Mechanism of insulin fibrillation: the structure of insulin under amyloidogenic conditions resembles a protein-folding intermediate

Insulin undergoes aggregation-coupled misfolding to form a cross-beta assembly. Such fibrillation has long complicated its manufacture and use in the therapy of diabetes mellitus. Of interest as a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Here, we describe the solution structure of human insulin under amyloidogenic conditions (pH 2.4 and 60 degrees C). Use of an enhanced sensitivity cryogenic probe at high magnetic field avoids onset of fibrillation during spectral acquisition. A novel partial fold is observed in which the N-terminal segments of the A- and B-chains detach from the core. Unfolding of the N-terminal alpha-helix of the A-chain exposes a hydrophobic surface formed by native-like packing of the remaining alpha-helices. The C-terminal segment of the B-chain, although not well ordered, remains tethered to this partial helical core. We propose that detachment of N-terminal segments makes possible aberrant protein-protein interactions in an amyloidogenic nucleus. Non-cooperative unfolding of the N-terminal A-chain alpha-helix resembles that observed in models of proinsulin folding intermediates and foreshadows the extensive alpha --> beta transition characteristic of mature fibrils.     

S-硫甲基型胰岛素A及B链相互作用的200MHz~1H NMR研究

前已报道,S-硫甲基型胰岛素A及B链相混后,α螺旋等有序二级结构含量增加,部分酪氨酸残基由相对亲水的环境逐渐转移到相对疏水的环境,整个过程在1.5～2小时达到稳定,说明在还原型A、B链能重组成有活力的胰岛素的相同条件下,两肽链间确实存在着次级键相互作用。本文报道以200兆赫NMR~1H谱所测S-硫甲基型A及B链的图谱,对其主要谱峰作了初步识别。在pH10.8时,A及B链以克分子比1.5:1相混后,Tyr的(2,6)位及(3,5)位质子峰较之单独链有低场位移,提示Tyr的酚羟基解离减少,Tyr转移至相对疏水的环境;也可能是肽链构象变化,使Tyr受去屏蔽效应所致。在A、B链相混后,每半小时累加的结果表明,不仅Tyr的两个低场峰随时间有低场位移,甲基峰随时间也有连续的低场位移,在1.5小时后达到稳定。此时间过程与前者报道的紫外差光谱、园二色性的观察结果一致,说明构象的调整涉及整个肽链。不同pH下的~1H谱测定显示,在一段pH区间都存在着A、B链间的次级键相互作用。上述结果表明,或许正是这种链间的次级键相互作用,才使A、B链之巯基有可能正确配对,重组成有生物活力的胰岛素分子。     

Deciphering the Hidden Informational Content of Protein Sequences: FOLDABILITY OF PROINSULIN HINGES ON A FLEXIBLE ARM THAT IS DISPENSABLE IN THE MATURE HORMONE*

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe^B1 in placental mammals) is variably positioned within crystal structures and exhibits ¹H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1–B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.     

Chiral mutagenesis of insulin's hidden receptor-binding surface: structure of an allo-isoleucine(A2) analogue

The hydrophobic core of vertebrate insulins contains an invariant isoleucine residue at position A2. Lack of variation may reflect this side-chain's dual contribution to structure and function: Ile(A2) is proposed both to stabilize the A1-A8 alpha-helix and to contribute to a "hidden" functional surface exposed on receptor binding. Substitution of Ile(A2) by alanine results in segmental unfolding of the A1-A8 alpha-helix, lower thermodynamic stability and impaired receptor binding. Such a spectrum of perturbations, although of biophysical interest, confounds interpretation of structure-activity relationships. To investigate the specific contribution of Ile(A2) to insulin's functional surface, we have employed non-standard mutagenesis: inversion of side-chain chirality in engineered monomer allo-Ile(A2)-DKP-insulin. Although the analogue retains native structure and stability, its affinity for the insulin receptor is impaired by 50-fold. Thus, whereas insulin's core readily accommodates allo-isoleucine at A2, its activity is exquisitely sensitive to chiral inversion. We propose that the Ile(A2) side-chain inserts within a chiral pocket of the receptor as part of insulin's hidden functional surface.     

鳜鱼传染性脾肾坏死病毒(ISKNV)PCR检测方法的建立及虹彩病毒新证据

利用真鲷虹彩病毒（ＲＳＩＶ）核苷酸还原酶小亚单位（ＲＮＲＳ）基因保守区设计的一对引物,建立了鳜鱼传染性脾肾坏死病毒（ＩＳＫＮＶ）特异的ＰＣＲ检测体系。运用该体系检测ＩＳＫＮＶ,具有简便、快速、敏感、特异等特点,为诊断与预防ＩＳＫＮＶ提供了一个重要的手段。通过对ＰＣＲ产物的克隆与序列分析,发现ＩＳＫＮＶ ＰＣＲ扩增产物与ＲＳＩＶ ＲＮＲＳ基因相应序列的同源性很高,达到９２．５％,进一步证明ＩＳＫＮＶ     

舒芬太尼和右美托咪定对胃癌术后镇痛效果对比及对术后快速康复的影响

目的:探讨舒芬太尼和右美托咪定对胃癌术后镇痛效果对比及对术后快速康复的影响.方法:选取我院2017年6月到2019年12月共收治的200例胃癌患者进行对比研究,所有患者在行胃癌根治术后均要求进行术后镇痛,依照随机分组法将患者分为A组(n=67)、B组(n=66)和C组(n=67),A组患者给予2 μg/kg舒芬太尼,B...     

