Posttranslational regulation of the GCN5 and PCAF acetyltransferases.

General control nonderepressible 5 protein (Gcn5) and its homologs, including p300/CBP-associated factor (PCAF), are lysine acetyltransferases that modify both histone and non-histone proteins using acetyl coenzyme A as a donor substrate. While decades of studies have uncovered a vast network of cellular processes impacted by these acetyltransferases, including gene transcription and metabolism, far less is known about how these enzymes are themselves regulated. In this review, we summarize the type and functions of posttranslational modifications proposed to control Gcn5 in both yeast and human cells. We further outline common themes, open questions, and strategies to guide future work.

Dynamic regulation of Pif1 acetylation is crucial to the maintenance of genome stability.

PIF1 family helicases are evolutionarily conserved among prokaryotes and eukaryotes. These enzymes function to support genome integrity by participating in multiple DNA transactions that can be broadly grouped into DNA replication, DNA repair, and telomere maintenance roles. However, the levels of PIF1 activity in cells must be carefully controlled, as Pif1 over-expression in Saccharomyces cerevisiae is toxic, and knockdown or over-expression of human PIF1 (hPIF1) supports cancer cell growth. This suggests that PIF1 family helicases must be subject to tight regulation in vivo to direct their activities to where and when they are needed, as well as to maintain those activities at proper homeostatic levels. Previous work shows that C-terminal phosphorylation of S. cerevisiae Pif1 regulates its telomere maintenance activity, and we recently identified that Pif1 is also regulated by lysine acetylation. The over-expression toxicity of Pif1 was exacerbated in cells lacking the Rpd3 lysine deacetylase, but mutation of the NuA4 lysine acetyltransferase subunit Esa1 ameliorated this toxicity. Using recombinant proteins, we found that acetylation stimulated the DNA binding affinity, ATPase activity, and DNA unwinding activities of Pif1. All three domains of the helicase were targets of acetylation in vitro, and multiple lines of evidence suggest that acetylation drives a conformational change in the N-terminal domain of Pif1 that impacts this stimulation. It is currently unclear what triggers lysine acetylation of Pif1 and how this modification impacts the many in vivo functions of the helicase, but future work promises to shed light on how this protein is tightly regulated within the cell.

Lysine acetylation regulates the activity of nuclear Pif1.

In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.

Quantification of In Vitro Protein Lysine Acetylation by Reversed Phase HPLC.

Protein lysine acetylation is a reversible posttranslational modification that is catalyzed by a group of enzymes that are collectively referred to as lysine (K) acetyltransferases (KATs). These enzymes catalyze the transfer of the acetyl group from acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine amino acid. Protein lysine acetylation plays a critical role in the regulation of important cellular processes and it is therefore paramount that we understand the catalytic mechanisms of these enzymes. While there is a variety of methods that have been developed to analyze the enzymatic properties of KATs, majority of the proposed methods have considerable limitations. We describe here a reversed phase HPLC based method that monitors substrate consumption and product formation simultaneously. This method is highly reproducible and optimally suited for the determination of accurate kinetic parameters of KATs.

Analysis of DNA Processing Enzyme FEN1 and Its Regulation by Protein Lysine Acetylation.

Cellular proteins are modified by lysine acetylation wherein an acetyltransferase transfers an acetyl group from acetyl co enzyme A onto the e-amino group of lysine residues. This modification is extremely dynamic and can be reversed by a deacetylase that removes the acetyl group. Addition of acetyl group to the lysine residue neutralizes its positive charge, thereby functioning as a molecular switch in regulating the enzymatic functions of the protein, its stability, and it cellular localization. Since this modification is extremely dynamic within the cell, biochemical studies characterizing changes in protein function are imperative to understand how this modification alters protein function in a specific cellular pathway. This unit describes in detail expression and purification of a recombinant nuclease and acetyltransferase, in vitro acetylation of the recombinant protein and biochemical assays to study the changes in enzymatic activity of the in vitro acetylated nuclease.