家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase, PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。     

LncRNA63及其共表达基因Hr3在家蚕中的表达和功能分析

【目的】本研究旨在通过对一个新的长链非编码RNA (long non-coding RNA, lncRNA)及其共表达基因在家蚕Bombyx mori中的表达和功能进行分析,进一步阐明lncRNA在调控家蚕变态发育中的分子机制。【方法】基于前期家蚕脂肪体转录组测序数据,我们发现在蜕皮激素20E(2 μg/μL)处理5龄幼虫早期的脂肪体中lncRNA63表达显著上调,家蚕激素受体基因Hr3与之共表达。利用qRT-PCR对lncRNA63和Hr3在家蚕不同发育时期(4-5龄幼虫、蛹和成虫)、5龄幼虫不同组织(头、体壁、血淋巴、中肠、马氏管、脂肪体、丝腺、精巢和卵巢)和2 μg/μL 20E处理2, 6, 12和24 h后5龄幼虫脂肪体中的表达谱进行分析;采用核质分离检测和原位杂交技术检测lncRNA63在家蚕BmN细胞中的定位;利用dsRNA干涉家蚕5龄幼虫个体中lncRNA63的表达,并利用qRT-PCR检测RNA干涉lncRNA63对头、丝腺、脂肪体和体壁中Hr3表达的影响。【结果】qRT-PCR结果表明,lncRNA63和Hr3在家蚕羽化前的蛹末期有一个表达高峰;二者在家蚕5龄幼虫头、体壁和丝腺中都有较高表达;lncRNA63和Hr3在2 μg/μL 20E处理2, 6和12 h的家蚕5龄幼虫脂肪体中较对照组(无水乙醇处理组)都显著上调,24 h下降到与对照组相当水平,二者的表达趋势完全一致。LncRNA63主要定位于BmN细胞核,但20E可诱导lncRNA63的核质迁移。利用dsRNA干涉lncRNA63表达后,Hr3的表达显著下调。【结论】20E不仅可调控lncRNA63的表达,还可诱导lncRNA63的核质迁移;lncRNA63可通过调控共表达基因Hr3的表达参与家蚕的变态发育过程。     

家蚕丝氨酸蛋白酶抑制剂20的表达特征分析

【目的】原核表达家蚕Bombyx mori丝氨酸蛋白酶抑制剂20(Serine proteinase inhibitor 20,Bm Serpin-20),分析Bm Serpin-20基因和蛋白组织表达分布特点,以及不同外源病原物刺激家蚕后Bm Serpin-20基因的表达模式。【方法】利用p ET-28a表达载体在大肠杆菌Transetta(DE3)中融合表达Bm Serpin-20蛋白,利用纯化的重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体,利用实时定量PCR技术及Western blot方法分别分析Bm Serpin-20基因和蛋白的组织表达水平,以及病原物免疫刺激后的表达模式。【结果】在大肠杆菌中正确表达和纯化了融合Bm Serpin-20蛋白,并制备了多克隆抗体;Bm Serpin-20基因和蛋白在脂肪体、中肠、血细胞、马氏管、表皮、睾丸、卵巢中均有表达,且在表皮中表达量最高;家蚕5龄幼虫经核型多角体病毒(Nuclear polyhedrosis viruses)、藤黄微球菌Micrococcus luteus、大肠杆菌Escherichia coli、白僵菌Beauveria bassiana处理后,Bm Serpin-20基因表达量先下调而后上调,但不同病原物在不同时间的影响不尽相同。【结论】研究了Bm Serpin-20基因和蛋白的表达模式,为下一步研究其在家蚕免疫系统中的作用奠定了基础。     

