-Physiol.)2000;279:G998-G1002;Waldegger et al.Cloning and characterization of a putativehuman serine/threonine protein kinase transcriptionallymodified during anisotonic and isotonic alterations of cellvolume.Proc Natl Acad Sci USA 1997;94:4440-4445;Waldegger etal.h-sgk Serine-Threonine protein kinase gene as earlytranscriptional target of TGF-β in human intestine.Gastroenterology 1999;116:1081-1088]。
由于hsgk1很可能以还有待于阐明的方式调节很多其它信号转导途径或这些途径的组分这一事实,hsgk1和它的人同系物应该具有诊断很多疾病的重要潜力。尤其是,由DE 197 08 173 A1明显可知,hsgk1可以用于诊断很多疾病,如高钠血症、低钠血症、糖尿病、肾功能不全、分解代谢过度、肝性脑病和微生物或病毒感染,其中细胞容积的改变起重要的病理生理作用。
WO 00/62781报道了hsgk1活化内皮Na+通道,导致肾Na+再吸收增加。由于肾Na+再吸收的这种增加伴随有高血压,因此这个文件中推测hsgk1表达的增加将导致高血压,而hsgk1表达的降低将最终导致低血压。
DE 100 421 37也报道了人同系物hsgk2和hsgk3的过表达或活动过强与ENaC过度活化、由此导致的肾Na+再吸收增加和由此发展的高血压之间的类似关系。此外,这个文件已经探讨了激酶hsgk2和hsgk3对于动脉高压的诊断潜力。
WO02/074987A2公开了hsgk1基因中各个核苷酸的两个不同多态性(单核苷酸多态性(SNP))的存在和高血压的遗传决定的倾向性之间的关系。这些多态性是hsgk1基因中内含子6中的多态性(T→C)和外显子8中的多态性(C→T)。
因为sgk1在很多组织中表达,并且因为sgk1大概具有很多仍然未知的底物,所以人们预期sgk家族的人同系物、尤其是hsgk1基因(NM 005627)、hsgk2基因和hsgk3基因(AF169035)的作用和其它疾病的发展之间将有进一步的相关性。牵涉sgk1的这些其它特殊疾病的揭露将可以产生含有sgk家族人同系物基因的多态性区域(该区域影响相应sgk蛋白的功能或表达)的核酸,该核酸用于诊断这些其它疾病的倾向。
因此本发明的目的是找到sgk家族人同系物的功能和新疾病之间的更多相关性,并因此提供含有sgk家族人同系物基因的多态性区域的核酸用于诊断用途的新可能性。
这个目的是通过hsgk1和hsgk3强烈刺激葡萄糖转运蛋白Glut1的惊奇发现而实现的(见图1)。特别是,葡萄糖转运蛋白Glut1调节眼睛的各种细胞对葡萄糖的吸收,尤其是[Busik et al.Glucose-induced activation of glucose uptake in cells from theinner and outer blood-retinal barrier.Invest Ophthalmol VisSci.2002;43:2356-63;TakataK,Ka Sahara T,Kasahara M,EzakiO,Hirano H.Ultracytochemical localization of theerythrocyte/HepG2-type glucose transporter (GLUT1)in theciliary body and iris of the rat eye.Invest Ophthalmol Vis Sc.1991;32:1659-66]。水跟随葡萄糖渗透,意思指Glut1活性增加将引起细胞肿胀。因此,Glut1活性增加可以引起白内障的发展[Gong et al.Development of cataractous macrophthalmia in mice expressingan active MEK1 in the lens.Invest Ophthalmol Vis Sci.2001;42:539-48]。除此之外,已经表明Glut1的过表达会促进结缔组织蛋白的形成和沉积[Ayo et al.Increased extracellular matrixsynthesis and mRNA in mesangial cells grown in high-glucosemedium.Am J Physiol.1991;260:F185-191;Heilig et al.Overexpression of glucose transporters in rat mesangial cellscultured in a normal glucose milieu mimics the diabeticphenotype.J Clin Invest.1995;96:1802-1814]。这种结缔组织蛋白的沉积妨碍了眼部液体的流失,导致眼内压力增加,并因此导致视网膜的损害[Fingert et al.Evaluation of the myocilin(MYOC)glaucoma gene in monkey and human steroid-induced ocularhypertension.Invest Ophthalmol Vis Sci.2001;42(1):145-52,Ueda et al.Distribution of myocilin and extracellular matrixcomponents in the juxtacanalicular tissue of human eyes.InvestOphthalmol Vis Sci.2002;43:1068-76]。刺激SGK1表达的糖皮质激素(参见上面)确实同时引起青光眼的发展[Fingert et al.2001]。然而,以前从未推测hsgk1是病因。
上述失调将在与hsgk1活性增加有关的任何情况下发生,也就是说在上述任何激素过量存在的情况下将发生。与血压增加有关的hsgk1基因的特殊多态性[Busjahn et al.Serum-andglucocorticoid-regulated kinase(SGK1)gene and blood pressure.Hypertension 40(3):256-260,2002]可以同时引起白内障和青光眼发生率的增加。该基因的相同改变也应该与过早出现的白内障和/或青光眼有关。
本发现揭示了葡萄糖转运蛋白Glut1调节中的一种全新机制。因此hsgk1活性的增加应该会引起细胞对葡萄糖的摄入增加。血清[Webster et al.1993]、糖皮质激素[Brenan & Fuller 2000,Websteret al.1993]、盐皮质激素[Chen et al.1999,Naray-Fejes-Toth etal.1999,Shigaev et al.2000,Brennan and Fuller 2000,Cowlingand Birnboim 2000]、促性腺激素[Alliston et al.Folliclestimulating hormone-regulated expression of serum/glucocorticoid-inducible kinase in rat ovarian granulosa cells:a functional role for the Spl family in promoter activity.MolEndocrinol.1997;11:1934-1949;Alliston et al.Expression andlocalization of serum/glucocorticoid-induced kinase in the ratovary:relation to follicular growth and differentiation.Endocrinology.2000;141:385-395;Gonzalez-Robayna et al.Follicle-Stimulating hormone(FSH)stimulates phosphorylationand activation of protein kinase B(PKB/Akt)and serum andglucocorticoid-Induced kinase (Sgk):evidence for Akinase-independent signaling by FSH in granulosa cells.MolEndocrinol.2000;14:1283-1300,Richards et al.Ovarian celldifferentiation:a cascade of multiple hormones,cellularsignals,and regulated genes.Recent Prog Horm Res.1995;50:223-254]和很多细胞因子[Lang & Cohen 2001],尤其是TGF-β[Fillon S.et al.Expression of the Serine/Threonine kinasehSGK1 in chronic viral hepatitis.Cell Physiol Biochem2002;12:47-54;Lang et al.2000,Waldegger et al.1999,WrntgesS et al.Excessive transcription of the human serum andglucocorticoid dependent kinase hSGK1 in lung fibrosis.CellPhysiol Biochem 2002,12:135-142]会刺激hsgk1的转录。除此之外,细胞皱缩也会增加hsgk1的转录,如已经引用的Waldegger et al.1997论文所示。葡萄糖浓度的增加,如糖尿病中显示的,会通过细胞皱缩和/或通过TGF-β形成的增加来刺激hsgk1的表达[Lang et al.2000]。所表达的hsgk1是通过胰岛素样生长因子IGF1、胰岛素或氧化应力来活化的[Kobayashi & Cohen 1999,Park et al.1999,Kobayashi et al.1999]。
