Liang P, Amons R, Clegg J S, MacRae T H
Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1, Canada.
J Biol Chem. 1997 Jul 25;272(30):19051-8. doi: 10.1074/jbc.272.30.19051.
Molecular chaperones protect cells during stress by limiting the denaturation/aggregation of proteins and facilitating their renaturation. In this context, brine shrimp embryos can endure a wide variety of stressful conditions, including temperature extremes, prolonged anoxia, and desiccation, thus encountering shortages of both energy (ATP) and water. How the embryos survive these stresses is the subject of continuing study, a situation true for other organisms facing similar physiological challenges. To approach this question we cloned and sequenced a cDNA for p26, a molecular chaperone specific to oviparous Artemia embryos. p26 is the first representative of the small heat shock/alpha-crystallin family from crustaceans to be sequenced, and it possesses the conserved alpha-crystallin domain characteristic of these proteins. The secondary structure of this domain was predicted to consist predominantly of beta-pleated sheet, and it appeared to lack regions of alpha-helix. Unique properties of the nonconserved amino terminus, which showed weak similarity to nucleolins and fibrillarins, are enrichments in both glycine and arginine. The carboxyl-terminal tail is the longest yet reported for a small heat shock/alpha-crystallin protein, and it is hydrophilic, a common attribute of this region. Site-specific differences between amino acids from p26 and other small heat shock/alpha-crystallin proteins bring into question the functions proposed for some of these residues. Probing of Southern blots disclosed a multi-gene family for p26, whereas two size classes of p26 mRNA at 0.7 and 1.9 kilobase pairs were seen on Northern blots, the larger probably representing nonprocessed transcripts. Examination of immunofluorescently stained samples with the confocal microscope revealed that a limited portion of intracellular p26 is found in the nuclei of encysted embryos and that it resides within discrete compartments of this organelle. The results in this paper demonstrate clearly that p26 is a novel member of the small heat shock/alpha-crystallin family of proteins. These data, in concert with its restriction to embryos undergoing oviparous development, suggest that p26 functions as a molecular chaperone during exposure to stress, perhaps able to limit protein degradation and thus ensure a ready supply of functional proteins when growth is reinitiated.
分子伴侣通过限制蛋白质的变性/聚集并促进其复性来在应激期间保护细胞。在这种情况下，卤虫胚胎能够耐受多种应激条件，包括极端温度、长时间缺氧和干燥，从而面临能量（ATP）和水的短缺。胚胎如何在这些应激中存活是持续研究的主题，对于面临类似生理挑战的其他生物也是如此。为了解决这个问题，我们克隆并测序了p26的cDNA，p26是卵生卤虫胚胎特有的分子伴侣。p26是甲壳类动物小热休克/α-晶状体蛋白家族中第一个被测序的代表，它具有这些蛋白质特有的保守α-晶状体蛋白结构域。该结构域的二级结构预计主要由β-折叠组成，并且似乎缺乏α-螺旋区域。与核仁素和原纤维蛋白有弱相似性的非保守氨基末端的独特性质是甘氨酸和精氨酸的富集。羧基末端尾巴是小热休克/α-晶状体蛋白中报道的最长的，并且它是亲水的，这是该区域的共同特征。p26与其他小热休克/α-晶状体蛋白的氨基酸之间的位点特异性差异使人们对其中一些残基所提出的功能产生质疑。Southern印迹杂交显示p26是一个多基因家族，而在Northern印迹上看到了0.7和1.9千碱基对的两种大小类别的p26 mRNA，较大的可能代表未加工的转录本。用共聚焦显微镜检查免疫荧光染色的样品发现，细胞内p26的有限部分存在于包囊胚胎的细胞核中，并且它位于该细胞器的离散隔室内。本文的结果清楚地表明p26是小热休克/α-晶状体蛋白家族的一个新成员。这些数据，连同其对卵生发育胚胎的限制，表明p26在暴露于应激期间作为分子伴侣发挥作用，也许能够限制蛋白质降解，从而在重新开始生长时确保有现成的功能性蛋白质供应。