外源激素处理后家蚕吡哆醛激酶和磷酸吡哆醇氧化酶转录水平的变化

【目的】研究家蚕 Bombyx mori 经蜕皮激素(20-hydroxyecdysone, 20-E)和保幼激素类似物(juvenile hormone analogue, JHA)处理后引起吡哆醛激酶(pyridoxal kinase, PLK)和磷酸吡哆醇氧化酶(pyridoxine-5′-phosphate oxidase, PNPO)的转录水平变化,为进一步研究激素对蚕体营养代谢等工作奠定基础。【方法】以20-E和JHA分别喂食不同发育时期(5龄第1, 3和5天)的家蚕幼虫,以喂食蒸馏水的家蚕为对照,采用实时荧光定量PCR(real-time quantitative PCR)方法在处理后24 和48 h对各组幼虫后部丝腺中PLP合成酶PLK和PNPO的转录水平进行分析。【结果】5龄第1天幼虫经20-E处理24和48 h后,PLK和PNPO的转录水平出现上调且与对照的差异达到极显著 (P＜0.01);5龄第3天幼虫经20-E处理,PLK的转录水平在48 h出现下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24 和48 h均出现上调且与对照的差异达到极显著 (P＜0.01);5龄第5天幼虫经20-E处理后PLK和PNPO的转录水平无变化。5龄第1天幼虫经JHA处理后PLK和PNPO的转录水平未受到影响;5龄第3天幼虫经JHA处理后,PLK的转录水平在48 h出现显著下调且与对照的差异达到显著(P＜0.05),PNPO的转录水平在24和48 h后均出现显著下调且与对照的差异达到极显著(P＜0.05);5龄第5天幼虫经JHA处理24和48 h后,PLK和PNPO的转录水平出现下调且与对照的差异达到极显著 (P＜0.01)。【结论】20-E和JHA显著影响家蚕5龄幼虫PLK和PNPO的转录水平,20-E提高5龄前期家蚕PLK和PNPO的转录水平,JHA降低5龄后期它们的转录水平,为深入研究激素对VB₆的调控奠定基础。     

苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究

【目的】丝氨酸蛋白酶(Serine protease,SP)是以丝氨酸为活性中心的重要的蛋白水解酶。在昆虫中,丝氨酸蛋白酶参与消化、发育、先天免疫反应和组织重建等重要的生理过程。本试验以苜蓿夜蛾Heliothis viriplaca为材料,克隆其丝氨酸蛋白酶基因的cDNA序列,再对该基因进行原核表达并对表达产物进行活性测定研究。【方法】从苜蓿夜蛾中肠中提取总RNA,通过RT-PCR和RACE技术,扩增获得丝氨酸蛋白酶基因cDNA全长序列,用大肠杆菌E.coli表达系统进行表达;再对表达的重组蛋白进行变性、纯化与复性,并以BTEE为底物进行活性测定。【结果】克隆得到的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为Hv SP,该基因已登录Gen Bank,登录号为KT907053。该基因全长1 017 bp,开放阅读框为886 bp,编码295个氨基酸,分子量约为30.8 ku,等电点为8.27,推导的氨基酸序列与其他昆虫丝氨酸蛋白酶氨基酸序列相似性在46%~92%之间。在Tris-HCl缓冲液中,p H为8.5时,复性的重组蛋白活性最高,为28.7 U/m L。荧光定量PCR结果表明,Hv SP基因的m RNA在苜蓿夜蛾的多个组织中特异性表达,且在中肠中表达量最高,但在唾腺中未检测到Hv SP的m RNA表达。【结论】该研究克隆了一个新的苜蓿夜蛾丝氨酸蛋白酶基因的cDNA序列,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性,为进一步探索丝氨酸蛋白酶在昆虫体内的生理生化功能奠定了基础。     

家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase, PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。     

