Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20
Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20
复制标题
DOI：
10.1042/bcj20190752
复制
发表时间：
2020-01-01
影响因子：
4.1
通讯作者：
Veit, Michael
中科院分区：
文献类型：
--
作者：
Gadalla, Mohamed Rasheed;Abrami, Laurence;Veit, Michael
血凝素（HA）是甲型流感病毒的一种糖蛋白，其质子通道M2被脂肪酸位点特异性修饰。尽管HA的短胞质尾区中的两个半胱氨酸仅含有棕榈酸酯，但硬脂酸酯仅与位于跨膜区（TMR）的胞质边界处的一个半胱氨酸连接。M2在TMR附近的两亲性螺旋中的半胱氨酸处被棕榈酰化。催化HA和M2酰化的酶尚未鉴定，但含锌指DHHC结构域（ZDHHC）棕榈酰转移酶是候选者。我们使用siRNA文库敲低HA表达HeLa细胞中23种人ZDHHC中每一种的表达。针对ZDHHC 2和8的siRNA对HA的酰化具有最强的作用，如通过Acyl-RAC所证明并通过3 H-棕榈酸酯标记所证实的。在HAP 1细胞中ZDHHC 2和8的CRISPR/Cas9敲除，以及遗传学相关的ZDHHC 15和20的CRISPR/Cas9敲除，强烈降低了组1和组2 HA以及M2的酰化，但个体ZDHHC表现出略微不同的底物偏好。这些ZDHHC与HA共定位在人肺细胞系中的胞吐途径的膜上。ZDHHC 2、8、15和20对于在一个跨膜半胱氨酸处仅含有硬脂酸盐的丙型流感病毒的HA-酯酶融合蛋白的酰化不是必需的。敲除这些ZDHHC也不损害在其胞质尾区中含有两个棕榈酰化半胱氨酸的B型流感病毒HA的酰化。结果进行了讨论相对于确定的ZDHHC的酰基偏好和可能的基板识别功能。
Hemagglutinin (HA), a glycoprotein of Influenza A viruses and its proton channel M2 are site-specifically modified with fatty acids. Whereas two cysteines in the short cytoplasmic tail of HA contain only palmitate, stearate is exclusively attached to one cysteine located at the cytoplasmic border of the transmembrane region (TMR). M2 is palmitoylated at a cysteine positioned in an amphiphilic helix near the TMR. The enzymes catalyzing acylation of HA and M2 have not been identified, but zinc finger DHHC domain-containing (ZDHHC) palmitoyltransferases are candidates. We used a siRNA library to knockdown expression of each of the 23 human ZDHHCs in HA-expressing HeLa cells. siRNAs against ZDHHC2 and 8 had the strongest effect on acylation of HA as demonstrated by Acyl-RAC and confirmed by 3H-palmitate labeling. CRISPR/Cas9 knockout of ZDHHC2 and 8 in HAP1 cells, but also of the phylogenetically related ZDHHCs 15 and 20 strongly reduced acylation of group 1 and group 2 HAs and of M2, but individual ZDHHCs exhibit slightly different substrate preferences. These ZDHHCs co-localize with HA at membranes of the exocytic pathway in a human lung cell line. ZDHHC2, 8, 15 and 20 are not required for acylation of the HA-esterase-fusion protein of Influenza C virus that contains only stearate at one transmembrane cysteine. Knockout of these ZDHHCs also did not compromise acylation of HA of Influenza B virus that contains two palmitoylated cysteines in its cytoplasmic tail. Results are discussed with respect to the acyl preferences and possible substrate recognition features of the identified ZDHHCs.