铁硫蛋白的生物合成及相关疾病

铁硫蛋白是以铁硫簇为辅基,相对分子质量较小的一类蛋白质．它广泛存在于各种生物体内,参与电子传递、能量代谢以及基因表达调控等重要生理过程．其生物合成过程复杂,并且从细菌到人类高度保守．在真核细胞内,铁硫蛋白的组装由线粒体铁硫簇组装系统（mitochondrial iron sulfur cluster assembly system,mitochondrial ISC assembly system）和细胞质铁硫簇组装器（cytosolic iron sulfur cluster assembly,CIA）完成．研究发现,铁硫蛋白的合成异常可导致弗里德赖希共济失调(friedreich ataxia,FRDA)、遗传性肌病和铁粒幼细胞性贫血等多种罕见疾病,这些疾病严重影响个体的生活质量和寿命．因此,深入了解铁硫蛋白的结构和生物合成过程,对研究其生物学功能与相关疾病的诊断和治疗有重要意义．     

嗜酸氧化亚铁硫杆菌亚硫酸还原酶的α亚基黄素蛋白(cysJ)的表达、纯化及活性测定

亚硫酸还原酶是嗜酸氧化亚铁硫杆菌硫氧化还原系统电子传递链的重要组分之一.本文以嗜酸氧化亚铁硫杆菌ATCC23270基因组为模板,通过基因重组技术在大肠杆菌中表达,用一步亲和层析法纯化出浓度和纯度都较高的亚硫酸还原酶的a亚基黄素蛋白(cysJ).通过SDS-聚丙烯酰胺凝胶电泳、紫外可见分光光度法等方法确定其性质并成功测定了活性,为进一步研究亚硫酸还原酶的功能及其应用提供了条件.     

铁氧还素(Fd)和Fd-NADP~+还原酶的抗体血清对光合电子传递的影响

非血红铁硫蛋白Fd和含黄素蛋白Fd-NADP~+还原酶,都是光合电子传递链上的电子递体,它们催化NADP~+光还原形成NADPH,为非循环光合电子传递。我们近来的研究结果证明:DCMU(3-(3,4-二氯苯)-1,1-二甲脲)在 Fd-NADP~+还原酶的部位     

D-氨基酸氧化酶研究进展

D-氨基酸氧化酶(D-amino acid oxidase:oxidoreductase, DAAO, EC 1.4.3.3)是一种以黄素腺嘌呤(FAD)为辅基的典型黄素蛋白酶类,可氧化D-氨基酸的氨基生成相应的酮酸和氨。在体内D-氨基酸的代谢中起着重要作用。主要介绍了D-氨基酸氧化酶的生理功能和应用、表达条件优化及通过定点突变对酶学性质的研究。     

谷氧还蛋白2与人类健康

谷氧还蛋白2(Glutaredoxin 2,GLRX2)是一种相对分子质量较小的氧化还原酶,属于硫氧还蛋白家族成员,以谷胱甘肽为辅基调节细胞的氧化还原内环境。在非应激条件下,GLRX2结合铁硫簇,以二聚体形式存在,可能参与铁硫簇的转运或运输;当氧化压力增加时,铁硫簇解聚,GLRX2二聚体转化为GLRX2单体,利用单巯基或双巯基机制,发挥抗氧化应激和抗细胞凋亡的功能。GLRX2与人类健康和疾病,如心血管疾病、神经退行性疾病、白内障、肿瘤细胞生长与分化和精子成熟等密切相关。因此,对GLRX2的深入研究将有助于设计针对氧化应激的药物,为治疗和预防由此产生的疾病或健康问题带来新的希望。     

嗜酸氧化亚铁硫杆菌ISC铁硫簇系统的合成

【目的】铁硫簇是最古老的一种氧化还原中心,它普遍存在于所有生命体内,在光合作用、呼吸作用和固氮作用这三个地球生命最基本的代谢途径中扮演着重要的角色。【方法】以嗜酸氧化亚铁硫杆菌(A.ferrooxidans ATCC 23270)基因组为模板,克隆表达其ISC铁硫簇组装的3个核心蛋白,IscS(半胱氨酸脱硫酶蛋白)、IscU(支架蛋白)和IscA(铁供体蛋白)。【结果】研究发现IscS能催化半胱氨酸脱硫,为铁硫簇的组装提供硫,支架蛋白IscU不具备结合铁的能力,IscA具有较强的铁结合能力。【结论】铁硫簇体外组装证明Fe-IscA在体外能将结合的铁传递给IscS,并在IscU上进行铁硫簇的组装。     