根据本发明的发现,hsgk1的表达增加会提高葡萄糖转运蛋白Glut-1的活性。结果,更多葡萄糖被吸收进细胞中,随后的通过渗透进入的水引起细胞肿胀。这样水掺入角膜和晶状体的程度增加,这样,由于透明度降低,将引起
白内障[Gong et al..2001]。
此外,
青光眼也可以以类似方式发展,此外也可由干结缔组织的掺入而发展[Fingert et al.2001]。
也怀疑细胞肿胀是
糖尿病性神经病变的原因[Burg et al.,Sorbitol,osmoregulation,and the complications of diabetes.J Clin Invest 1988;81:635-40]。然而,不仅仅在糖尿病的情况下,而且在糖皮质激素的影响下或在显示遗传决定的hsgk1活性过强的患者中,预计Glut1活性增加[Busjahn et al.,Serum-andglucocorticoid-regulated kinase(SGK1)gene and blood pressure.Hypertension 40(3):256-260,2002]。糖皮质激素确实会引起青光眼[Fingert et al.2001]。以前并不清楚与糖皮质激素给药有关的青光眼发展的机制。尤其是,以前并不清楚hsgk1在这个机制中起作用、因此适合用作诊断和治疗青光眼的靶蛋白。
因此,根据本发明的观察意外地证明了hsgk1和hsgk3会增加非上皮葡萄糖转运以及增加上皮Na+通道。结果,hsgk1和hsgk3显示全新的病理生理性重要性,因而具有重要的诊断和治疗/预防效果。
因此本发明涉及hsgk1蛋白或hsgk3蛋白的功能抑制剂或者hsgk1基因或hsgk3基因的负转录调节剂用于降低细胞肿胀的用途。
此外,本发明涉及hsgk1蛋白或hsgk3蛋白的功能抑制剂或者hsgk1基因或hsgk3基因的负转录调节剂用于制备治疗和/或预防白内障、青光眼或糖尿病性神经病变的药物的用途。
hsgk1蛋白或hsgk3蛋白的功能抑制剂可以是抑制hsgk1蛋白或hsgk3蛋白的正常生理活性的任何性质的化学物质。优选该hsgk1蛋白或hsgk3蛋白的功能抑制剂是低分子量化学物质(“小分子”)或蛋白质或肽。hsgk1蛋白或hsgk3蛋白的功能抑制剂尤其可以是这些酶的拮抗剂,其阻断hsgk1蛋白或hsgk3蛋白的底物结合位点,但同时不受hsgk1或hsgk3的任何催化转化影响。优选地,在这种情况下合适的拮抗剂是在结构上类似于hsgk1蛋白或hsgk3蛋白的天然底物的那些分子,也就是说,尤其是在结构上类似于可磷酸化的氨基酸丝氨酸和苏氨酸的那些分子。
星孢素和白屈菜赤碱是两种已知的hsgk1功能抑制剂。因此在特别优选的实施方案中,星孢素或白屈菜赤碱用作hsgk1或hsgk3的功能抑制剂,用于治疗和/或预防疾病白内障、青光眼和糖尿病性神经病变中的至少一种疾病。
hsgk1基因或hsgk3基因的负转录调节剂定义为在转录水平活化hsgk1基因或hsgk3基因表达的物质。
除实际的活性物质即hsgk1或hsgk3的功能抑制剂或者hsgk1或hsgk3的负转录调节剂之外,根据本发明的用于治疗和/或预防白内障、青光眼或糖尿病性神经病变的药物还可以包含稳定剂和/或载体物质,如淀粉、乳糖、硬脂酸、脂肪、蜡、醇类,或其它添加剂,如防腐剂、颜料或调味剂。
可以以任何方式施用药物,特别是以片剂、粒剂或胶囊或作为溶液形式口服。其它特别合适的施用形式涉及以软膏、酊剂或喷雾直接施用(例如在皮肤或眼睛上)或任何注射类型形式(例如皮下或静脉内)或输注。
此外,本发明涉及包含根据登记号NM_005627的hsgk1序列或其一个片段的单链或双链核酸用于诊断发展白内障、青光眼和/或糖尿病性神经病变的倾向性的用途。在这一点上,该单链或双链核酸中可以包含的hsgk1片段的长度是至少10个核苷酸/碱基对,优选长度至少15个核苷酸/碱基对,特别是长度至少20个核苷酸/碱基对。
在这一点上,优选该单链或双链核酸包含hsgk1基因的至少一个多态性核苷酸,尤其是hsgk1基因的单核苷酸多态性(SNP)。
在特别优选的实施方案中,所述单链或双链核酸包含hsgk1基因的下列SNPs中的至少一个:
-在hsgk1基因内含子2中732/733位置处的G插入,
-在hsgk1基因内含子6中2071位置处的T/C置换(WO02/074987 A2),
-在hsgk1基因外显子8中2617位置处的T/C置换(WO02/074987 A2)。
优选地,可通过下列方法将上述单链或双链核酸用于检测患者基因组DNA或cDNA中hsgk1基因的上述SNPs:
-通过使用上述核酸对基因组DNA或cDNA直接测序,
-通过将基因组DNA或cDNA与上述核酸特异性杂交,
-通过PCR寡核苷酸延伸试验或通过连接试验。
在这一点上,优选患者的基因组DNA或cDNA是从取自患者的身体样品中分离的,尤其是从唾液、血液、组织或细胞中分离的。
推测表达的hsgk1基因的活性取决于患者的hsgk1基因中该多态性的形式,因此含有这些多态性中至少一种的核酸尤其很适于诊断发展白内障、青光眼和/或糖尿病性神经病变的倾向性。
此外,本发明涉及包含根据登记号AF169035的hsgk3序列或其一个片段的单链或双链核酸用于诊断发展白内障、青光眼和/或糖尿病性神经病变的倾向性的用途。在这一点上,所述单链或双链核酸可以包含的hsgk3片段的长度是至少10个核苷酸/碱基对,优选长度至少15个核苷酸/碱基对,特别是长度至少20个核苷酸/碱基对。
在这一点上,优选所述单链或双链核酸包含hsgk3基因的至少一个多态性核苷酸,尤其是hsgk3基因的单核苷酸多态性(SNP)。
除上述的单链或双链核酸之外,针对sgk家族人同系物的底物,尤其是针对hsgk1和hsgk3的底物的抗体也适于诊断发展白内障、青光眼和糖尿病性神经病变中的至少一种疾病的倾向性。优选这些诊断抗体针对的是sgk家族人同系物(尤其是hsgk1和hsgk3)的表位,其中含有磷酸化形式或未磷酸化形式的底物磷酸化位点。
例如,由于hsgk1基因的个体遗传组成造成的hsgk1蛋白过表达可以引起hsgk底物的转变增加,即hsgk1对底物的酶促磷酸化增加。同时,hsgk1蛋白的过表达将引起葡萄糖转运蛋白Glut1的刺激,最终导致眼睛细胞内葡萄糖的高水平摄入,继而通过渗透作用引起水的高水平摄入,结果最终导致发展白内障、青光眼和糖尿病性神经病变的倾向性。因此,通过针对含有磷酸化形式或未磷酸化形式之hsgk1磷酸化位点的感兴趣底物区域的抗体而检测更频繁的hsgk1底物磷酸化可代表诊断发展白内障、青光眼和糖尿病性神经病变的倾向性的方法。
在优选实施方案中,泛素蛋白连接酶Nedd4-2(登记号BAA23711)被用作sgk家族人同系物的底物。这个泛素蛋白连接酶是被sgk家族的人同系物特异性磷酸化的蛋白[Debonneville et al.,Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+)channel cell surface expression.EMBO J.,2001;20:7052-7059;Snyder et al.,Serum and glucocorticoid-regulated kinasemodulates Nedd4-2-mediated inhibition of the epithelial Na(+)channel.J.Biol.Chem.,2002,277:5-8]。hsgk1的磷酸化位点具有共有序列(RXRXXS/T),其中R是精氨酸,S是丝氨酸,T是苏氨酸,X是任何任意氨基酸。在Nedd4-22(登记号BAA23711)中,存在上述共有序列匹配的两个hsgk1潜在磷酸化位点,即在382位氨基酸处的丝氨酸和在468位氨基酸处的丝氨酸。
因此,优选用于诊断发展白内障、青光眼和糖尿病性神经病变中至少一种疾病的倾向性的上述抗体针对的是底物Nedd4-2,特别优选针对的是具有hsgk1潜在磷酸化位点序列、即共有序列(RXRXXS/T)的Nedd4-2蛋白的区域。特别是,这些抗体针对的是Nedd4-2蛋白区域,该区域包含两个潜在磷酸化位点中的至少一个,即382位氨基酸处的丝氨酸和/或468位氨基酸处的丝氨酸。
此外,本发明涉及诊断白内障、青光眼和糖尿病性神经病变之一的试剂盒,该试剂盒包含至少一种下列成分:
-针对hsgk1或hsgk3的抗体,
-能够在严格条件下与登记号NM_005627的hsgk1基因或登记号AF169035的hsgk3基因杂交的单链或双链核酸;尤其是包含hsgk1基因或hsgk3基因的多态性核苷酸、特别是“SNPs”的那些单链或双链核酸,
-针对sgk家族人同系物的底物的抗体;优选针对磷酸化或未磷酸化形式的该底物的磷酸化位点的抗体;尤其是针对磷酸化或未磷酸化形式的Nedd4或Nedd4-2磷酸化位点的抗体。
本发明还涉及从许多测试物质中鉴定和表征治疗活性物质的筛选方法,该治疗活性物质用于治疗和/或预防选自包括白内障、青光眼和糖尿病性神经病变的组中的至少一种疾病,所述方法包括下列步骤:
a)在细胞中异源性地共表达