家蚕BmlncR2036调控P25基因启动子活性及参与中肠发育调控

【目的】长链非编码RNA(long non-coding RNA, lncRNA)对家蚕Bombyx mori发育具有重要调控作用。我们在前期研究中发现一个位于家蚕丝素蛋白基因P25附近的lncRNA BmlncR2036。本研究旨在进一步探索BmlncR2036调控家蚕P25基因表达的分子机制。【方法】qPCR检测BmlncR2036在5龄第3天家蚕幼虫不同组织(体壁、脑、神经、精巢、卵巢、丝腺、马氏管、血淋巴、脂肪体和中肠)中和不同发育阶段前丝腺、中丝腺和后丝腺中的表达谱。预测能够同时靶向P25和BmlncR2036的miRNA,利用荧光素酶检测法验证miRNA与P25互作关系。荧光素酶检测法测定BmlncR2036对P25基因启动子转录活性的影响。在家蚕5龄第3天幼虫中注射dsRNA敲低BmlncR2036的表达,观察丝腺及中肠表型的变化。【结果】qPCR结果显示,BmlncR2036在家蚕5龄第3天幼虫和5龄熟蚕的后丝腺中高表达,与P25基因呈现一致的表达趋势;BmlncR2036在家蚕5龄第3天幼虫的精巢和中肠中高表达,在其他组织中表达量较低,暗示其功能的多样性。miR-2739和miR-279a既能够与BmlncR2036匹配,也可能靶向P25基因的3′UTR。但荧光素酶检测法结果显示P25不是miR-2739和miR-279a的真实靶标。BmlncR2036负调控P25启动子活性,共转染pcDNA3.1(+)［BmlncR2036］和pGL3-Enhancer［P25-promoter］载体后,细胞荧光素酶活性极显著下降了52%。在家蚕5龄第3天幼虫中敲低BmlncR2036表达后出现体色变暗,中肠残留大量内容物,直至死亡的现象,表明BmlncR2036可能参与家蚕中肠的发育调控。【结论】发现lncRNA BmlncR2036可调控P25基因启动子活性,还可能参与调控家蚕中肠的发育。本研究为进一步探索家蚕lncRNA的功能提供了实验依据。     

美国白蛾丝氨酸蛋白酶基因HcSP1的克隆、时空表达及对取食不同寄主植物的表达响应

【目的】为探究美国白蛾Hyphantria cunea在寄主转换过程中的消化生理机制奠定基础。【方法】通过筛选美国白蛾cDNA文库,克隆美国白蛾丝氨酸蛋白酶基因。荧光定量PCR检测该基因在美国白蛾不同发育阶段的表达特性;半定量RT-PCR和荧光定量PCR分别检测该基因在美国白蛾5龄幼虫体内不同组织中的分布及表达特性;荧光定量PCR检测取食不同寄主植物(美洲黑杨Populus deltoides,日本晚樱Cerasus serrulata var.lannesiana,山樱花Cerasus serrulata,喜树Camptotheca acuminata和法国梧桐Platanus orientalis)叶片后美国白蛾4龄幼虫中该基因的表达量。【结果】克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1(GenBank登录号:MH663425),开放阅读框长882 bp,编码293个氨基酸,预测分子量为30.5 kD,理论等电点预测为9.86。编码蛋白N末端疏水区包含15个氨基酸组成的信号肽;具有丝氨酸蛋白酶的典型特征,即氨基酸序列中具有组氨酸(His)、天门冬氨酸(Asp)以及丝氨酸(Ser)残基组成的酶活性催化中心三元件;具有明显的胰蛋白酶前体的特征,即具有信号肽、激活肽以及胰蛋白酶N末端保守的起始氨基酸序列(IVGG)。NCBI BLAST比对结果表明美国白蛾HcSP1与其他鳞翅目昆虫丝氨酸蛋白酶的氨基酸序列一致性在50%～70%之间。荧光定量PCR结果显示,HcSP1在美国白蛾幼虫不同发育阶段的相对表达量呈现动态的变化,并随着幼虫虫龄的增长呈现上升趋势。半定量RT-PCR及荧光定量PCR结果显示,HcSP1在美国白蛾5龄幼虫头部、唾液腺、中肠、脂肪体、表皮、马氏管和血淋巴等组织中均有表达且在幼虫中肠中表达量极高。与取食其他寄主植物叶片相比,美国白蛾取食喜树叶片后HcSP1的相对表达量明显升高,并显著高于取食其他寄主植物。【结论】本研究克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1,检测了其在美国白蛾不同发育阶段、不同组织以及取食不同寄主植物叶片后的表达量,为探究美国白蛾在寄主转换过程中消化生理的机制奠定基础,也为美国白蛾的防治提供新的思路。     

