Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK
Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK
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DOI：
10.1016/j.biocel.2006.11.006
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发表时间：
2007-01-01
影响因子：
4
通讯作者：
Blatch, G. L.
中科院分区：
文献类型：
--
作者：
Nicoll, W. S.;Botha, M.;Blatch, G. L.
原核和真核细胞都含有多种热休克蛋白40（Hsp 40）和热休克蛋白70（Hsp 70）蛋白，它们作为分子伴侣合作以确保蛋白质生物合成的所有阶段的保真度。热休克蛋白40的特征结构域，J-结构域，是热休克蛋白40与伴侣热休克蛋白70结合所必需的，也可能在结合的特异性中发挥作用。通过创建嵌合Hsp 40蛋白的原核Hsp 40（DnaJ）的J-结构域的替代，我们已经测试了J-结构域的功能等效性，从一些不同的Hsp 40的哺乳动物和寄生虫来源（疟疾Pfj 1和Pfj 4，锥虫Tcj 3，人ERj 3，ERj 5，和Hsj 1，和鼠ERj 1）。使用体内功能测定来测试嵌合蛋白的功能性，所述嵌合蛋白基于其逆转dnaJ cbpA突变体大肠杆菌菌株（OD 259）的热敏性的能力。含有来源于可溶性（胞浆或内质网（ER）-内腔）Hsp 40的J结构域的Hsp 40嵌合体能够逆转E. coliOD 259。在所有情况下，这些嵌合蛋白的修饰衍生物在J结构域的HPD基序中含有His到Gln的取代，不能逆转E. coliOD 259。这表明这些J结构域通过与E. coli Hsp70、DnaK.有趣的是，含有ERj 1的J结构域的Hsp 40嵌合体，一个完整的膜结合ER Hsp 40，不能逆转E. coliOD 259中，表明该J结构域不能与DnaK发生功能性相互作用。在J-结构域的所有四个螺旋（I-IV）和环区域中进行保守氨基酸残基和基序的取代，并且使用体内测定测试修饰的嵌合Hsp 40的功能性。发现J结构域的螺旋II中高度保守的碱性残基的取代破坏了所有测试的J结构域的体内功能。我们建议，螺旋II和HPD基序的J-结构域的热休克蛋白40与热休克蛋白70的相互作用所需的结合表面的基本要素，并且该表面已被保存在哺乳动物，寄生虫和细菌系统。(c)2007年由Elsevier Ltd.出版
Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosonnal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gin substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I-IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems. (c) 2007 Published by Elsevier Ltd.