i)葡萄糖转运蛋白Glut1和
ii)hsgk1和/或hsgk3
b)在每种情况下,在至少一种测试物质存在的情形下,培养A1至AX至少一种细胞等分试样,其中每种情况下所述至少一种测试物质根据细胞等分试样的下标1至X而不同,并在缺乏任何测试物质的情况下培养对照细胞等分试样B,
c)与对照细胞等分试样B中葡萄糖转运蛋白Glut1的活性相比较,测定细胞等分试样A1至AX中葡萄糖转运蛋白Glut1的活性。
物质文库,优选小分子文库,或蛋白文库等等,均可以用作所述“多种测试物质”。
在a)步骤中,使用标准方法,如电穿孔法、CaPO4沉淀法、脂质转染法等等,用合适的表达载体转染合适的细胞,优选哺乳动物细胞或细胞系,尤其是人细胞或细胞系,该表达载体含有表达Glut1和hsgk1和/或hsgk3的合适表达盒。该表达盒含有处于合适启动子控制下的相关靶基因(Glut1、hsgk1或hsgk3)的基因组DNA或cDNA,所述启动子在感兴趣的细胞类型中有活性,并能够以合适的量表达靶基因。该表达载体可以另外含有选择标记。
然后,在能够使靶基因i)和ii)被表达的条件下培养转染的细胞。
在b)步骤中,来自a)的转染细胞被分成不同的细胞等分试样A1至AX以及对照细胞等分试样B。在每种情况下,在至少一种测试物质存在时培养细胞等分试样A1至AX。每种情况下添加到细胞等分试样A1至AX的测试物质彼此不同(依各细胞等分试样A1至AX的下标1至X而定)。另一方面,在缺乏任何测试物质的情形下培养对照细胞等分试样B。
在c)步骤中,与对照细胞等分试样B中葡萄糖转运蛋白Glut1的活性相比较,定量测定细胞等分试样A1至AX中葡萄糖转运蛋白Glut1的活性。能够在功能上抑制hsgk1或hskg3或者降低它们的表达的测试物质应该已被加入到其中Glut1的活性经测定比对照细胞等分试样B中测定的活性明显较低的细胞等分试样A1至AX中。这种物质可能适于治疗白内障、青光眼和糖尿病性神经病变中的至少一种疾病。
在可供选择的实施方案中,根据本发明的用于从许多测试物质中鉴定和表征治疗活性物质的筛选方法包括下列步骤,其中所述治疗活性物质可用于治疗和/或预防选自包括白内障、青光眼和糖尿病性神经病变的组中的至少一种疾病:
d)在细胞的至少一个等分试样A1至AX中异源性地共表达
i)葡萄糖转运蛋白Glut1和
ii)hsgk1和/或hsgk3,并且
在细胞的至少一个等分试样B1至BX中异源性地表达
i)葡萄糖转运蛋白Glut1,
e)在每种情况下,在存在至少一种测试物质的情形下培养A1至AX和B1至BX细胞等分试样,其中每种情况下所述至少一种测试物质依细胞等分试样的下标1至X而不同,
f)进行细胞等分试样A1至AX和细胞等分试样B1至BX中葡萄糖转运蛋白Glut1的活性的比较性测定。
上面关于a)至c)各个程序步骤给出的说明以相应方式适合于根据本发明的可供选择的筛选方法中的d)至f)程序步骤。
通过下图1更详细解释本发明。
图1的纵座标A上绘制了2-脱氧葡萄糖的摄入(以pmol/1/10分钟/卵母细胞)(算术平均数±SEM)。给Xenopus laevis卵母细胞注射Glut-1 cRNA(有或没有SGK1、SGK2、SGK3或蛋白激酶B(PKB)cRNA)(见实施例1)。
图1显示了与自身表达Glut1的卵母细胞相比较,除Glut1之外还表达hsgk1或hsgk3的卵母细胞中发生的2-脱氧葡萄糖摄入的增加。因此,这证明了hsgk1和hsgk3的功能有效地刺激了葡萄糖转运蛋白Glut1的活性。在表达hsgk2或PKBmut而不是hsgk1或hsgk3的卵母细胞的情况下,没有看到类似结果。
通过下列实施例1更详细解释本发明。
实施例1:在Xenopus laevis卵母细胞中的表达和双电极电压
钳
体外合成正常SGK1 cRNA[Waldegger S,Barth P,Raber G,LangF:Cloning and characterization of a putative humanserine/threonine protein kinase transcriptionally modifiedduring anisotonic and isotonicalterations of cell volume.ProcNatl Acad Sci USA 1997;94:4440-4445]和组成型活性SGK1(S422DSGK1)cRNA[Kobayashi & Cohen 1999],以及正常Glut1 cRNA[IserovichP,Wang D,Ma L,Yang H,Zuniga FA,Pascual JM,Kuang K,De VivoDC,Fischbarg J.Changes in glucose transport and waterpermeability resulting from the T310I pathogenic mutation inGlut1 are consistent with two transport channels per monomer.J Biol Chem.2002;277:30991-7]。已经详细描述了Xenopus laevis卵巢的解剖和卵母细胞的收集与处理[Wagner CA,Friedrich B,Setiawan I,Lang F,Brer S:The use of Xenopus laevis oocytesfor the functional characterization of heterologouslyexpressed membrane proteins.Cell Physiol Biochem2000;10:1-12]。给卵母细胞注射5ng人Glut1、7.5ng人S422DSGK1和/或5ng蟾蜍(Xenopus)Nedd4-2。给对照卵母细胞注射水。注射各个cRNAs后2天,在室温下测定放射性标记的葡萄糖的摄入。对照浴液含有96mM NaCl、2mM KCl、1.8mM CaCl2、1mM MgCl2和5mM HEPES,pH 7.4。所有物质以所给浓度使用。用HCl或NaOH使最终溶液滴定至pH 7.4。
计算
以算术平均值±SEM的形式给出数据;n是被检测的卵母细胞数。对至少三个不同组的卵母细胞进行所有实验。使用Student’s t检验来检验结果的显著性差异。仅P<0.05的结果被认为有统计学显著性。
序列表
<110>Prof.Dr.Lang,Florian
<120>sgk基因家族用于诊断和治疗白内障和青光眼的用途
<130>L62135
<140>DE 103 05 212.7
<141>2003-02-07
<160>3
<170>PatentIn version 3.1
<210>1
<211>5719
<212>DNA
<213>人hsgk1基因,NM_005627
<220>
<221>变异
<222>(732)..(733)
<223>在内含子2中位置732/733处插入额外的G(SNP)
<220>
<221>变异
<222>(2071)..(2071)
<223>在内含子6中位置2071处的T/C交换(SNP)
<220>
<221>变异
<222>(2617)..(2617)
<223>在外显子8中位置2617处的C/T交换(SNP)
<400>1
ggccgagcgc gcggcctggc gcacgatacg ccgagccggt ctttgagcgc taacgtcttt 60
ctgtctcccc gcggtggtga tgacggtgaa aactgaggct gctaagggca ccctcactta 120
ctccaggatg aggggcatgg tggcaattct catcggtgag tgcaggaatc ttgcgggact 180
tctgctccag gagacgcaaa gtggaaattt tttgaaagtc ccggatcaga ttagtgtgtg 240
tggcgccggg acgttatgaa gccgtctaaa cgtttcttta tttctcctcc ttctatccac 300
agctttcatg aagcagagga ggatgggtct gaacgacttt attcagaaga ttgccaataa 360
ctcctatgca tgcaaacagt aagttcagac cggattgagg aaataactag tatagtttga 420
atttgccagc ggtaaacatt ctcatcacgg cgtttatcgg gaaggcgaag acttcttctg 480
gggtggggat ctcatttctc cttaaattct aatatatttg acacatttta aacattaaag 540
ttaatttgct gatttggctt gaactggaga tgtaagataa atggttcgtg ttggccgaat 600
tcacgctttc tccatgagca acaatcctta tttctgtatt taatggggtt tattattttc 660
tttaactgac taatgtattg gggtattttc agtttaaaca gtgaattatc gggtagaagt 720
cggtagagcc aggaaactca cttttgatgt tggtgtgccc cctagtggcg agctggattc 780
taaatcgtgc cctttattcc ctgcagccct gaagttcagt ccatcttgaa gatctcccaa 840
cctcaggagc ctgagcttat gaatgccaac ccttctcctc cagtaagttt ttgtatgtgc 900
cgtgcatctg tggagaactg taagggagtc agttagtatt cctacattaa tggattaaaa 960