表达乙型肝炎病毒表面抗原基因又形成多角体的杆状病毒

用形成包含体(OCC~+)并能利用人工合成启动序列和多角体XIV启动子表达外源基因的转移载体质粒pSXIVVI~+X3,将乙型肝炎病毒表面抗原(HBsAg)基因和多角体基因同时插入无包含体的粉纹夜蛾核型多角体病毒TnNPV-SVI-G基因组中,得到表达HBsAg基因又形成包含体(多角体)的重组毒侏TnNPV-HBs85-OCC~+。与利用野生型多角体启动子表达HBsAg基因的无包含体毒株TnNPV-HBsD4不同,TnNPV-HBs85-OCC~+由于具包含体,能以口服方式大规模感染粉纹夜蛾(Trichoplusia ni)幼虫,且HBsAg基因在草地夜蛾(Spodoptera frugiperda)离体细胞中的表达量要比前者高约37％,在虫体中的表达则更高。     

芽孢杆菌发酵条件的优化及其在室内条件下提高原油采收率的初步研究

筛选到一株可以在厌氧条件下以原油为唯一碳源生长的芽孢杆菌L - 2 3。对其中试发酵条件进行了优化 ,确定了L - 2 3的最适发酵条件 ,用补料的方法控制葡萄糖浓度在 0 .6% ,在 16h内 ,发酵液的菌体浓度可以达到 5 .2× 10 9cell mL。把 5 %的L - 2 3发酵液加入到原油中 ,在 4 5℃条件下培养 2 4h ,能够使原油粘度降低 2 8%。在室内条件下 ,L - 2 3可以使原油的采收率提高 11%以上。实验证明 ,用L - 2 3进行微生物采油有很好的应用前景。     

太行山山地森林群落植物区系与地理格局——基于植物群落清查数据

太行山区位于黄土高原与华北平原之间,是我国生物多样性保护的重要优先区之一.本文以广义太行山涉及的108个行政县域为研究区域,基于太行山山地森林群落植物清查数据,系统分析了太行山山地森林群落的科属特征、区系组成、植物多样性地理格局及其丰富度热点地区.结果表明: 调查的778个样地得到太行山山地森林群落种子植物共计100科447属963种,其中,裸子植物3科7属12种,被子植物97科440属951种,生活型以草本植物占优势(71.1%);科的分布区类型以热带分布(38%)和温带分布(24%)为主,属的分布区类型以温带成分占主导(68.7%);太行山山地森林群落植物多样性的水平分布格局呈由西南向东北逐渐递增的趋势,群落物种多样性与经纬度均呈正相关关系,但不同生活型植物的多样性格局不相一致,草本植物多样性与经纬度呈正相关,而木本植物多样性与经纬度则无明显相关性;在垂直梯度上,太行山山地森林群落植物丰富度呈单峰分布,集中分布在400~1800 m的低中海拔地带,在1000~1200 m丰富度最高;基于群落清查数据绘制太行山山地森林群落植物丰富度分布图,鉴别出小五台山、云台山、太岳山、王屋山、中条山等山地为植物丰富度热点地区,应列入太行山优先保护的重点规划区域.     

重组表达猪圆环病毒2型衣壳蛋白的抗原特性分析

将猪圆环病毒2型(PCV2 )去核定位信号衣壳蛋白(Nuclearlocalizationsignal_defectedcapsidprotein ,dCap)与谷胱甘肽_S_转移酶(GST)融合,在大肠杆菌中表达,经纯化和凝血酶剪切分别获得纯化的GST_dCap融合蛋白和dCap蛋白,Westernblot结果表明二者都能与猪抗PCV2血清发生特异性反应。dCap蛋白免疫小鼠制备的单克隆抗体,不仅能特异地与GST_dCap融合蛋白、dCap蛋白和纯化的PCV2粒子发生反应,而且能特异地与PK_15细胞内的PCV2病毒颗粒发生反应,其中抗dCap蛋白的单克隆抗体4C4、3F6和2G7具有阻止病毒感染细胞的能力。表明原核表达的dCap蛋白完全或部分正确模拟了PCV2天然衣壳蛋白的构像,PCV2衣壳蛋白存在阻止PCV2病毒感染细胞的功能性表位。同时重组PCV2dCap蛋白的获得为进一步研究Cap蛋白晶体结构和将重组的dCap蛋白作为抗原建立血清学诊断试剂及疫苗研究提供了基础