The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains - PubMed (original) (raw)

Ataxin-3 preferentially cleaves longer chains and Lys63linkages. A, ataxin-3 (Gln22) cleavage of ubiquitinated CHIP generated in vitro with the E2/E3 pair, UbcH5c/CHIP. For the indicated times at 37 °C, GST-ataxin-3 was incubated with a quenched CHIP auto-ubiquitination reaction, and then analyzed by immunoblotting with anti-CHIP (top panel) and anti-ubiquitin (bottom panel) antibodies. B, time course of ataxin-3 (Gln22) cleavage of Lys48-linked (top panel) or Lys63-linked (bottom panel) Ub6. GST or GST-ataxin-3 (1 μ

m

) was incubated with Ub6 chains (250 n

m

) for the indicated times and then analyzed by anti-ubiquitin immunoblotting. _C_and D, concentration curve of ataxin-3 (Gln22) cleavage of Lys48-linked Ub6 (C) or Lys63-linked Ub6 (D). Increasing concentrations of GST-ataxin-3 were incubated with Ub6 chains (250 n

m

) at 37 °C for 2 h and then analyzed by anti-ubiquitin immunoblotting. E, quantification of ataxin-3 cleavage of Lys48- and Lys63-linked Ub6. Shown is the percentage of the total ubiquitin signal in each lane that is lower molecular weight reaction product (<Ub6) (_n_ = 2; means ± S.D.). _F_, requirement of catalytic cysteine for both Lys63-Ub6 cleavage and activity against HMW ubiquitin chain complexes (>6). Normal (WT) or catalytically inactive (C14A) ataxin-3 (Gln22) (1μ

m

) was incubated with Lys63-linked Ub6 for the indicated times. G, chain length dependence of ataxin-3 activity. Performed as in B, except GST-ataxin-3 (100 n

m

) was incubated with the indicated lengths of ubiquitin chains. H, quantification of chain length dependant ataxin-3 activity from G. Shown is the percentage of the total ubiquitin signal in each lane that is lower molecular weight reaction product (n = 2; means ± S.D.). Cleavage at 24 h of Ub6 was greater than of Ub3, 4, and 5 (*, p < 0.01), and cleavage of Ub5 was greater than of Ub3 (**, p < 0.05).