tagcatttct agaaattagt atcaaggcag gaatgcttca ttatgcataa cagtgatata 1020
aatatttaag tattgagtca gagtattatt tttatttttt tcctgggcat attttacctc 1080
aagtggttat tttaaaaggc atatttcata aaaaggtttt atctgtctga aacaacatga 1140
ctgtgtgcag tttccatact catttgaaat gtgatgaaat gtagttttga atgtttatag 1200
atgtatggtc atttgcatca gtcatttgta gatgtaacat tttctacatc gtttatgtta 1260
tagatgtctt cctttgaagc aatggtatta aaagaaattc tttttttttt tttctagcca 1320
agtccttctc agcaaatcaa ccttggcccg tcgtccaatc ctcatgctaa accatctgac 1380
tttcacttct tgaaagtgat cggaaagggc agttttggaa aggtaatttc aaatctgaag 1440
atcttttggt acacttcctt catgtcctct tttatattct ccctggatga ggatcgaaaa 1500
atgatttttt taaattgaaa tttcaggttc ttctagcaag acacaaggca gaagaagtgt 1560
tctatgcagt caaagtttta cagaagaaag caatcctgaa aaagaaagag gtgagatgtg 1620
cttgatgggg ctggcattgg cggtagacac tccttgaata atcttgattc tggaatgttg 1680
gtgccagttg aacatgccac taaatctgaa tcgtcatttt cctaggagaa gcatattatg 1740
tcggagcgga atgttctgtt gaagaatgtg aagcaccctt tcctggtggg ccttcacttc 1800
tctttccaga ctgctgacaa attgtacttt gtcctagact acattaatgg tggagaggtg 1860
agcagggggg atagaagtca actcttagtg tctctgcaca gcctgctttg ttttagtttg 1920
agaaaaaagt tttcaaagat ttttggtggg gagaatgtta ccagaattag catttccttc 1980
aacctgtcag gttatagtta atagattact tggggccact tcctgcagtt gttcttttgc 2040
tgtgtatgtc aaaactaatt aaattacatt gtgcaaccca gaatgacttt gttctgtctc 2100
ctgcagttgt tctaccatct ccagagggaa cgctgcttcc tggaaccacg ggctcgtttc 2160
tatgctgctg aaatagccag tgccttgggc tacctgcatt cactgaacat cgtttatagg 2220
taagcctgag agctcttcag gctaccagtt ttggtataaa ggagacgtag cactggctgt 2280
ttcatagggc cttaaaataa tttgtgttta tttgcaactt ggttcgctaa aaccagatcc 2340
cctagcacgt gagctggctt gacttaagtg ccaaggggga acagccaagt aggattgtgc 2400
ctaatccaga atagatgagc agaacaaggg ctcctttttt cttcactaca caactacagt 2460
gaacctaaat gcctctaata ccttagcaat tatctttaag aggatatctt atgaagtgaa 2520
attaacttgt gcaactactt ttctattcac ttttttacag agacttaaaa ccagagaata 2580
ttttgctaga ttcacaggga cacattgtcc ttactgactt cggactctgc aaggagaaca 2640
ttgaacacaa cagcacaaca tccaccttct gtggcacgcc ggaggtaggc gctgtcttgg 2700
tttggtgcct ggtttacccc cgccttccaa gagagagatg tacaatcatg cacttaacta 2760
ccaaaaagag taaactcctc tcagagactt cttaatacag ttcagtgcaa ataaaataca 2820
tttgctgttt gatgtagcat gagaaatccc aagtccttct gttcctttac tgaaaagtag 2880
ctgtttgtaa gtaagatctg catcataaaa actttctaat cctaagtaag agatatcaag 2940
tgccagcagt ttcctaaatg tcagtacaca taggtagcca gtcaccctca aaaagtccag 3000
cagttttatc aggaaggaat ctaaagatat ctatcttcca agctggctct gggtctctca 3060
gctttttcaa actaaatgtg tggtcgtggg attgcttgct ttcgcaggtt ctaaacgctg 3120
tttccctggt ctgtttttca gtatctcgca cctgaggtgc ttcataagca gccttatgac 3180
aggactgtgg actggtggtg cctgggagct gtcttgtatg agatgctgta tggcctggtg 3240
agtggcacat tgggaaccac tggaacactg cctgctccct acaatattgc cttcacacag 3300
caaaagcagc taagaggcat attggttatt ttatagttca taagaataat cacttacctg 3360
gttcttttgt gcatttcaca ttttactaga taggaccaca ttgaacctgt gtggtggtga 3420
aaaactacca cttattaaca tctaccccct accctccaca cacacacaca caaacacaca 3480
cacgggttgc aaagtagaca cttaaatagc aagggaaaag aaagcattga ggtggggaga 3540
gtttctcaaa tcgagcctaa tatttattgc cgtttatatc tttttctcta ctggtaatgt 3600
gtgccatatg aaacttccaa ttaagtctaa agtaattttc cccttctttc agccgccttt 3660
ttatagccga aacacagctg aaatgtacga caacattctg aacaagcctc tccagctgaa 3720
accaaatatt acaaattccg caagacacct cctggagggc ctcctgcaga aggacaggac 3780
aaagcggctc ggggccaagg atgacttcgt gagtgatgtt ttcctgtcct cctgggccgg 3840
ccgggacgtg cactagacct ccctgccctt attgaatgca cctgtctaaa ttaatcttgg 3900
gtttcttatc aacagatgga gattaagagt catgtcttct tctccttaat taactgggat 3960
gatctcatta ataagaagat tactccccct tttaacccaa atgtggtgag tatctgtctc 4020
tcttctaagt atagagaagc caagcgattt attttaattc agaattgtct gggggagggt 4080
tggaaggaat acattggcag atgttttctc cataaacctg ttattttacc tacatagaca 4140
catttatcaa ttcgaagcac caaaaggcaa caagtgaaca ttattcttat gtttaactgt 4200
gtgtagcctt ttgagatttt gtgcttgaag tgggtgatta tggaagttga tataagactt 4260
aaacttggta tttaaagcct ggtcaagatt tccctgtcct gtgtctagtg tgagttcttg 4320
acaagagtgt ttttcccttc ccgtcacaga gtgggcccaa cgagctacgg cactttgacc 4380
ccgagtttac cgaagagcct gtccccaact ccattggcaa gtcccctgac agcgtcctcg 4440
tcacagccag cgtcaaggaa gctgccgagg ctttcctagg cttttcctat gcgcctccca 4500
