The Skaggs Institute for Chemical Biology and Department of Molecular Biology, Scripps Research Institute, BCC-379, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2011 Feb 15;108(7):2723-8. doi: 10.1073/pnas.1019400108. Epub 2011 Feb 1.
The toxicity of mistranslation of serine for alanine appears to be universal, and is prevented in part by the editing activities of alanyl-tRNA synthetases (AlaRSs), which remove serine from mischarged tRNA(Ala). The problem of serine mistranslation is so acute that free-standing, genome-encoded fragments of the editing domain of AlaRSs are found throughout evolution. These AlaXps are thought to provide functional redundancy of editing. Indeed, archaeal versions rescue the conditional lethality of bacterial cells harboring an editing-inactive AlaRS. In mammals, AlaXps are encoded by a gene that fuses coding sequences of a homolog of the HSP90 cochaperone p23 (p23(H)) to those of AlaXp, to create p23(H)AlaXp. Not known is whether this fusion protein, or various potential splice variants, are expressed as editing-proficient proteins in mammalian cells. Here we show that both p23(H)AlaXp and AlaXp alternative splice variants can be detected as proteins in mammalian cells. The variant that ablated p23(H) and encoded just AlaXp was active in vitro. In contrast, neither the p23(H)AlaXp fusion protein, nor the mixture of free p23(H) with AlaXp, was active. Further experiments in a mammalian cell-based system showed that RNAi-directed suppression of sequences encoding AlaXp led to a serine-sensitive increase in the accumulation of misfolded proteins. The results demonstrate the dependence of mammalian cell homeostasis on AlaXp, and implicate p23(H) as a cis- and trans-acting regulator of its activity.
丝氨酸错译为丙氨酸的毒性似乎是普遍存在的，部分原因是丙氨酰-tRNA 合成酶（AlaRS）的编辑活性，它可以从错误负载的 tRNA（Ala）中去除丝氨酸。丝氨酸错译的问题非常严重，以至于在整个进化过程中都发现了独立的、基因组编码的 AlaRS 编辑结构域片段。这些 AlaXps 被认为提供了编辑功能的冗余。事实上，古菌版本拯救了携带无编辑活性 AlaRS 的细菌细胞的条件致死性。在哺乳动物中，AlaXps 由一个基因编码，该基因融合了 HSP90 伴侣蛋白 p23（p23(H)）的同源物的编码序列与 AlaXp 的编码序列，从而产生 p23(H)AlaXp。目前尚不清楚这种融合蛋白或各种潜在的剪接变体是否作为具有编辑功能的蛋白质在哺乳动物细胞中表达。在这里，我们表明，p23(H)AlaXp 和 AlaXp 替代剪接变体都可以作为蛋白质在哺乳动物细胞中检测到。缺失 p23(H)并仅编码 AlaXp 的变体在体外是活跃的。相比之下，p23(H)AlaXp 融合蛋白和游离的 p23(H)与 AlaXp 的混合物都没有活性。在基于哺乳动物细胞的系统中的进一步实验表明，针对 AlaXp 编码序列的 RNAi 介导的抑制导致错误折叠蛋白积累的丝氨酸敏感性增加。结果表明 AlaXp 依赖于哺乳动物细胞内稳态，并暗示 p23(H) 是其活性的顺式和反式调节因子。
