The residual pro-part of cathepsin C fulfills the criteria required for an intramolecular chaperone in folding and stabilizing the human proenzyme
The residual pro-part of cathepsin C fulfills the criteria required for an intramolecular chaperone in folding and stabilizing the human proenzyme
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DOI：
10.1021/bi0008837
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发表时间：
2000-10-10
期刊：
影响因子：
2.9
通讯作者：
Pain, RH
中科院分区：
文献类型：
--
作者：
Cigic, B;Dahl, SW;Pain, RH
前肽的13.5 kDa N-末端部分在蛋白水解活化和切除活化肽后仍与成熟组织蛋白酶C相关。从重组酶中分离的该残留前体部分自发且快速地折叠成具有二级结构和稳定的三级相互作用的稳定的紧凑单体。折叠和展开动力学与完整的二硫化物的残留前部分是复杂的，并观察到瞬态中间体的积累。从天然人组织蛋白酶C分离的原部分的切割形式也折叠，表明完整形式包含两个折叠结构域。对于天然酶，两个二硫键的连接已经确定为30-118和54-136。天然二硫键弯曲可以通过氧化重折叠从完全还原和变性状态重新形成，导致在光谱上与原始重折叠的残留前体部分无法区分的结构域。两种二硫化物都是溶剂暴露的，并且可以在不存在变性剂的情况下被还原。还原形式保留了大部分或全部的天然三级结构，其稳定性仅比氧化形式低约2 kcal.mol（-1）。相对于生物合成的速率，它快速折叠成与氧化形式相同的构象。折叠和二硫化物的形成是连续的。这些结果表明，酶原折叠顺序在体内和残留的前部分构成了一个快速和独立的折叠结构域，稳定成熟的酶。因此，它符合分子内伴侣所需的标准。它也可能参与稳定成熟酶的四聚体结构。
The 13.5 kDa N-terminal part of the propeptide remains associated with mature cathepsin C after proteolytic activation and excision of the activation peptide. This residual pro-part, isolated from the recombinant enzyme, folds spontaneously and rapidly to a stable, compact monomer with secondary structure and stable tertiary interactions. Folding and unfolding kinetics of the residual pro-part with intact disulfides are complex, and accumulation of transient intermediates is observed. The cleaved form of the pro-part isolated from natural human cathepsin C also folds, suggesting that the intact form comprises two folding domains. The linkages of the two disulfide bridges have been established as 30-118 and 54-136 for the native enzyme. The native disulfide bends can be re-formed from the fully reduced and denatured state by oxidative refolding, resulting in a domain that is spectroscopically indistinguishable from the original refolded residual pro-part. Both disulfides are solvent-exposed and can be reduced in the absence of denaturant. The reduced form retains most or all of the native tertiary structure and is only approximate to 2 kcal.mol(-1) less stable than the oxidized form. It folds fast relative to the rate of biosynthesis, to the same conformation as the oxidized form. Folding and disulfide formation are sequential. These results indicate that the proenzyme folds sequentially in vivo and that the residual pro-part constitutes a rapidly and independently folding domain that stabilizes the mature enzyme. It thus fulfills the criteria required of an intramolecular chaperone. It may also be involved in stabilizing the tetrameric structure of the mature enzyme.