cggactcttt cctctgaacc ctgttagggc ttggttttaa aggattttat gtgtgtttcc 4560
gaatgtttta gttagccttt tggtggagcc gccagctgac aggacatctt acaagagaat 4620
ttgcacatct ctggaagctt agcaatctta ttgcacactg ttcgctggaa ttttttgaag 4680
agcacattct cctcagtgag ctcatgaggt tttcattttt attcttcctt ccaacgtggt 4740
gctatctctg aaacgagcgt tagagtgccg ccttagacgg aggcaggagt ttcgttagaa 4800
agcggacctg ttctaaaaaa ggtctcctgc agatctgtct gggctgtgat gacgaatatt 4860
atgaaatgtg ccttttctga agagattgtg ttagctccaa agcttttcct atcgcagtgt 4920
ttcagttctt tattttccct tgtggatatg ctgtgtgaac cgtcgtgtga gtgtggtatg 4980
cctgatcaca gatggatttt gttataagca tcaatgtgac acttgcagga cactacaacg 5040
tgggacattg tttgtttctt ccatatttgg aagataaatt tatgtgtaga cttttttgta 5100
agatacggtt aataactaaa atttattgaa atggtcttgc aatgactcgt attcagatgc 5160
ctaaagaaag cattgctgct acaaatattt ctatttttag aaagggtttt tatggaccaa 5220
tgccccagtt gtcagtcaga gccgttggtg tttttcattg tttaaaatgt cacctgtaaa 5280
atgggcatta tttatgtttt tttttttgca ttcctgataa ttgtatgtat tgtataaaga 5340
acgtctgtac attgggttat aacactagta tatttaaact tacaggctta tttgtaatgt 5400
aaaccaccat tttaatgtac tgtaattaac atggttataa tacgtacaat ccttccctca 5460
tcccatcaca caactttttt tgtgtgtgat aaactgattt tggtttgcaa taaaaccttg 5520
aaaaatattt acatatattg tgtcatgtgt tattttgtat attttggtta agggggtaat 5580
catgggttag tttaaaattg aaaaccatga aaatcctgct gtaatttcct gcttagtggt 5640
ttgctccaac agcagtggtt tctgactcca gggagtatag gatggcttaa gccaccacgt 5700
ccaggccttt agcagcatt 5719
<210>2
<211>2391
<212>DNA
<213>人,hsgk3 mRNA,AF169035
<400>2
ggtgtgctct tgagggatta aatgcaaaga gatcacacca tggactacaa ggaaagctgc 60
ccaagtgtaa gcattcccag ctccgatgaa cacagagaga aaaagaagag gtttactgtt 120
tataaagttc tggtttcagt gggaagaagt gaatggtttg tcttcaggag atatgcagag 180
tttgataaac tttataacac tttaaaaaaa cagtttcctg ctatggccct gaagattcct 240
gccaagagaa tatttggtga taattttgat ccagatttta ttaaacaaag acgagcagga 300
ctaaacgaat tcattcagaa cctagttagg tatccagaac tttataacca tccagatgtc 360
agagcattcc ttcaaatgga cagtccaaaa caccagtcag atccatctga agatgaggat 420
gaaagaagtt ctcagaagct acactctacc tcacagaaca tcaacctggg accgtctgga 480
aatcctcatg ccaaaccaac tgactttgat ttcttaaaag ttattggaaa aggcagcttt 540
ggcaaggttc ttcttgcaaa acggaaactg gatggaaaat tttatgctgt caaagtgtta 600
cagaaaaaaa tagttctcaa cagaaaagag caaaaacata ttatggctga acgtaatgtg 660
ctcttgaaaa atgtgaaaca tccgtttttg gttggattgc attattcctt ccaaacaact 720
gaaaagcttt attttgttct ggattttgtt aatggagggg agcttttttt ccacttacaa 780
agagaacggt cctttcctga gcacagagct aggttttacg ctgctgaaat tgctagtgca 840
ttgggttact tacattccat caaaatagta tacagagact tgaaaccaga aaatattctt 900
ttggattcag taggacatgt tgtcttaaca gattttgggc tttgtaaaga aggaattgct 960
atttctgaca ccactaccac attttgtggg acaccagagt atcttgcacc tgaagtaatt 1020
agaaaacagc cctatgacaa tactgtagat tggtggtgcc ttggggctgt tctgtatgaa 1080
atgctgtatg gattgcctcc tttttattgc cgagatgttg ctgaaatgta tgacaatatc 1140
cttcacaaac ccctaagttt gaggccagga gtgagtctta cagcctggtc cattctggaa 1200
gaactcctag aaaaagacag gcaaaatcga cttggtgcca aggaagactt tcttgaaatt 1260
cagaatcatc ctttttttga atcactcagc tgggctgacc ttgtacaaaa gaagattcca 1320
ccaccattta atcctaatgt ggctggacca gatgatatca gaaactttga cacagcattt 1380
acagaagaaa cagttccata ttctgtgtgt gtatcttctg actattctat agtgaatgcc 1440
agtgtattgg aggcagatga tgcattcgtt ggtttctctt atgcacctcc ttcagaagac 1500
ttatttttgt gagcagtttg ccattcagaa accattgagc aaaataagtc tatagatggg 1560
actgaaactt ctatttgtgt gaatatattc aaatatgtat aactagtgcc tcatttttat 1620
atgtaatgat gaaaactatg aaaaaatgta ttttcttcta tgtgcaagaa aaatagggca 1680
tttcaaagag ctgttttgat taaaatttat attcttgttt aataagctta tttttaaaca 1740
atttaaaagc tattattctt agcattaacc tatttttaaa gaaacctttt ttgctattga 1800
ctgttttttc cctctaagtt tacactaaca tctacccaag atagactgtt ttttaacagt 1860
caatttcagt tcagctaaca tatattaata cctttgtaac tctttgctat ggcttttgtt 1920
atcacaccaa aactatgcaa ttggtacatg gttgtttaag aagaaaccgt atttttccat 1980
gataaatcac tgtttgaaat atttggttca tggtatgatc gaaatgtaaa agcataatta 2040
acacattggc tgctagttaa caattggaat aactttattc tgcagatcat ttaagaagta 2100
acaggccggg cgcggtggct cacgcctgta atcccagcac tttgggaggc tgaggcgggc 2160
agatcacctg aggtcaggag ttggagacca gcctgaccaa catggacaaa ccccgtctct 2220
actaaaaata caaaattggc agggtgtggt ggcacatgcc tataatccca gctacttggg 2280