沙葱萤叶甲丝氨酸蛋白酶基因GdSP的克隆及对温度胁迫的响应

【目的】丝氨酸蛋白酶(serine protease,SP)是一类以丝氨酸为活性中心的重要的蛋白水解酶。本研究旨在克隆获得沙葱萤叶甲Galeruca daurica丝氨酸蛋白酶基因,分析其对温度胁迫的响应,以期为进一步揭示沙葱萤叶甲耐温性的调控机制及其他生理功能奠定基础。【方法】根据沙葱萤叶甲2龄幼虫转录组数据,采用RACE技术克隆得到沙葱萤叶甲丝氨酸蛋白酶基因的cDNA全长序列,并进行生物信息学分析;应用qPCR技术检测其在不同温度(-10,-5,0,5,25和35℃)下处理1 h后及25℃下恢复30 min后在沙葱萤叶甲2龄幼虫中的表达量变化。【结果】自沙葱萤叶甲克隆获得一个丝氨酸蛋白酶基因,命名为GdSP(GenBank登录号:MG797556)。该基因全长1 110 bp,开放阅读框969 bp,编码322个氨基酸;蛋白预测分子量35.41 kD,等电点5.61;编码蛋白具有丝氨酸蛋白酶的典型特征,具有一个跨膜结构,无信号肽。同源序列比对和系统发育分析表明,GdSP与光肩星天牛Anoplophora glabripennis SP的同源性最高,氨基酸序列一致性为30.53%。qPCR测定结果表明,不同温度处理间2龄幼虫中GdSP表达量差异不显著,但对各高低温(-10℃除外)胁迫处理回温后GdSP表达量显著上升。【结论】快速冷驯化对沙葱萤叶甲丝氨酸蛋白酶基因表达无显著影响,而回温可诱导其上调表达。     

烟草甲clip丝氨酸蛋白酶基因的克隆及其对外源激素和免疫胁迫的表达响应(英文)

【目的】作为细胞外信号级联通路的重要组成部分,含有clip结构域的丝氨酸蛋白酶(clip-domain serine proteases, CLIPs)在昆虫发育和先天免疫过程中起着重要作用。本研究旨在克隆烟草甲Lasioderma serricorne CLIP基因,解析其在烟草甲不同发育阶段和幼虫不同组织中的表达模式,分析其在外源激素20-羟基蜕皮酮(20E)和免疫胁迫后的表达特征,为进一步研究其生理功能奠定基础。【方法】采用RT-PCR技术克隆获得烟草甲两个CLIPs基因(LsCLIP1和LsCLIP2)全长cDNA序列,并利用生物信息学软件预测其编码蛋白的结构和特征,利用MEGA 6.06构建昆虫CLIPs系统发育树;利用实时荧光定量PCR(quantitative real-time PCR, qPCR)研究这两个基因在不同发育阶段［低龄幼虫(卵孵化后24 h内)、高龄幼虫(4龄以上)、蛹(化蛹后48 h以上)、早期成虫(化蛹后24 h内)和晚期成虫(化蛹后7 d)］、5龄幼虫不同组织(表皮、脂肪体、肠道和剩余组织)中以及注射20E(120 ng/幼虫)和来源于大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus的肽聚糖(0.2 μL)后4龄幼虫中的表达模式。【结果】克隆获得烟草甲LsCLIP1和LsCLIP2基因的cDNA全序列,其开放阅读框长度均为1 194 bp,编码397个氨基酸。序列分析显示,其氨基酸序列各自具有一个clip结构域和胰蛋白酶结构域。系统发育分析表明,CLIP1和CLIP2都属于subfamily C CLIPs。qPCR结果表明,LsCLIP1和LsCLIP2基因在所检测的各发育阶段和幼虫各组织中均有表达,分别尤以蛹期和表皮中表达量最高;经20E和肽聚糖诱导后,烟草甲幼虫体内LsCLIP1和LsCLIP2基因的表达量明显提高。【结论】推测LsCLIP1和LsCLIP2可能参与了烟草甲的蜕皮发育和对免疫胁迫的应激响应。本研究将为后续研究昆虫CLIPs的分子调控提供参考。     

