Structural and functional consequences of chaperone site deletion in αA-crystallin.
Structural and functional consequences of chaperone site deletion in αA-crystallin.
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αA-晶状体中伴侣位点缺失的结构和功能后果。
DOI：
10.1016/j.bbapap.2016.08.006
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发表时间：
2016-11
影响因子：
3.2
通讯作者：
Sharma, Krishna K.
中科院分区：
文献类型：
--
作者：
Santhoshkumar, Puttur;Karmakar, Srabani;Sharma, Krishna K.
αA-晶状体蛋白的伴侣样活性在维持晶状体透明度方面起着重要作用。在此之前，我们发现70-88位残基是αA-晶体蛋白中的伴侣位点。在本研究中，我们删除了伴侣位点残基以产生αAΔ70-76和αAΔ70-88突变体，并研究了αA晶体蛋白中是否存在额外的底物结合位点。这两种突变蛋白在大肠杆菌中表达时形成包涵体，在溶解和复性时表现出相似的结构性质，摩尔质量比野生型蛋白的摩尔质量增加2-3倍。缺失突变体的稳定性不如野生型αA-晶状体蛋白。从功能上讲，αAΔ70-88作为伴侣完全没有活性，而αAΔ70-76对乙醇脱氢酶的抗聚集活性降低了40%-50%。在生理温度下，70-88位残基的缺失取消了αA-晶状体蛋白中的ADH结合部位。在45℃时，隐匿的ADH结合位点(S)暴露出来，这对αAΔ70-88的伴侣活性起到了微妙的作用。在53℃时，两个缺失突变体都完全不能抑制βL-晶体蛋白的聚集。突变体在保护人视网膜色素上皮细胞株ARPE-19和原代人晶状体上皮细胞免受氧化应激的同时，完全失去了αA-晶体蛋白所具有的抗凋亡特性。我们的研究表明，αA-晶体蛋白中的70-88残基是主要的底物结合部位，占总伴侣活性的大部分。βA-晶体蛋白中的β3和α4链由70-88个残基组成，在维持结构和防止变性蛋白聚集方面发挥着重要作用。
The chaperone-like activity of αA-crystallin has an important role in maintaining lens transparency. Previously we identified residues 70–88 as a chaperone site in αA-crystallin. In this study, we deleted the chaperone site residues to generate αAΔ70–76 and αAΔ70–88 mutants and investigated if there are additional substrate-binding sites in αA-crystallin. Both mutant proteins when expressed in E. coli formed inclusion bodies, and on solubilizing and refolding, they exhibited similar structural properties, with a 2- to 3-fold increase in molar mass compared to the molar mass of wild-type protein. The deletion mutants were less stable than the wild-type αA-crystallin. Functionally αAΔ70–88 was completely inactive as a chaperone, while αAΔ70–76 demonstrated a 40–50% reduction in anti-aggregation activity against alcohol dehydrogenase (ADH). Deletion of residues 70–88 abolished the ADH binding sites in αA-crystallin at physiological temperature. At 45 °C, cryptic ADH binding site(s) became exposed, which contributed subtly to the chaperone-like activity of αAΔ70–88. Both of the deletion mutants were completely inactive in suppressing aggregation of βL-crystallin at 53 °C. The mutants completely lost the anti-apoptotic property that αA-crystallin exhibits while they protected ARPE-19 (a human retinal pigment epithelial cell line) and primary human lens epithelial (HLE) cells from oxidative stress. Our studies demonstrate that residues 70–88 in αA-crystallin act as a primary substrate binding site and account for the bulk of the total chaperone activity. The β3 and β4 strands in αA-crystallin comprising 70–88 residues play an important role in maintenance of the structure and in preventing aggregation of denaturing proteins.
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影响因子：
4.8
作者：
Das, KP;Petrash, JM;Surewicz, WK
通讯作者：
Surewicz, WK
DOI：
10.1016/j.bbadis.2011.11.011
发表时间：
2012-02-01
影响因子：
6.2
作者：
Nagaraj, Ram H.;Nahomi, Rooban B.;Biswas, Ashis
通讯作者：
Biswas, Ashis
DOI：
10.1006/bbrc.1997.6950
发表时间：
1997-07-18
影响因子：
3.1
作者：
Das, BK;Liang, JJN
通讯作者：
Liang, JJN
DOI：
10.1073/pnas.79.7.2360
发表时间：
1982-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
INGOLIA, TD;CRAIG, EA
通讯作者：
CRAIG, EA
影响因子：
56.9
作者：
BLOEMENDAL, H
通讯作者：
BLOEMENDAL, H