Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants
Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants
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DOI：
10.1074/jbc.m113.487231
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发表时间：
2013-07-26
影响因子：
4.8
通讯作者：
Green, William N.
中科院分区：
文献类型：
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作者：
Antinone, Sarah E.;Ghadge, Ghanashyam D.;Green, William N.
铜锌超氧化物歧化酶(MtSOD1)基因突变可导致家族性肌萎缩侧索硬化症(FALS)，这是一种由运动神经元变性引起的神经退行性疾病。在这里，我们证明了野生型SOD1(WtSOD1)经历了棕榈酰化，这是一种可逆的翻译后修饰，可以调节蛋白质的结构、功能和定位。通过酰基-生物素交换、点击化学、半胱氨酸诱变和质谱学等多种技术证实了SOD1的棕榈酰化。质谱学和半胱氨酸诱变结果表明，半胱氨酸残基6是棕榈酰化的主要位点。FALS连接的mtSOD1s(A4V和G93A)的棕榈酰化水平显著高于在HEK细胞和运动神经元细胞系中表达的wtSOD1s。转基因小鼠脊髓中FALS连接的mtSOD1s(G93A和G85R)的棕榈酰化程度也高于wtSOD1s。我们发现SOD1的棕榈酰化水平与膜相关的SOD1的水平相关，提示棕榈酰化在将SOD1靶向到膜上的过程中发挥作用。我们进一步观察到棕榈酰化主要发生在二硫键还原而不是二硫键的SOD1上，这表明未成熟的SOD1是主要的棕榈酰化物种。随着SOD1表达的铜伴侣蛋白的增加，SOD1二硫键的增加和成熟度的增加导致wtSOD1棕榈酰化的减少。铜伴侣蛋白过表达对A4V棕榈酰化的影响小于wtSOD1，对G93A mtSOD1棕榈酰化的影响不大。这些发现表明，在SOD1成熟过程中，SOD1棕榈酰化发生在二硫键之前，当二硫键延迟或减少时，棕榈酰化增加，正如在几个mtSOD1s观察到的那样。
Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.