Unraveling the hidden catalytic activity of vertebrate class IIa histone deacetylases

Abstract

Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-Nepsilon2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this "gain of function phenotype" could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro. Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.

Fig. 1.

Sequence motif conservation and active-site geometry. (A) Class-specific sequence motif completely conserved in class I. Conservation is generally high also within class II, with the notable exception of class IIa. (B) Active-site region of a bacterial class II homolog. To substitute Y as transition-state stabilizer, the H has to move closer to the active site. The LEGGY motif is indicated, as is the interaction between the Y and a representative hydroxamic acid moiety (INH).

Fig. 2.

The HDAC3 Y(298)H substitution generates a loss-of-function mutant. (A) Enzymatic activities of WT and Y(298)H-mutated HDAC3-FLAG (HDAC3-F) proteins on pepH4-AcK16. (B) Western blot of HDAC3-FLAG immunocomplexes assayed in A by using anti-FLAG (HDAC3-F) or anti-Gal4 (GAL4-DAD) antibodies. Ten, 20, and 40 nM WT and mutant HDAC3 were analyzed.

Fig. 3.

HDAC4 compounds binding assays. (A) IC₅₀ values of LAQ824 and apicidin on HDAC4-FLAG and HDAC3-FLAG activities and structures of the corresponding linkerized/biotinylated derivatives. A negative control lacking the HDACi moiety is also shown. The IC₅₀ values of the modified compounds for HDAC4-FLAG (HDAC4-F) are reported. (B) HDAC4-FLAG pull-down on streptavidin-coated magnetic beads preabsorbed with each of the three biotinylated compounds shown in A. A representative Coomassie blue-stained SDS/PAGE gel loaded with the unbound (U) and bound (B) fractions is shown. (C) UV cross-linking of HDAC4-FLAG to each of the three compounds described in A. A representative ExtrAvidin Western blot is shown. A control lane developed with anti-FLAG antibodies after a mock UV treatment is also shown.

Fig. 4.

Functional characterization of the isolated HDAC4-CD purified from E. coli. (A) Pull-down of HDAC4-CD on biotinylated LAQ824-coated beads after preincubation with the indicated HDACis [LAQ824 (Laq), apicidin (Api), or MS275 (MS)]. In lane C, control beads coated with the compound lacking the HDACi moiety were used. A representative Coomassie blue-stained SDS/PAGE gel is shown. (B) Enzymatic activity of HDAC4-CD on acetylated histones. One micromolar protein was needed to measure significant activity levels. Deacetylation was assayed in the absence or presence of 10 μM apicidin (inactive) or LAQ824 (active).

Fig. 5.

The HDAC4 H976Y substitution generates a gain-of-function mutant. (A) Comparison of the enzymatic activities exhibited by WT, H976F-mutated, and H976Y-mutated HDAC4-FLAG proteins immunopurified from mammalian cells. (B) Comparison of the enzymatic activities of WT and H976Y-mutated HDAC4-CD proteins purified from E. coli. Data obtained on acetylated histones are shown.

Fig. 6.

Trifluoroacetyl-lysine is a class IIa-specific substrate in vitro. (A) The fluorogenic derivative of the trifluoroacetyl-lysine compound is shown. (B) The catalytic activities of flag-tagged HDACs representative of class I, IIa, and IIb were measured on the trifluoroacetyl-lysine substrate shown in A.