aggctaaggc aggagaatcg cttgaacccg ggaggcggag gttgcagtga gccgagatcg 2340
caccattgca ctcctgcctg ggcaacaaga gtgaaactcc atctccaaaa a 2391
<210>3
<211>995
<212>PRT
<213>人,Nedd 4-2蛋白,BAA23711
<400>3
Pro Gly Gly Trp Leu Arg Arg Ala Leu Pro Gly Arg Glu Arg Leu Gln
1 5 10 15
Ser Pro Val His Ala Val Pro Pro Gln His Gly Thr Ser His Ser Arg
20 25 30
Leu Leu Val Thr Trp Pro Gly Ala Gly Arg Asp Gln Asp Phe Ser Ser
35 40 45
Pro Pro Leu Leu Leu Leu Gly Glu Thr Asp His Leu His Leu Asp Leu
50 55 60
Pro Leu Ser Pro Leu Pro Thr Ser Asp Glu Leu Phe Leu Pro Gly Ile
65 70 75 80
Cys Asp Pro Tyr Val Lys Leu Ser Leu Tyr ValAla Asp Glu Asn Arg
85 90 95
Glu Leu Ala Leu Val Gln Thr Lys Thr Ile Lys Lys Thr Leu Asn Pro
100 105 110
Lys Trp Asn Glu Glu Phe Tyr Phe Arg Val Asn Pro Ser Asn His Arg
115 120 125
Leu Leu Phe Glu Val Phe Asp Glu Asn Arg Leu Thr Arg Asp Asp Phe
130 135 140
Leu Gly Gln Val Asp Val Pro Leu Ser His Leu Pro Thr Glu Asp Pro
145 150 155 160
Thr Met Glu Arg Pro Tyr Thr Phe Lys Asp Phe Leu Leu Arg Pro Arg
165 170 175
Ser His Lys Ser Arg Val Lys Gly Phe Leu Arg Leu Lys Met Ala Tyr
180 185 190
Met Pro Lys Asn Gly Gly Gln Asp Glu Glu Asn Ser Asp Gln Arg Asp
195 200 205
Asp Met Glu His Gly Trp Glu Val Val Asp Ser Asn Asp Ser Ala Ser
210 215 220
Gln His Gln Glu Glu Leu Pro Pro Pro Pro Leu Pro Pro Gly Trp Glu
225 230 235 240
Glu Lys Val Asp Asn Leu Gly Arg Thr Tyr Tyr Val Asn His Asn Asn
245 250 255
Arg Thr Thr Gln Trp His Arg Pro Ser Leu Met Asp Val Ser Ser Glu
260 265 270
Ser Asp Asn Asn Ile Arg Gln Ile Asn Gln Glu Ala Ala His Arg Arg
275 280 285
Phe Arg Ser Arg Arg His Ile Ser Glu Asp Leu Glu Pro Glu Pro Ser
290 295 300
Glu Gly Gly Asp Val Pro Glu Pro Trp Glu Thr Ile Ser Glu Glu Val
305 310 315 320
Asn Ile Ala Gly Asp Ser Leu Gly Leu Ala Leu Pro Pro Pro Pro Ala
325 330 335
Ser Pro Gly Ser Arg Thr Ser Pro Gln Glu Leu Ser Glu Glu Leu Ser
340 345 350
Arg Arg Leu Gln Ile Thr Pro Asp Ser Asn Gly Glu Gln Phe Ser Ser
355 360 365
Leu Ile Gln Arg Glu Pro Ser Ser Arg Leu Arg Ser Cys Ser Val Thr
370 375 380
Asp Ala Val Ala Glu Gln Gly His Leu Pro Pro Pro Ser Val Ala Tyr
385 390 395 400
Val His Thr Thr Pro Gly Leu Pro Ser Gly Trp Glu Glu Arg Lys Asp
405 410 415
Ala Lys Gly Arg Thr Tyr Tyr Val Asn His Asn Asn Arg Thr Thr Thr
420 425 430
Trp Thr Arg Pro Ile Met Gln Leu Ala Glu Asp Gly Ala Ser Gly Ser
435 440 445
Ala Thr Asn Ser Asn Asn His Leu Ile Glu Pro Gln Ile Arg Arg Pro
450 455 460
Arg Ser Leu Ser Ser Pro Thr Val Thr Leu Ser Ala Pro Leu Glu Gly
465 470 475 480
Ala Lys Asp Ser Pro Val Arg Arg Ala Val Lys Asp Thr Leu Ser Asn
485 490 495
Pro Gln Ser Pro Gln Pro Ser Pro Tyr Asn Ser Pro Lys Pro Gln His
500 505 510
Lys Val Thr Gln Ser Phe Leu Pro Pro Gly Trp Glu Met Arg Ile Ala
515 520 525
Pro Asn Gly Arg Pro Phe Phe Ile Asp His Asn Thr Lys Thr Thr Thr
530 535 540
Trp Glu Asp Pro Arg Leu Lys Phe Pro Val His Met Arg Ser Lys Thr
545 550 555 560
Ser Leu Asn Pro Asn Asp Leu Gly Pro Leu Pro Pro Gly Trp Glu Glu
565 570 575
Arg Ile His Leu Asp Gly Arg Thr Phe Tyr Ile Asp His Asn Ser Lys
580 585 590
Ile Thr Gln Trp Glu Asp Pro Arg Leu Gln Asn Pro Ala Ile Thr Gly
595 600 605
Pro Ala Val Pro Tyr Ser Arg Glu Phe Lys Gln Lys Tyr Asp Tyr Phe
610 615 620
Arg Lys Lys Leu Lys Lys Pro Ala Asp Ile Pro Asn Arg Phe Glu Met
625 630 635 640
Lys Leu His Arg Asn Asn Ile Phe Glu Glu Ser Tyr Arg Arg Ile Met
645 650 655
Ser Val Lys Arg Pro Asp Val Leu Lys Ala Arg Leu Trp Ile Glu Phe
660 665 670
Glu Ser Glu Lys Gly Leu Asp Tyr Gly Gly Val Ala Arg Glu Trp Phe
675 680 685
Phe Leu Leu Ser Lys Glu Met Phe Asn Pro Tyr Tyr Gly Leu Phe Glu
690 695 700
Tyr Ser Ala Thr Asp Asn Tyr Thr Leu Gln Ile Asn Pro Asn Ser Gly
705 710 715 720
Leu Cys Asn Glu Asp His Leu Ser Tyr Phe Thr Phe Ile Gly Arg Val
725 730 735
Ala Gly Leu Ala Val Phe His Gly Lys Leu Leu Asp Gly Phe Phe Ile
740 745 750
Arg Pro Phe Tyr Lys Met Met Leu Gly Lys Gln Ile Thr Leu Asn Asp
755 760 765
Met Glu Ser Val Asp Ser Glu Tyr Tyr Asn Ser Leu Lys Trp Ile Leu
770 775 780
Glu Asn Asp Pro Thr Glu Leu Asp Leu Met Phe Cys Ile Asp Glu Glu
785 790 795 800
Asn Phe Gly Gln Thr Tyr Gln Val Asp Leu Lys Pro Asn Gly Ser Glu
805 810 815
Ile Met Val Thr Asn Glu Asn Lys Arg Glu Tyr Ile Asp Leu Val Ile
820 825 830
Gln Trp Arg Phe Val Asn Arg Val Gln Lys Gln Met Asn Ala Phe Leu
