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.
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Left: In breast cancer cells TFEB (orange) is activated in the presence of doxorubicin, resulting in nuclear localization (top: vehicle treated, bottom: doxorubicin treated). Right: Knockdown of TFEB results in increased sensitivity to doxorubicin induced DNA damage as measured by H2A.X foci (yellow, top: vehicle treated, bottom: doxorubicin treated). Centre: In breast cancer, TFEB activation by DNA damage promotes expression of genes involved in apoptosis inhibition, DNA repair, and cell cycle regulation. Inhibition of TFEB causes increased DNA damage, interferon- and apoptosis signalling, leading to cell death. For more information see the article by Slade and colleagues on pp. 137–160. Image courtesy of Thomas Pulinilkunnil.
Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20
Mohamed Rasheed Gadalla, Laurence Abrami, F. Gisou van der Goot, Michael Veit; Hemagglutinin of Influenza A, but not of Influenza B and C viruses is acylated by ZDHHC2, 8, 15 and 20. Biochem J 17 January 2020; 477 (1): 285–303. doi: https://doi.org/10.1042/BCJ20190752
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