The hydrophobic effect contributes to polyubiquitin chain recognition
The hydrophobic effect contributes to polyubiquitin chain recognition
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DOI：
10.1021/bi972514p
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发表时间：
1998-03-03
期刊：
影响因子：
2.9
通讯作者：
Pickart, CM
中科院分区：
文献类型：
--
作者：
Beal, RE;Toscano-Cantaffa, D;Pickart, CM
泛素-蛋白酶体降解途径中使用的主要靶向信号是均聚的K48连接的聚泛素链：该链被26 S蛋白酶体的19 S调节复合物中的特定因子识别，而底物被20 S催化复合物降解。我们以前提出的证据暗示L 8，I44的侧链。和V70在识别K48连接链中的作用。在tetraubiquitin的晶体结构中，这些侧链形成重复的、表面暴露的疏水斑块。为了检验涉及该补丁的紧密堆积相互作用对于链识别是重要的这一假设，将残基8突变为一系列较小的脂肪族氨基酸（G、A、V）。这些突变的影响首先在兔网织红细胞组分II中进行了研究;即使是最短的截短突变（L 8 G）对模型底物（I-125-乳白蛋白）的降解也只有适度的抑制作用。我们表明，这些稳态降解数据大大低估了这些突变对链识别的蛋白酶体的有害影响，因为识别步骤并不有助于在馏分II系统的速率限制。当使用纯化的26 S蛋白酶体在竞争测定中测量链结合时，观察到更强的抑制，并且结合自由能的变化线性地依赖于侧链的表面积。这种行为与疏水效应做出有利贡献的结合模式一致;即，当链与19 S复合物结合时，一个或多个L 8侧链与溶剂隔离。对于与称为S5 a的19 S亚基的链结合，观察到结合能与侧链面积的类似线性依赖性（如使用与镍珠结合的重组S5 a测定的）。八-泛素（K-0.5 = 6 μ M）结合S5 a 4.2倍更紧密地比四-泛素，这是类似的因子的5.8倍相关的亲和力相同的两条链的26 S蛋白酶体。总之，这些发现表明K48连接的链与19 S复合物的相互作用基本上类似于链与分离的S5 a的相互作用。结果进一步表明，疏水补丁是一个最小的元素，允许由26 S蛋白酶体的聚泛素降解信号的特异性识别的一部分。
The principal targeting signal used in the ubiquitin-proteasome degradation pathway is a homopolymeric, K48-linked polyubiquitin chain: the chain is recognized by a specific factor(s) in the 19S regulatory complex of the 26S proteasome, while the substrate is degraded by the 20S catalytic complex. We have previously presented evidence implicating the side chains of L8, I44. and V70 in the recognition of K48-linked chains. In the crystal structure of tetraubiquitin, these side chains form a repeating, surface-exposed hydrophobic patch. To test the hypothesis that a close-packing interaction involving this patch is important for the chain recognition, residue 8 was mutated to a series of smaller aliphatic amino acids (G, A, V). The effects of these mutations were first investigated in rabbit reticulocyte fraction II; even the severest truncating mutation (L8G) had only a modest inhibitory effect on the degradation of a model substrate (I-125-lactalbumin). We show that these steady-state degradation data substantially underestimate the deleterious effects of these mutations on chain recognition by the proteasome, because the recognition step does not contribute to rate limitation in the fraction II system. Much stronger inhibition was observed when chain binding was measured in a competition assay using purified 26S proteasomes, and the change in binding free energy depended linearly on the surface area of the side chain. This behavior is consistent with a mode of binding in which the hydrophobic effect makes a favorable contribution; i.e., one or more L8 side chains is shielded from solvent when the chain binds to the 19S complex. A similar linear dependence of binding energy an side chain area was observed for chain binding to the 19S subunit known as S5a (as assayed using recombinant S5a bound to nickel beads). Octa-ubiquitin (K-0.5 = 6 mu M) bound to S5a 4.2-fold more tightly than tetra-ubiquitin; this is similar to the factor of 5.8-fold relating the affinities of the same two chains for the 26S proteasome. Altogether, these findings indicate that the interaction of K48-linked chains with the 19S complex is substantially similar to the interaction of chains with isolated S5a. The results further suggest that the hydrophobic patch is part of a minimum element which allows for specific recognition of the polyubiquitin degradation signal by the 26S proteasome.