Fig. 6: K50 and K51 are critical ubiquitination sites of cpzA3H. | Nature Communications

Fig. 6: K50 and K51 are critical ubiquitination sites of cpzA3H.

From: HIV-1 vif mediates ubiquitination of the proximal protomer in the APOBEC3H dimer to induce degradation

Fig. 6

a The schematic overview of Vif-induced ubiquitination of A3H. ARIH2 is responsible for transferring the first Ub molecule onto A3H. The UbcH3 then elongates the Ub chain. b In vitro ubiquitination of cpzA3H by NL4-3 Vif WT (lanes 2–5) and the R41A variant (lanes 6–9). Ubiquitinated cpzA3H proteins were detected by Western blotting using an anti-A3H rabbit polyclonal Abs. c Ubiquitinated lysine (red) of cpzA3H, detected by mass spectrometry, are highlighted in the amino acid sequence of cpzA3H. d Comparison of K50, K51, and K52 ubiquitination. The frequency (%) with which peptides containing ubiquitinated K50, K51, K52, and their combinations are detected in the trypsin-digested K-ɛ-GG-enriched sample is shown in the pie chart. e cpzA3H-7KR contains K27R, K50R, K51R, K52R, K153R, K161R, and K168R mutations and protects cpzA3H from both NL4-3 Vif (left panel) and LAI Vif (right panel) induced degradation (lane 2). Each mutation was reversed within cpzA3H-7KR to generate cpzA3H-7KR-R27K, -R50K, -R51K, -R52K, -R153K, -R161K and -R168K (lanes 3 to 9). The 7KR + R50K (lane 4) and 7KR + R51K (lane 5) variants showed protein degradation efficiency equivalent to wild-type cpzA3H (lane 1). All proteins were expressed without any tag and detected by anti-A3H rabbit polyclonal Abs.

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