835 840 845
Glu Gly Phe Thr Glu Leu Leu Pro Ile Asp Leu Ile Lys Ile Phe Asp
850 855 860
Glu Asn Glu Leu Glu Leu Leu Met Cys Gly Leu Gly Asp Val Asp Val
865 870 875 880
Asn Asp Trp Arg Gln His Ser Ile Tyr Lys Asn Gly Tyr Cys Pro Asn
885 890 895
His Pro Val Ile Gln Trp Phe Trp Lys Ala Val Leu Leu Met Asp Ala
900 905 910
Glu Lys Arg Ile Arg Leu Leu Gln Phe Val Thr Gly Thr Ser Arg Val
915 920 925
Pro Met Asn Gly Phe Ala Glu Leu Tyr Gly Ser Asn Gly Pro Gln Leu
930 935 940
Phe Thr Ile Glu Gln Trp Gly Ser Pro Glu Lys Leu Pro Arg Ala His
945 950 955 960
Thr Cys Phe Asn Arg Leu Asp Leu Pro Pro Tyr Glu Thr Phe Glu Asp
965 970 975
Leu Arg Glu Lys Leu Leu Met Ala Val Glu Asn Ala Gln Gly Phe Glu
980 985 990
Gly Val Asp
995
Claims (15)
1.hsgk1蛋白或hsgk3蛋白的功能抑制剂或者hsgk1基因或hsgk3基因的负转录调节剂用于降低细胞肿胀的用途。
2.hsgk1蛋白或hsgk3蛋白的功能抑制剂或者hsgk1基因或hsgk3基因的负转录调节剂用于制备治疗和/或预防白内障、青光眼或糖尿病性神经病变的药物的用途。
3.如权利要求1或2的用途,其特征在于hsgk1蛋白或hsgk3蛋白的功能抑制剂是星孢素或白屈菜赤碱。
4.一种包含hsgk1蛋白或hsgk3蛋白的功能抑制剂或者hsgk1基因或hsgk3基因的负转录调节剂的药物,其用于治疗和/或预防白内障、青光眼或糖尿病性神经病变的药物。
5.包含根据Acc No.NM_005627的hsgk1序列或其一个片段的单链或双链核酸用于诊断发展白内障、青光眼和/或糖尿病性神经病变的倾向性的用途。
6.如权利要求5的用途,其特征在于所述单链或双链核酸包含hsgk1基因的至少一个多态性核苷酸,尤其是hsgk1基因的“SNP”。
7.如权利要求6的用途,其特征在于所述hsgk1基因的SNP选自下述SNPs组中:在hsgk1基因内含子2的732/733位置处的G插入,在hsgk1基因内含子6的2071位置处的T/C置换和在hsgk1基因外显子8中2617位置处的T/C置换。
8.包含根据Acc No.AF169035的hsgk3序列或其一个片段的单链或双链核酸用于诊断发展白内障、青光眼和/或糖尿病性神经病变的倾向性的用途。
9.如权利要求8的用途,其特征在于所述单链或双链核酸包含hsgk3基因的至少一个多态性核苷酸,尤其是hsgk3基因的“SNP”。
10.针对sgk家族人同系物的底物的抗体用于诊断发展白内障、青光眼和糖尿病性神经病变中的至少一种疾病的倾向性的用途,该抗体针对的是含有磷酸化形式或未磷酸化形式的磷酸化位点的人同系物表位。
11.如权利要求10的用途,其特征在于所述sgk家族人同系物的底物是Acc No.BAA23711的Nedd4-2。
12.一种用于诊断白内障、青光眼和糖尿病性神经病变之一的试剂盒,其中包含针对hsgk1或hsgk3的抗体,或者包含在严格条件下能够与根据Acc No.NM_005627的hsgk1基因或根据Acc No.AF169035的hsgk3基因相杂交的核酸,或者组合性地包含这些抗体和核酸。
13.如权利要求12的试剂盒,其特征在于,在严格条件下,所述核酸能够与根据Acc No.NM_005627的hsgk1基因或根据Acc No.AF169035的hsgk3基因的DNA区域相杂交,所述DNA区域包含hsgk1基因或hsgk3基因的多态性核苷酸,尤其是“SNPs”。
14.一种从许多测试物质中鉴定和表征治疗活性物质的筛选方法,该治疗活性物质用于治疗和/或预防选自包括白内障、青光眼和糖尿病性神经病变的组中的至少一种疾病,其中所述方法包括下列步骤:
a)在细胞中异源性地共表达
i)葡萄糖转运蛋白Glut1和
ii)hsgk1和/或hsgk3
b)在每种情况下,在存在至少一种测试物质的情形下,培养A1至AX至少一种细胞等分试样,其中每种情况下的所述至少一种测试物质依细胞等分试样的下标1至X而不同,并在缺乏任何测试物质的情况下培养对照细胞等分试样B,
c)与对照细胞等分试样B中葡萄糖转运蛋白Glut1的活性相比较,测定细胞等分试样A1至AX中葡萄糖转运蛋白Glut1的活性。
15.一种从许多测试物质中鉴定和表征治疗活性物质的筛选方法,该治疗活性物质用于治疗和/或预防选自包括白内障、青光眼和糖尿病性神经病变的组中的至少一种疾病,所述方法包括下列步骤:
d)在细胞的至少一个等分试样A1至AX中异源性地共表达
i)葡萄糖转运蛋白Glut1和
ii)hsgk1和/或hsgk3,和
在细胞的至少一个等分试样B1至BX中异源性地共表达
i)葡萄糖转运蛋白Glut1
e)在每种情况下,在存在至少一种测试物质的情形下培养A1至AX和B1至BX的细胞等分试样,其中每种情况下的所述至少一种测试物质依细胞等分试样的下标1至X而不同,
f)在细胞等分试样A1至AX和细胞等分试样B1至BX中进行葡萄糖转运蛋白Glut1活性的比较性测定。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10305212A DE10305212A1 (de) | 2003-02-07 | 2003-02-07 | Verwendung der sgk-Genfamilie zur Diagnose und zur Therapie von Katarakt und Glaukom |
DE10305212.7 | 2003-02-07 |
Publications (1)
Publication Number | Publication Date |
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CN1771039A true CN1771039A (zh) | 2006-05-10 |
Family
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CNA2004800070348A Pending CN1771039A (zh) | 2003-02-07 | 2004-02-05 | sgk基因家族用于诊断和治疗白内障和青光眼的用途 |
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EP (1) | EP1663246A2 (zh) |
JP (1) | JP2006519189A (zh) |
KR (1) | KR20050114214A (zh) |
CN (1) | CN1771039A (zh) |
AU (1) | AU2004210416A1 (zh) |
BR (1) | BRPI0407300A (zh) |
CA (1) | CA2514703A1 (zh) |
DE (1) | DE10305212A1 (zh) |
MX (1) | MXPA05008394A (zh) |
PL (1) | PL378399A1 (zh) |
RU (1) | RU2005127808A (zh) |
WO (1) | WO2004069258A2 (zh) |
ZA (1) | ZA200506280B (zh) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
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CA2559141A1 (en) * | 2004-03-11 | 2005-10-13 | Merck Patent Gesellschaft Mit Beschraenkter Haftung | Methods for interfering with fibrosis |
RU2006135654A (ru) * | 2004-03-11 | 2008-09-10 | Мерк Патент ГмбХ (DE) | Способы модуляции глутаматных рецепторов для лечения нейропсихиатрических расстройств, включающие применение модуляторов сывороточных и индуцируемых глюкокортикоидами киназ |
DE102004059781A1 (de) * | 2004-12-10 | 2006-06-22 | Sanofi-Aventis Deutschland Gmbh | Verwendung der Serum-/Glucocorticoid regulierten Kinase |
US20060293378A1 (en) * | 2005-06-28 | 2006-12-28 | Mcintire Gregory | Method of lowering intraocular pressure |
