Figure 1.
HSP-associated mutations affect the expression and catalytic activity of DDHD2. (A) Domain diagram of human DDHD2 protein with locations of HSP-associated and catalytic (S351A) mutations indicated above the domain map bar. (B) Representative anti-FLAG immunoblots of lysates from HEK293T cells transfected with the indicated N-terminal FLAG-DDHD2 constructs. Note that frameshift and nonsense mutants result in bands of lower molecular weight that generally match the predicted sizes of truncated products from the DDHD2 sequence. (C) Quantification of anti-FLAG immunoblotting signals in panel B. Data represent average values ± the standard error of the mean (SEM) (N = 4 biological replicates). (D) ABPP of lysates from HEK293T cells transfected with the indicated N-terminal FLAG-DDHD2 constructs using the DDHD2-directed probe HT-01 (1 μM, 30 min). HT-01-treated samples were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and in-gel fluorescence scanning (fluorescent gel shown in grayscale). (E) Quantification of HT-01 labeling from panel D. Data represent average values ± SEM (N = 4 biological replicates). (F) TAG hydrolysis activity of soluble lysates from HEK293T cells transfected with the indicated N-terminal FLAG-DDHD2 constructs. C18:1/C18:1/C18:1 TAG (167 μM) was used as a substrate, and the production of C18:1 FFA was measured by liquid chromatography and mass spectrometry. Data represent average values ± SEM (N = 4 biological replicates). For panels A—F, DDHD2 mutants are represented using the single-letter amino acid abbreviation of the affected position. Mutations that result in a stop codon are denoted by*. The number following fs* represents the number of amino acids after a frameshift mutation until the first stop codon.