Identification of serine protease,serine protease homolog and prophenoloxidase genes in Spodoptera frugiperda (Lepidoptera: Noctuidae)

In insects, proteolytic cascades medicated by serine proteases (SPs), serine protease homologs (SPHs) and prophenoloxidases (PPOs) control several physiological processes, notably the innate immunity. However, no attempts have been made to identify and characterize these genes in Spodoptera frugiperda, one of the most destructive agricultural pests. In this study, 83 SPs, 26 SPHs and four PPOs were respectively identified in S. frugiperda genome based on homology blast against those of other insects. We then analyzed the domain organization of these proteins and assigned them into different groups by phylogenetic reconstruction. Furthermore, the mRNA levels of clip-domain SPs/SPHs (cSPs/cSPHs) and PPOs were quantified in response to a mixed infection of Micrococcus luteus and Escherichia coli, and obvious accumulations were recorded in immune tissues, including hemocytes and fat body. In the latter study, we profiled the expression patterns of highly expressed cSPs and PPOs in different developmental stages, including egg, larva, pupa, female and male adults. It was shown that most cSPs were abundantly expressed in adults, while PPOs were detected at high levels in both egg and larval stages. These current findings substantially add to our understanding of the roles of S. frugiperda SPs, SPHs and PPOs in immune regulation and further lay a solid foundation for uncovering the interaction mechanisms between insects and pathogens.     

(p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases

p-(Amidinophenyl)methanesulfonyl fluoride (p-APMSF) has been synthesized and shown to be a specific, irreversible inhibitor of the class of plasma serine proteases which demonstrate substrate specificity for the positively charged side chains of the amino acid lysine or arginine. In equimolar concentration, this compound causes immediate and complete irreversible inhibition of bovine trypsin and human thrombin. A 5-10-fold molar excess of reagent over enzyme is required to achieve complete irreversible inhibition of bovine Factor Xa, human plasmin, human C1-r, and human C1-s. the Ki of p-APMSF for all of the above-mentioned proteases is between 1 and 2 microM. In contrast, p-APMSF in large molar excess does not inactivate chymotrypsin or acetylcholinesterase. The unique reactivity of p-APMSF has been further shown in comparison with the related compound p-nitrophenyl (p-amidinophenyl)methanesulfonate which is an active-site titrant for thrombin but reacts poorly with Factor Xa, C1-r, and C1-s and is not hydrolyzed by bovine trypsin or human plasmin. Similarly, (p-amidinophenyl)methanesulfonate has a Ki of 30 microM for thrombin but is a poor inhibitor of trypsin, Factor Xa, C1-r, C1-s, and plasmin. Studies with bovine trypsin have demonstrated that the inhibitory activity of p-APMSF is the result of its interaction with the diisopropyl fluorophosphate reactive site. The unique reactivity of this inhibitor classifies it as one of the most effective active site directed reagents for this class of serine proteases. Collectively, these results suggest that the primary substrate binding site of these enzymes, which share a high degree of structural homology, do in fact significantly differ from each other in their ability to interact with low molecular weight inhibitors and synthetic substrates.     

Specificity comparison of a serine endopeptidase (SH1) and a serine thiol endopeptidase (STH2) purified from human urine

In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].     

跨膜丝氨酸蛋白酶6与贫血

跨膜丝氨酸蛋白酶6基因（transmembrane serine protease 6 gene,TMPRSS6）是一种主要在肝脏中表达,编码II型跨膜丝氨酸蛋白酶的基因,它的突变可导致严重贫血的发生。TMPRSS6突变后可造成铁调节激素肝杀菌肽在机体缺铁状态下也表达增加,这造成小肠对食物铁的吸收减少而出现缺铁性贫血。TMPRSS6突变与贫血关系的发现一方面有利于对铁代谢调节机制的深入理解,另一方面也为铁代谢紊乱相关疾病如贫血等的治疗带来了希望。     

Sequential activation of caspases and serine proteases (serpases) during apoptosis

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.