Aquilina J Andrew, Benesch Justin L P, Ding Lin Lin, Yaron Orna, Horwitz Joseph, Robinson Carol V
Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, United Kingdom.
J Biol Chem. 2005 Apr 15;280(15):14485-91. doi: 10.1074/jbc.M500135200. Epub 2005 Feb 7.
The small heat shock protein, alpha-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of alphaA-crystallin involving the truncation of 5 C-terminal residues (alphaA(1-168)). Using novel mass spectrometry approaches and established biophysical techniques, we show that alphaA(1-168) forms oligomeric assemblies with a lower average molecular mass than wild-type alphaA-crystallin (alphaA(WT)). Also apparent from the mass spectra of both alphaA(WT) and alphaA(1-168) assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for alphaA(1-168). To examine the rate of exchange of subunits between assemblies, we mixed alphaB crystallin with either alphaA(WT) or alphaA(1-168) and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when alphaA(1-168) is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both alphaA(WT) and alphaA(1-168). Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian alphaA-crystallin.
小分子热休克蛋白α-晶体蛋白通过陪伴结构受损的蛋白质，在维持晶状体透明度方面发挥关键作用。这在人类晶状体中尤为重要，因为蛋白质在个体一生中会经历翻译后修饰。在此，我们研究了αA-晶体蛋白一种特定修饰的结构和功能后果，该修饰涉及5个C末端残基的截断（αA(1-168)）。使用新型质谱方法和成熟的生物物理技术，我们表明αA(1-168)形成的寡聚体组装体的平均分子量低于野生型αA-晶体蛋白（αA(WT)）。从αA(WT)和αA(1-168)组装体的质谱中也明显看出，含有偶数个亚基的寡聚体占主导；有趣的是，这种偏好对αA(1-168)更为明显。为了研究组装体之间亚基交换的速率，我们将αB晶体蛋白与αA(WT)或αA(1-168)混合，并在实时质谱实验中监测异源寡聚体的形成。结果表明，当涉及αA(1-168)时，交换速率显著降低。然而，这些降低的交换动力学对伴侣效率没有影响，发现αA(WT)和αA(1-168)的伴侣效率非常相似。因此，总体而言，我们的结果使我们能够得出结论，与植物、酵母和细菌中类似蛋白质所确立的机制不同，亚基交换速率不是决定哺乳动物αA-晶体蛋白有效伴侣行为的关键参数。
