Henics T, Nagy E, Oh H J, Csermely P, von Gabain A, Subjeck J R
Department of Medical Microbiology and Immunology, University Medical School of Pécs, H-7643 Pécs, Hungary.
J Biol Chem. 1999 Jun 11;274(24):17318-24. doi: 10.1074/jbc.274.24.17318.
In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.
在本研究中，对哺乳动物70 kDa热休克蛋白（Hsp）家族成员的体外RNA结合情况进行了检测。我们发现，Hsp/Hsc70和Hsp110蛋白在体外优先结合富含AU的RNA。ATP对RNA结合的抑制作用表明N端ATP结合结构域参与其中。通过使用Hsp110蛋白（一种分化的Hsp70家族成员）的缺失突变体，将RNA结合定位到该分子的N端ATP结合结构域。C端肽结合结构域不结合RNA，但其与肽底物的结合会消除该蛋白N端的RNA结合。有趣的是，去除肽结合结构域下游Hsp110特有的C端α螺旋结构或α环结构域（但不是两者都去除），与野生型蛋白相比，会导致RNA结合显著增加。最后，从人淋巴细胞胞质蛋白与富含AU的RNA在体外形成的RNA-蛋白复合物中免疫沉淀出一种70 kDa的活性物质。这些发现支持了这样一种观点，即某些热休克蛋白可能在体内作为RNA结合实体，指导RNA底物进行适当折叠，以用于后续的调控过程，如mRNA降解和/或翻译。