JPWO2007037560A1 (ja) * | 2005-09-30 | 2009-04-16 | リンク・ジェノミクス株式会社 | Sgk2遺伝子の治療的又は診断的用途 |
CA2630668C (en) * | 2005-11-22 | 2016-07-12 | Mcgill University | Intraocular pressure-regulated early genes and uses thereof |
US20080153903A1 (en) * | 2006-12-22 | 2008-06-26 | Alcon Manufacturing, Ltd. | Inhibitors of protein kinase c-delta for the treatment of glaucoma |
DE102008029072A1 (de) * | 2008-06-10 | 2009-12-17 | Lang, Florian, Prof. Dr.med. | Sgk3 als therapeutisches und diagnostisches Target für Alterserkrankungen |
CA2824067C (en) * | 2011-01-25 | 2021-02-02 | Monell Chemical Senses Center | Compositions and methods for providing or modulating sweet taste and methods of screening therefor |
KR102357260B1 (ko) * | 2020-12-10 | 2022-02-08 | 주식회사 레피겐엠디 | 마이크로rna를 이용한 당뇨병성 신경병증의 예측 및 진단 방법 및 이를 위한 키트 |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
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AU3352495A (en) * | 1994-08-30 | 1996-03-22 | Barbara Ann Spruce | Agents for inducing apoptosis and applications of said agents in therapy |
AU6263996A (en) * | 1995-06-07 | 1996-12-30 | Ligand Pharmaceuticals Incorporated | Method for screening for receptor agonists and antagonists |
US5925523A (en) * | 1996-08-23 | 1999-07-20 | President & Fellows Of Harvard College | Intraction trap assay, reagents and uses thereof |
DE19708173A1 (de) * | 1997-02-28 | 1998-09-03 | Dade Behring Marburg Gmbh | Zellvolumenregulierte humane Kinase h-sgk |
AU3991499A (en) * | 1998-05-15 | 1999-12-06 | Joslin Diabetes Center | Independent regulation of basal and insulin-stimulated glucose transport |
EP1141003B9 (en) * | 1998-12-14 | 2008-07-02 | The University of Dundee | Methods of activation of SGK by phosphorylation. |
US6399655B1 (en) * | 1998-12-22 | 2002-06-04 | Johns Hopkins University, School Of Medicine | Method for the prophylactic treatment of cataracts |
DE19917990A1 (de) * | 1999-04-20 | 2000-11-02 | Florian Lang | Arzneimittel enthaltend Hemmstoffe der zellvolumenregulierten humanen Kinase h-sgk |
AU5840300A (en) * | 1999-07-14 | 2001-01-30 | University Of Lausanne | Glutx polypeptide family and nucleic acids encoding same |
US6416759B1 (en) * | 1999-09-30 | 2002-07-09 | The Regents Of The University Of California | Antiproliferative Sgk reagents and methods |
US20030236246A1 (en) * | 2002-04-30 | 2003-12-25 | Brazzell Romulus Kimbro | Method for decreasing capillary permeability in the retina |
DE10225844A1 (de) * | 2002-06-04 | 2003-12-18 | Lang Florian | sgk und nedd als diagnostische und therapeutische targets |
-
2003
- 2003-02-07 DE DE10305212A patent/DE10305212A1/de not_active Withdrawn
-
2004
- 2004-02-05 MX MXPA05008394A patent/MXPA05008394A/es unknown
- 2004-02-05 JP JP2006501737A patent/JP2006519189A/ja active Pending
- 2004-02-05 CN CNA2004800070348A patent/CN1771039A/zh active Pending
- 2004-02-05 PL PL378399A patent/PL378399A1/pl not_active Application Discontinuation
- 2004-02-05 AU AU2004210416A patent/AU2004210416A1/en not_active Abandoned
- 2004-02-05 RU RU2005127808/15A patent/RU2005127808A/ru not_active Application Discontinuation
- 2004-02-05 CA CA002514703A patent/CA2514703A1/en not_active Abandoned
- 2004-02-05 WO PCT/EP2004/001048 patent/WO2004069258A2/de active Search and Examination
- 2004-02-05 KR KR1020057014582A patent/KR20050114214A/ko not_active Application Discontinuation
- 2004-02-05 EP EP04708350A patent/EP1663246A2/de not_active Withdrawn
- 2004-02-05 BR BR0407300-2A patent/BRPI0407300A/pt not_active IP Right Cessation
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Also Published As
Publication number | Publication date |
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WO2004069258A3 (de) | 2005-02-24 |
PL378399A1 (pl) | 2006-04-03 |
CA2514703A1 (en) | 2004-08-19 |
KR20050114214A (ko) | 2005-12-05 |
BRPI0407300A (pt) | 2006-02-07 |
EP1663246A2 (de) | 2006-06-07 |
WO2004069258A2 (de) | 2004-08-19 |
ZA200506280B (en) | 2006-05-31 |
AU2004210416A1 (en) | 2004-08-19 |
MXPA05008394A (es) | 2005-10-05 |
RU2005127808A (ru) | 2006-05-27 |
JP2006519189A (ja) | 2006-08-24 |
DE10305212A1 (de) | 2004-08-19 |
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