真核细胞中铁硫簇的组装机制及相关铁硫蛋白疾病

铁硫簇是一类古老而功能众多的蛋白质辅基,在细胞中参与电子传递过程、酶促反应及感知内环境的变化而调节基因的表达等。虽然铁硫簇的组成元素和结构都较为简单,但是铁硫簇的组装是需多种组装蛋白参与、有序进行的催化反应。直至近几年,人们才逐渐阐明了在生命体中铁硫簇是如何组装并结合到未成熟的铁硫蛋白中的。如果线粒体中铁硫簇组装及转运过程发生障碍,将严重影响细胞内铁的稳态及铁硫蛋白的功能,由此可见,线粒体中铁硫簇的组装功能使得线粒体成为细胞中必不可少的一类细胞器。该文重点概述了近十年来真核生物中铁硫簇组装机制的研究进展并阐述线粒体铁硫簇组装在人体中的重要作用及其组装障碍所引起的疾病。     

生物磁受体蛋白MagR/IscA研究进展

铁硫簇蛋白是一类重要的线粒体功能蛋白,在细胞能量代谢、电子传递、底物结合与激活、铁/硫存储、酶促反应、基因表达调控等诸多过程中均发挥了关键作用.铁硫簇蛋白质组装及转运过程一旦发生障碍,必将严重影响细胞内铁的稳态及铁硫蛋白的功能.分子质量约11 ku的铁硫簇蛋白IscA,是铁硫蛋白亚家族hesB高保守性成员之一,能结合铁离子及[2Fe-2S]簇,参与铁硫簇蛋白质合成,因此IscA在铁硫簇组装蛋白与级联反应系统中具有重要的作用.更值得关注的是,2015年谢灿和张生家两个研究组发现IscA1具有磁受体(MagR/MAR)作用,此外,谢灿课题组揭示MagR能与Cry形成磁感应复合物行使磁感应器(magnetic sensor,MagS)功能.尤为重要的是,体内实验表明通过外磁场刺激活化MagR能调控相关磁基因表达,影响神经活动及行为定位.鉴于MagR磁受体的独特功能,张生家等将磁受体的基因定位与远程磁刺激相结合,发明了一种非损伤性的神经调控方法,称之为磁遗传学.本文简要介绍MagR/IscA及其同源基因的始初发现与鉴定历程、进化保守性、独特的生理生物学功能,并凝练出磁遗传假说机制调控模型,以解释MagR/IscA的磁遗传学功能.     

低表达线粒体蛋白ISCA2对线粒体功能影响的机制研究

该文探讨了ISCA2蛋白低表达对细胞内铁硫蛋白和能量代谢的影响。在HeLa细胞内将ISCA2基因敲低,通过免疫印迹、顺乌头酸酶胶内酶活性分析法分析线粒体内外铁硫蛋白水平和活性改变;用紫外–可见分光光度法检测细胞线粒体内氧化磷酸化复合体活性;用海马能量分析仪分析细胞内能量代谢的改变。结果表明,ISCA2蛋白低表达后,氧化磷酸化复合体中各铁硫蛋白亚基都出现不同程度的下调,且对于线粒体[4Fe-4S]型铁硫蛋白亚基影响显著,但对线粒体[2Fe-2S]型铁硫蛋白亚基以及胞质[4Fe-4S]型铁硫蛋白亚基影响较小。同时,ISCA2蛋白低表达后对线粒体复合体活性和线粒体能量代谢影响显著,细胞有氧呼吸降低,细胞外乳酸含量增加。这些结果表明,ISCA2蛋白低表达后抑制铁硫簇的组装,导致铁硫蛋白功能障碍,影响线粒体复合体活性和氧化磷酸化系统,使得细胞能量代谢紊乱。     

铜对线粒体中铁硫蛋白功能的毒性机制研究

该文探讨了铜对线粒体内铁硫蛋白的毒性机理。通过包装慢病毒将Hep G2细胞中铜转运蛋白ATP7B(ATPase copper transporting beta)基因敲低,并用铜离子处理构建高铜细胞模型。通过免疫印迹、胶内酶活、紫外–可见光分光光度法检测细胞线粒体内铁硫蛋白、非铁硫蛋白及铁硫簇组装蛋白量和活性的改变;用电镜观察高铜模型中线粒体的形态改变;用海马能量代谢分析仪检测铜离子对细胞能量代谢的影响。结果发现,高铜细胞模型线粒体内铁硫簇组装蛋白ISCA2(ironsulfur cluster assembly 2)及ISCU(iron-sulfur cluster assembly enzyme)水平下降,抑制了铁硫簇的组装,并进一步影响了线粒体内[2Fe-2S]型及[4Fe-4S]型铁硫蛋白功能,但并不影响非铁硫蛋白。高铜状态也影响了呼吸链复合体活性及线粒体能量代谢,并导致线粒体形态发生改变。这些结果表明,异常累积的铜离子也会通过抑制线粒体中铁硫簇的组装,影响线粒体内铁硫蛋白的功能。     

6-Methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone from Salvia aegyptiaca

From the whole plant of Salvia aegyptiaca, 6-methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone have been isolated and characterized, mainly by spectroscopic means. In addition to these novel diterpenoids, the known compounds 3beta-hydroxy-olean-12-en-28-oic acid, 3beta-hydroxy-oleana-11,13(18)-dien-28-oic acid, sitosterol-3beta-glucoside, sitosterol, stigmasterol, 5-hydroxy-7,3',4'-trimethoxyflavone and 5, 6-dihydroxy-7,3',4'-trimethoxyflavone were isolated.     

