A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state
A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state
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DOI：
10.1093/emboj/16.3.659
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发表时间：
1997-02-03
期刊：
影响因子：
11.4
通讯作者：
Vierling, E
中科院分区：
文献类型：
--
作者：
Lee, GJ;Roseman, AM;Vierling, E
最近有报道称小分子热休克蛋白（sHSPs）在体外具有分子伴侣活性，然而，这种活性的机制还不清楚。我们发现，HSP 18.1，一种来自豌豆的十二聚体sHSP，阻止了加热到45 ℃的苹果酸脱氢酶（MDH）和甘油醛-3-磷酸脱氢酶的聚集。在HSP 18.1阻止底物聚集的条件下，尺寸排阻色谱和电子显微镜显示变性底物包被HSP 18.1十二聚体以形成扩展的复合物，分离的复合物的SDS-PAGE证明每个HSP 18.1十二聚体可以结合12个MDH单体的当量，表明HSP 18.1与其他已知的分子伴侣相比具有大的非天然底物能力，疏水探针1，1 '-双（4-苯胺基）萘-5，5 '-二磺酸（bis-ANS）进入HSP 18.1保守的C-末端区域的能力随着温度的升高而可逆地增加，但被MDH的预先结合所阻断，这表明bis-ANS掺入到底物结合区附近，并且底物-HSP 18.1相互作用是疏水性的。我们还表明，热变性的萤火虫荧光素酶结合到HSP 18.1，热聚集的荧光素酶相比，可以重新激活的存在下，兔网织红细胞或小麦胚芽提取物中的ATP依赖性的过程中，这些数据支持的模型中，sHSP防止蛋白质聚集，并促进基板重折叠与其他分子伴侣。
The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes, SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones, Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process, These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.