Activity-based probes for proteomic profiling of histone deacetylase complexes - PubMed (original) (raw)
Activity-based probes for proteomic profiling of histone deacetylase complexes
Cleo M Salisbury et al. Proc Natl Acad Sci U S A. 2007.
Abstract
Histone deacetylases (HDACs) are key regulators of gene expression that require assembly into larger protein complexes for activity. Efforts to understand how associated proteins modulate the function of HDACs would benefit from new technologies that evaluate HDAC activity in native biological systems. Here, we describe an active site-directed chemical probe for profiling HDACs in native proteomes and live cells. This probe, designated SAHA-BPyne, contains structural elements of the general HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), as well as benzophenone and alkyne moieties to effect covalent modification and enrichment of HDACs, respectively. Both class I and II HDACs were identified as specific targets of SAHA-BPyne in proteomes. Interestingly, multiple HDAC-associated proteins were also enriched by SAHA-BPyne, even after denaturation of probe-labeled proteomes. These data indicate that certain HDAC-associated proteins are directly modified by SAHA-BPyne, placing them in close proximity to HDAC active sites where they would be primed to regulate substrate recognition and activity. We further show that SAHA-BPyne can be used to measure differences in HDAC content and complex assembly in human disease models. This chemical proteomics probe should thus prove valuable for profiling both the activity state of HDACs and the binding proteins that regulate their function.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Design and synthesis of the HDAC activity-based probe SAHA-BPyne. (A) Structures of the general HDAC inhibitor SAHA and SAHA-BPyne. (B) Synthetic scheme for preparation of SAHA-BPyne.
Fig. 2.
ABPP of melanoma cell proteomes with the SAHA-BPyne probe. (A) Soluble proteomes from the melanoma lines MUM2B and MUM2C were incubated with 100 nM SAHA-BPyne probe in the presence or absence of excess SAHA (10 μM) as a competitor. Probe targets were detected by UV-irradiation, followed by click chemistry with a rhodamine-azide tag, SDS/PAGE analysis, and in-gel fluorescence scanning (fluorescent gel shown in grayscale). Multiple SAHA-sensitive targets were detected (arrows). These proteins were identified as HDACs 1 and 2 (60-kDa doublet) and MBD3 (38-kDa band). (B) Confirmation that SAHA-BPyne targets both HDACs (HDAC2) and HDAC-associated (CoREST, MTA2) proteins. Shown are Western blots of proteins enriched from melanoma proteomes by treatment with SAHA-BPyne (or SAHA-BPyne plus excess SAHA), click conjugation to biotin-azide, and enrichment on avidin beads.
Fig. 3.
Alterations in HDACs and HDAC complexes between aggressive and nonaggressive melanoma lines. (A) ABPP-MudPIT with SAHA-BPyne identified lower and higher levels of HDAC6 and CoREST, respectively, in the aggressive melanoma line MUM2B compared with the less aggressive MUM2C line. P < 0.01, for levels of proteins between MUM2B and MUM2C cells. (B) Western blotting of soluble proteomes from melanoma cells confirmed that HDAC6 is more highly expressed in MUM2C cells (Upper). In contrast, equivalent levels of CoREST were observed in MUM2B and MUM2C soluble proteomes (Lower). (C) Western blotting analysis of avidin-enriched MUM2B and MUM2C proteomes treated with SAHA-BPyne revealed stronger CoREST signals in the former samples, indicating higher levels of CoREST in HDAC complexes from MUM2B cells.
Fig. 4.
Profiling HDAC complexes in living cancer cells with SAHA-BPyne. Cultured preparations of MDA-MB-231 cells were treated with 500 nM SAHA-BPyne probe in the presence or absence of excess SAHA (10 μM) and irradiated with UV light for various times. Cells were washed, scraped, and homogenized, followed by click chemistry with a rhodamine-azide tag, SDS/PAGE analysis, and in-gel fluorescence scanning (fluorescent gel shown in grayscale). Multiple SAHA-sensitive targets were detected, including those previously identified in in vitro preparations as HDAC1, HDAC2, and MBD3 (single arrowheads) and those that are more strongly labeled in living cells (double arrowheads). Labeling of the corresponding in vitro proteomic preparations of MDA-MB-231 cells is shown for reference.
Fig. 5.
Model for SAHA-BPyne labeling of HDACs and HDAC-associated proteins. Incubation of proteomes with SAHA-BPyne results in selective binding to HDACs, which exist as parts of large multiprotein complexes. The BP group of SAHA-BPyne rests on the outer rim of HDAC active sites, resulting in UV light-induced photocross-linking to both HDACs and proximally associated proteins (green). More distally associated proteins (yellow) do not react with the probe. Proteome denaturation and click chemistry with a biotin-azide tag enables identification of SAHA-BPyne-labeled proteins by ABPP-MudPIT methods (sequential avidin enrichment, on-bead trypsin digestion, and shotgun LC-MS/MS analysis).
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