Fig. 3. NAD-dependent ribosylation and deacetylation of proteins by TbSIR2RP1. (A) In vitro ribosylation reactions were performed with 1 µg of GST–TbSIR2RP1 or GST–TbSIR2RP1^H142Y, plus 5 µg of BSA and 5 µg of histones using [³²P]NAD as donor. The top panel illustrates a Coomassie-stained gel of reaction products resolved by 10% SDS–PAGE, whereas the bottom panel shows the autoradiograph of the gel. (B) Time curves of histone and BSA ribosylation by GST–TbSIR2RP1 at 37°C. Each point represents the ribosylation products of 5 µg of substrate by 0.5 µg of GST–TbSIR2RP1 using 5 µCi of [³²P]NAD at the indicated time. Reaction products were precipitated with 20% TCA (w/v), collected and washed on a GF/C glass fibre filter (Whatman), and then counted after addition of liquid scintillation fluid. (C) Ribosylation reactions were performed with 5 µg of histones, 0.5 µg of GST–TbSIR2RP1 and 5 µCi of [³²P]NAD (standard reaction, column St) or a double concentration of histone (10 µg, column 2× histones), GST–TbSIR2RP1 (1 µg, column 2× GST–TbSIR2RP1) and [³²P]NAD (10 µCi, column 2× NAD). (D) Comparison of TbSIR2RP1 ribosylation activity with that of other members of the SIR2-like family. Ribosylation reactions with 1 µg of GST–TbSIR2RP1, GST–TbSIR2RP1^H142Y, GST–SIR2 or GST–HST2 were performed with and without 5 µg of histones using [³²P]NAD as donor. The top panel illustrates a Coomassie-stained gel of the reaction products, and the bottom panel shows the autoradiograph of the gel. (E) Triton–acid–urea (TAU) gel of T.brucei histones treated with GST–TbSIR2RP1 or GST–TbHST1^H142Y and [³²P]NAD, respectively stained with Coomassie blue and autoradiographed. The histone types are indicated on the left. (F) Analysis of the NAD-dependent histone deacetylase activity. Histones were acetylated with [³H]acetyl-CoA by HAT1 and then treated with GST–TbSIR2RP1, GST– TbSIR2RP1^H142Y, GST–HST2 and GST–SIR2. The amount of acetate released was measured in the presence or absence of NAD.