Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
PLoS One. 2010 Nov 30;5(11):e15045. doi: 10.1371/journal.pone.0015045.
An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.
酰基转移酶/脱酰基酶循环对于维持翻译后修饰的蛋白质的亚细胞分布和生物活性的稳定状态是必需的。尽管已经在鉴定和表征棕榈酰转移酶（PAT）方面取得了进展，但对于参与蛋白质脱酰基化的硫酯酶知之甚少。在这项工作中，我们研究了生长相关蛋白-43（GAP-43）的脱酰基化，该蛋白在半胱氨酸残基 3 和 4 处发生双酰化。使用荧光融合构建体，我们测量了 GAP-43及其单酰化突变体在 CHO-K1 和人 HeLa 细胞中的体内脱酰基化速率。生化和活细胞成像实验表明，两种细胞类型中单酰化突变体的脱酰基化速率相似。通过 RT-PCR，我们观察到酰基蛋白硫酯酶 1（APT-1），唯一被证明在体内介导脱酰基化的真正硫酯酶，在 HeLa 细胞中表达，但在 CHO-K1 细胞中不表达。然而，APT-1 的过表达既不能增加单酰化 GAP-43 的脱酰基化速率，也不能影响双酰化 GAP-43 的亚细胞稳态分布，无论是在 CHO-K1 还是 HeLa 细胞中，这表明 GAP-43 的脱酰基化不是由 APT-1 介导的。因此，我们进行了生物信息学搜索，以鉴定具有酰基蛋白硫酯酶活性的潜在候选物。在几个候选物中，我们发现 APT-2 在 CHO-K1 和 HeLa 细胞中均有表达，其过表达增加了单酰化 GAP-43 的脱酰基化速率，并影响了二酰化 GAP-43 和 H-Ras 的稳态定位。因此，这些结果表明 APT-2 是参与 GAP-43 亚细胞分布的酰基转移酶/脱酰基酶循环中的蛋白硫酯酶。
