PCAF, SIRT1 and the regulation of substrate acetylation
Keywords: bromodomain
tandem mass spectrometry
High throughput screening
mass spectrometry
HDAC
MALDI-TOF-MS
cyclic peptide
HAT
PCAF
deacetylation
RapifleX
mRNA display
epigenetic crosstalk
acetylation
K14Me1
histone peptide
SIRT1
Epigenetics
MALDI
chemical probes
HIF1 alpha peptide

Levels of lysine acetylation (KAc) in cells are regulated by epigenetic enzymes with opposing roles; histone acetyl transferases (HATs) and histone deacetylases (HDACs). The HAT p300 CBP associating factor (PCAF) and the sirtuin HDAC SIRT1 have protein substrates in common, including histone 3 (H3) at lysine residues 9 and 14 (H3K9 and H3K14) and non histone protein hypoxia inducible factor 1α (HIF1α). Studying PCAF and SIRT1 in parallel helps understand how levels of acetylation are maintained by the two classes of proteins.
In this work, a matrix assisted laser desorption ionisation (MALDI) time of flight (TOF) mass spectrometry (MS) assay was developed and used to characterise the activity of PCAF and SIRT1 proteins for their reported histone substrates (H3(1-21) and H3(1-21)K9Ac respectively). MALDI TOF MS was used to effectively identify peptide and cofactor selectivity for the enzymes. Commercially available nonspecific HAT inhibitors were tested with MALDI-TOF MS, identifying anacardic acid as the most potent inhibitor available against PCAF HAT and HAT BRD catalytic acetylation activity.
Epigenetic enzymes operate in a context dependent manner in cells. Histone and non-histone proteins in cells often have multiple different post translational modifications (PTMs) at the same time (e.g. acetylation and methylation). These combinatorial PTMs are an important regulatory system affecting the activity of surrounding epigenetic enzymes, which is not often taken into account in peptide based assays. For this reason, a rapifleX MALDI-TOF MS high throughput screen (HTS) was developed to screen commercial libraries of histone peptides with combinatorial PTMs against PCAF and SIRT1 enzymes. This allows for mapping of PTM interactions with the enzymes, leading to changes in enzyme activity, referred to as PTM crosstalk. Instances of PTM crosstalk were identified, with a notable example being the activation of K18 acetylation on H3(1-21) by PCAF in the presence of a mono-methylation (Me1) PTM at K14 of the H3(1-21) substrate peptide. MALDI TOF/TOF MS was used to confirm site selectivity. Efforts to expand the method to screening of non-histone peptides using HIF1α as a protein template were not successful, however, other proteins may be more suited to screening through peptide segments.
There is a lack of selective and potent inhibitors targeting PCAF HAT. As PTM modified histone peptides were shown to have inhibitory and activating effects on the PCAF enzyme, cyclic peptides were thought to be a good avenue for developing chemical probes for PCAF HAT. mRNA display was used to screen a library of cyclic peptides against PCAF HAT and HAT BRD, however, no binding peptides were identified. It was determined that the screen failed due to nonspecific binding of PCAF to the hybrid mRNA/DNA tag used in the selection method.
In conclusion, an effective screening method has been developed to study the effects of PTM of histone peptides on epigenetic enzymes. The assay developed has potential for wider applications in screening epigenetic enzymes against a range of peptide libraries and epigenetic enzymes. In addition, the screening results provide insight into the biological activity of PCAF, and may provide a starting point for the development of PCAF selective chemical probes.