CAJ | 학술논문

Metacaspases（MCPs）是发现于植物、真菌、原生动物体内的一种结构上类似多细胞动物Caspase的半胱氨酸蛋白酶家族，其大多数成员的激活依赖于钙离子，但钙离子如何影响MCPs的激活机制有待深入研究。借助圆二色谱技术、Terbium／Stains‐all探针技术以及荧光光谱技术，以番茄Ⅱ型 Metacaspase （LeMCA1）重要位点突变的三种体外原核表达重组蛋白，包括LeMCA1C139A （活性催化位点突变体）、LeM‐CA1K223G （自降解位点突变体）以及预测的Ca2＋结合位点突变体LeMCA1D116A／D117A为研究材料，探究了MCPs与Ca2＋相互作用机制。实验结果表明，Ca2＋与LeMCA1之间既不存在强烈的结合作用，也不影响蛋白的二级结构，但Ca2＋通过相互作用改变LeMCA1的三级结构来实现对LeMCA1的酶原激活过程。其中，LeM‐CA1蛋白的Asp‐116，Asp‐117氨基酸残基作为预测的与Ca2＋作用的重要位点，其缺失将导致蛋白与Ca2＋相互作用能力下降。利用圆二色谱、荧光光谱结合离子探针技术研究了典型茄科植物番茄中Ca2＋与LeM‐CA1的相互作用特性，结合之前同源序列比对、位点突变结果确定了LeMCA1中的Asp‐116，Asp‐117氨基酸残基影响着Ca2＋与蛋白质的相互作用。该结果对后续LeMCA1的生化特性及晶体结构的解析研究有着重要的参考价值。
Metacaspases（MCPs）시발현우식물、진균、원생동물체내적일충결구상유사다세포동물Caspase적반광안산단백매가족，기대다수성원적격활의뢰우개리자，단개리자여하영향MCPs적격활궤제유대심입연구。차조원이색보기술、Terbium／Stains‐all탐침기술이급형광광보기술，이번가Ⅱ형 Metacaspase （LeMCA1）중요위점돌변적삼충체외원핵표체중조단백，포괄LeMCA1C139A （활성최화위점돌변체）、LeM‐CA1K223G （자강해위점돌변체）이급예측적Ca2＋결합위점돌변체LeMCA1D116A／D117A위연구재료，탐구료MCPs여Ca2＋상호작용궤제。실험결과표명，Ca2＋여LeMCA1지간기불존재강렬적결합작용，야불영향단백적이급결구，단Ca2＋통과상호작용개변LeMCA1적삼급결구래실현대LeMCA1적매원격활과정。기중，LeM‐CA1단백적Asp‐116，Asp‐117안기산잔기작위예측적여Ca2＋작용적중요위점，기결실장도치단백여Ca2＋상호작용능력하강。이용원이색보、형광광보결합리자탐침기술연구료전형가과식물번가중Ca2＋여LeM‐CA1적상호작용특성，결합지전동원서렬비대、위점돌변결과학정료LeMCA1중적Asp‐116，Asp‐117안기산잔기영향착Ca2＋여단백질적상호작용。해결과대후속LeMCA1적생화특성급정체결구적해석연구유착중요적삼고개치。
Metacaspases are cysteine‐dependent proteases found in protozoa ,fungi and plants and are distantly related to metazo‐an caspases .Most of MCPs activation are the calcium dependent ,but the mechanisms are still unknown .Based on the techniques of CD spectroscopy ,fluorescence spectroscopy ,and Terbium Stains‐all probe ,we selected three purified recombinant proteins from key residues mutated in tomato metacaspase (LeMCA1) ,including conserved catalytic site (C139A) mutant ,N‐sequenced cleaved site (K223G) mutant and the predicted Ca2+ binding sites (D116A/D117A ) mutant ,to explore the interaction mecha‐nism of LeMCA1 and Ca2+ .CD spectroscopy and Stains‐all probe results suggested that the intense binding does not exist be‐tween LeMCA1 and Ca2+ as well as Ca2+ has little effect on the secondary structure of LeMCA1 .However ,fluorescence spec‐troscopy and Tb3+ probe results showed that Ca2+‐induced the changes occur in the tertiary structure of LeMCA 1 ,which con‐tributes to the activation of zymogen .In addition ,predicted Ca2+ binding residues ,Asp‐116 and Asp117 ,are the key sites re‐sponsible for the Ca2+ interaction with LeMCA1 ,and the loss of these two residues resulted in decreased interaction .Our data firstly provided insight on the mechanism of the interaction between Ca2+ and recombinant purified Solanaceae type Ⅱ meta‐caspase by spectroscopy and molecular probe techniques .Combined the results we got before from sequence‐alignment and sites‐mutation ,the key residues Asp‐116 and Asp117 affect the Ca2+‐induced the changes of LeMCA1 tertiary structure .Our data provided information for the further biochemical and crystal assays of LeMCA 1 .