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Comprehensive and systematic study of the acetylation function of acetyltransferases in human cells

Final Report Summary - ACETO-SAGATAC-HUMAN (Comprehensive and systematic study of the acetylation function of acetyltransferases in human cells)

Introduction and objectives:

Lysine acetylation is a widespread post-translational modification conserved from prokaryotes to eukaryotes, which regulates protein activity. Aberrant lysine acetylation has been linked to many diseases such as cancer, metabolic syndrome (obesity, diabetes, and cardiovascular disease), and neurological disorders. While lysine acetylation has been recently shown to be part of many essential biological pathways beyond chromatin and transcription, little is known about its physiological role and mechanism by which it is regulated. Lysine acetylation is dynamically regulated by lysine acetyltransferases (KATs) formerly called histone acetyltransferases (HATs), which transfer the acetyl-group of acetyl-CoA of the epsilon-amino group of an internal lysine residues, and lysine deacetylases, which perform the reverse reaction. Around 20 KATs have been described in mammals; however, their physiological targets remain to be identified. This is especially the case for the KATs, Gcn5 (or KAT2a) and PCAF (or KAT2b) which perform the same catalytic activity in vitro, but show some specific role in vivo in mammals, more likely via the acetylation of non-histone proteins. In this study, our goal was to get more insights into functional role of KATs and acetylation in human cells in vivo. We were particularly interested in identifying in a comprehensive manner, targets of the KAT2 family containing Gcn5/PCAF and their containing protein complexes, Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex and Ada-two-A-containing (ATAC) complex and to understand the physiological role of acetylation regulated by Gcn5/PCAF.

Description of the work:

During the time-course of the fellowship, we have first identified in a comprehensive manner, acetylated proteins (called 'acetylome') by Gcn5/PCAF and their containing complexes, SAGA and ATAC. We have further investigated the physiological role of acetylation of some targets of interest. The acetylome characterisation was performed by shotgun proteomic analysis based on the MudPIT technology. MudPIT, which stands for multidimensional protein identification technology, combines multidimensional peptide separation by high-performance liquid chromatography (HPLC) with tandem mass spectrometry analysis. The multidimensional HPLC analysis allowed performing highly resolutive peptide separation for a comprehensive analysis of the complex sample, leading to of the identification of a high number of acetylated proteins. The tandem mass spectrometry analysis allowed mapping acetylated lysine on proteins targeted by our KATs of interest. This was a key aspect, for the understanding of the physiological role of acetylation on these proteins, when the acetylated lysine(s) were contained within functional domains. The Gcn5, SAGA and ATAC acetylome contained hundreds of novel proteins targeted by these KATs. These acetylated proteins were involved in diverse biological pathways, linked to the known physiological role of these KATs but also involved in novel biological pathways. This is very promising for the discovery of novel function for these KATs. Another interesting aspect is to link these protein targets with the physiological role of these KATs, to understand how these KATs act mechanistically on these pathways. The discovery of such mechanisms can act as novel drug targets for disease therapy. Following this idea, we have particularly focused on targets involved in cell cycle regulation and in the regulation of centriole duplication, which, when deregulated, is linked to cancer development and genomic instability.

Main results obtained:

1) The Gcn5/PCAF-, SAGA- and ATAC-dependent acetylomes and the discovery of novel biological targets / functions

The Gcn5/PCAF-, SAGA- and ATAC-dependent in vitro and in vivo acetylomes, were characterised after MudPIT analysis from which hundreds of novel protein targets were identified. These acetylated proteins belonged to diverse biological pathways such as transcription regulation but also cellular differentiation, cellular transport, regulation of apoptosis, response to stress, cell cycle regulation, nervous system development and cellular differentiation, which was linked to the physiological role of these KATs in mammals and for which the investigation of the physiological role of acetylation regulated by these KATs could provide mechanisms by which these KATs act in these pathways. The acetylome data contained also proteins involved in cellular transport and signal transduction which was very interesting, since these KATs were never described before to a role in these essential pathways. Moreover, the Gcn5-containing SAGA and ATAC complexes can acetylate specific non-histone targets. Organised in multiprotein complexes, KATs can have different target specificities based on their associated protein partners. By comparing proteins acetylated by SAGA and ATAC from our acetylome data, subsets of proteins specifically acetylated by one of the two complexes were isolated. These proteins belonged to common biological processes but had unique molecular functions. This is providing evidence that these complexes have a specific role and suggesting that they could act in synergy at different molecular levels to regulate essential biological pathways.

2) Role of Gcn5/ATAC in the regulation of centriole duplication

Gcn5 has been described to be involved in cell cycle regulation but mechanism by which this KAT is acting is still unclear. The Gcn5 acetylome contained proteins involved in cell cycle regulation, such as proteins involved in the regulation of centriole duplication. This finding was very appealing since aberration in the regulation of centriole duplication, such as centriole overduplication, has been shown to cause cancer and genomic instability. In this research project, we have investigated for the first time the role of Gcn5 and acetylation in the regulation of centriole duplication. We have shown that Gcn5-containing ATAC complex regulates centriole duplication in vivo by regulating the activity of main regulators of centriole duplication via lysine acetylation.

Besides the great fundamental aspect that this project represents, such as expanding functional role of KATs and better understanding of acetylation in human cells in vivo, it can be expanded to clinical applications such as disease therapy. As mentioned earlier, deregulation of lysine acetyltransferase or lysine deacetylase activity has been involved in cancerogenesis. HDAC inhibitors are currently in clinical trial for cancer therapy with great success. The more insights we will gain about the role and activity of these enzymes, the more we will improve the efficiency and application of cancer drug therapy which is a priority task and common interest of the international scientific community.