Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.
Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.
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DOI：
10.1016/s0021-9258(17)37352-0
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发表时间：
1994-03
期刊：
影响因子：
--
通讯作者：
Alberto Puig;H. Gilbert
中科院分区：
文献类型：
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作者：
Alberto Puig;H. Gilbert
还原的、变性的溶菌酶倾向于在中性pH下聚集，而生产性折叠和聚集之间的竞争大大降低了再折叠的效率（Goldberg, m.e., Rudolph， R., and Jaenicke， R. (1991) Biochemistry 30,2790 -2797）。蛋白质二硫异构酶（PDI）是蛋白质氧化折叠的催化剂，在还原变性蛋白质的氧化再折叠过程中对天然溶菌酶的产率有多种影响。根据溶菌酶的浓度、PDI的浓度以及加入溶菌酶和PDI启动折叠的顺序，与未催化的反应相比，PDI可以使天然溶菌酶的回收率大幅增加或大幅减少。在谷胱甘肽氧化还原缓冲液的存在下，变性溶菌酶（1-10微米）几乎均匀地在产生天然溶菌酶（50-63%）和非产生溶菌酶（包括形成二硫交联聚集体）之间进行分割。在较高的溶菌酶浓度（5-10微米）下，亚化学计量浓度的PDI（0.5-1微米）表现出“抗伴侣”活性；PDI积极地将大部分变性溶菌酶从生产折叠中转移出来，因此只有17 +/- 9%的溶菌酶被回收为天然酶。PDI的抗伴侣活性导致溶菌酶广泛的分子间二硫交联成大的，无活性的聚集体。另一方面，当变性溶菌酶被稀释以启动折叠时，如果PDI最初以大的摩尔过量（5-10倍）存在，PDI表现出一种类似伴侣的活性，可以阻止聚集体的形成并促进正确的折叠。当PDI的伴侣活性占主导地位时，几乎所有的变性溶菌酶都被正确折叠。PDI活性的精神分裂症伴侣/抗伴侣性质解释了体内蛋白质折叠的许多观察结果，包括在内质网中维持高浓度PDI的必要性，以及在含有二硫化物的蛋白质表达期间内质网中形成二硫化物交联聚集体(deSilva, a ., Braakman， I., and Helenius, a . (1993) J. Cell。生物学报，2000,647 -655)。
Reduced, denatured lysozyme tends to aggregate at neutral pH, and competition between productive folding and aggregation substantially reduces the efficiency of refolding (Goldberg, M.E., Rudolph, R., and Jaenicke, R. (1991) Biochemistry 30, 2790-2797). Protein disulfide isomerase (PDI), a catalyst of oxidative protein folding, has a variety of effects on the yield of native lysozyme during the oxidative refolding of the reduced, denatured protein. Depending on the concentration of lysozyme, the concentration of PDI, and the order in which lysozyme and PDI are added to initiate folding, PDI can produce a substantial increase or a substantial decrease in the recovery of native lysozyme, when compared with the uncatalyzed reaction. In the presence of a glutathione redox buffer, denatured lysozyme (1-10 microM) partitions almost equally between productive folding leading to native lysozyme (50-63%) and non-productive fates including the formation of disulfide cross-linked aggregates. At the higher lysozyme concentrations examined (5-10 microM), substoichiometric concentrations of PDI (0.5-1 microM) exhibit “anti-chaperone” activity; PDI actively diverts most of the denatured lysozyme away from productive folding so that only 17 +/- 9% of the lysozyme is recovered as native enzyme. PDI's anti-chaperone activity results in extensive intermolecular disulfide crosslinking of lysozyme into large, inactive aggregates. On the other hand, if PDI is initially present at a large molar excess (5-10-fold) when denatured lysozyme is diluted to initiate folding, PDI demonstrates a chaperone-like activity that prevents aggregate formation and promotes correct folding. When PDI's chaperone activity is dominant, virtually all of the denatured lysozyme is correctly folded. The schizophrenic chaperone/anti-chaperone nature of PDI activity accounts for a number of observations on in vivo protein folding, including the necessity for maintaining a high concentration of PDI in the endoplasmic reticulum and the formation of disulfide cross-linked aggregates in the endoplasmic reticulum during the expression of disulfide-containing proteins (deSilva, A., Braakman, I., and Helenius, A. (1993) J. Cell. Biol. 120, 647-655).