Butyrate is a critical cancer-preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high-turnover epithelia such as the colon. We used a 2-D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring-initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.

Cytokeratins 8 and 18 have recently been identified as acetylated. The acetylation of other cytoskeletal proteins has been reported as linked to stabilility. As colorectal cells exist bathed in pharmacologically active levels of the HDACi butyrate, we sought to apply state-of-the-art High Content Analytical approaches to identify the effect of butyrate upon the cytoskeleton of colorectal cells. We observed butyrate caused an increase in acetylation at three distinct residues of cytokeratin 8 (K10, K471, and K482) and that the kinetics of altered acetylation were distinct, implying either separate HDACs, or a heirachy of acetylation. This change in acetylation was associated with a breakdown in the cytokeratin cytoskeleton, implying a functional role for cytokeratin acetylation.

The intermediate filament cytoskeleton in epithelial tissues is formed of keratin heterodimers. Keratins are highly post-translationally modified proteins, with tyrosine phopsphorylation, serine phosphorylation, and glycosylation amongst reported modifications. We and others have recently reported multiple acetylation sites on keratin 8 and we have previously shown that these sites are responsive to butyrate. In this study, we report the application of cellomic approaches to demonstrate differential responses of three lysine acetylations (lys 10, lys 471 and lys 482) to different short-chain fatty acids. The data imply no fixed hierarchy of acetylation on keratin 8, and furthermore imply different ranges of histone deacetylase (HDAC) inhibitory specificities for short chain fatty acids (SCFA). Furthermore we have used the functionality of the High Content Analysis (HCA) platform to show that the acetylation sites are differentially modified in cells undergoing mitosis. Taken together the data imply distinct roles for keratin acetylations in function