Preparation of 6-deoxy-6-fluorocellulose

6-Deoxy-6-fluorocellulose was prepared from cellulose 2,3-diacetate (1) or cellulose 2,3-dibenzoate (2) in various solvents, and was characterized by ¹⁹F and ¹³C NMR measurements. The best product, having ds of 0.95 at C-6 and 0.04 at C-3, was prepared from cellulose 2,3-dibenzoate in nitrobenzene. Other combinations of starting material and solvent gave a lower (≈ 0.8) ds of fluorine at C-6 and higher (≈ 0.12) at C-2 or C-3. Substitution at C-2 was observed when the combination of 1 and 1,4-dioxane, or 2 and chloroform was used. The products substituted at C-2 by fluorine were relatively resistant to acid hydrolysis.     

New 6-butylamino-6-deoxycellulose and 6-deoxy-6-pyridiniumcellulose derivatives with highest regioselectivity and completeness of reaction

This paper investigates the nucleophilic substitution (S(N)) reactions of tosylcellulose with butylamine and pyridine, respectively. The S(N) reactions of tosylcellulose 1 (DS(Total) 2.02; DS(C-6) 1.0) with butylamine carried out at 25, 50, 75 and 100 degrees C in both dimethyl sulfoxide (DMSO) and pure butylamine showed that the regioselectivity of substitution at C-6 of cellulose is temperature dependent: the highest regioselectivity at C-6 can be reached at 25 and 50 degrees C; substitution at C-2 also occurred at 75 and 100 degrees C. The substitution speed in pure butylamine is greater than that in the presence of DMSO. A complete and regioselective substitution at C-6 with a DS of 1.0 was obtained under the conditions of 50 degrees C, 40 h in butylamine. The substitution reactions of 1 with pyridine carried out at 25, 50, 75 and 100 degrees C for 24h in DMSO did not occur. In contrast to this the S(N) reactions done in pure pyridine showed that a temperature- and steric-dependent, regioselective substitution took place at C-6 at temperatures from 25 to 145 degrees C. The highest regioselectivity and completeness at C-6 can be obtained at 100 degrees C for 90 h, whereas at 145 degrees C substitution also occurs at C-2. The results were proved by 1H NMR and 13C NMR spectroscopy.     

Facile synthesis of 6-amino-6-deoxycellulose

6-Amino-6-deoxycellulose (4) was synthesized from cellulose by three reaction steps, namely bromination at C-6, displacement of bromine by azide ion, and reduction of the azide group to amino group, in 67% overall yield. The 13C NMR spectrum of compound 4 supports the expected structure for 6-amino-6-deoxycellulose. The degree of substitution of compound 4 was 0.96.     

Syntheses of (+/-)-methyl 6'alpha-demethyl-6'alpha-cyanoabscisate and (+/-)-methyl 6'alpha-demethyl-6'alpha-methoxycarbonylabscisate

New abscisic acid analogs possessing a cyano or methoxycarbonyl group at the 6'alpha-position of methyl abscisate were synthesized by regioselective hydrocyanation. These compounds had weak activity in the rice second leaf sheath elongation test.     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Minimal Interleukin 6 (IL-6) Receptor Stalk Composition for IL-6 Receptor Shedding and IL-6 Classic Signaling

Signaling of the pleiotropic cytokine Interleukin-6 (IL-6) is coordinated by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The soluble IL-6R is mainly generated by ADAM10- and ADAM17-mediated ectodomain shedding. Little is known about the role of the 52-amino acid-residue-long IL-6R stalk region in shedding and signal transduction. Therefore, we generated and analyzed IL-6R stalk region deletion variants for cleavability and biological activity. Deletion of 10 amino acids of the stalk region surrounding the ADAM17 cleavage site substantially blocked IL-6R proteolysis by ADAM17 but only slightly affected proteolysis by ADAM10. Interestingly, additional deletion of the remaining five juxtamembrane-located amino acids also abrogated ADAM10-mediated IL-6R shedding. Larger deletions within the stalk region, that do not necessarily include the ADAM17 cleavage site, also reduced ADAM10 and ADAM17-mediated IL-6R shedding, questioning the importance of cleavage site recognition. Furthermore, we show that a 22-amino acid-long stalk region is minimally required for IL-6 classic signaling. The gp130 cytokine binding sites are separated from the plasma membrane by ∼96 Å. 22 amino acid residues, however, span maximally 83.6 Å (3.8 Å/amino acid), indicating that the three juxtamembrane fibronectin domains of gp130 are not necessarily elongated but somehow flexed to allow IL-6 classic signaling. Our findings underline a dual role of the IL-6R stalk region in IL-6 signaling. In IL-6 trans-signaling, it regulates proper proteolysis by ADAM10 and ADAM17. In IL-6 classic-signaling, it acts as a spacer to ensure IL-6·IL-6R·gp130 signal complex formation.