Acetyl-lysines

Determination of Optimal pH for SC-PEG Reactivity. To triethanolamine-borate buffer (0.3 M, 1 mL) at the appropriate pH, a stock solution of N,a-acetyl-lysine (NAL) in water (50 mM, 0.1 mL) was added followed by a stock solution of SC-activated mPEG-5000 in CH3CN (50 mM active acyl, 0.1 mL). The resultant solution was vortexed and incubated at 28 °C for 1 h. A mixture of the same components but leaving out SC-PEG was used as a control. The TNBS assay version of Snyder and Sobocinski (18) was used to determine the unreacted NAL. 

Hardy, P.M., Nicholls, A.C., and Rydon, H.N. (1976) The nature of the cross-linking of proteins by glutaraldehyde. Interaction of glutaraldehyde with the amino-groups of 6-ami-nohexanoic acid and of b-N-acetyl-lysine. J. Chem. Soc., Perk Trans. 1, 958. 

Zeng L, Zhou MM (2002) Bromodomain an acetyl-lysine binding domain. EEBS letters 513 124-128 Zhang Y, Reinberg D (2001) Transcription regulation by histone methylation interplay between different covalent modifications of the core histone tails. Genes Dev 15 2343-2360... 

Methylation plays an important role in transcriptional regulation and a lesser role in signal transduction. " Histones are heavily methylated proteins. Single, double, or triple methylated lysines play an important role on histones. Lysine methylation is a more subtle transcriptional control than acetylation. Lysine methylation has come to light in another protein known as p53. p53 is a protein expressed in low levels in the cell and stabilized by posttranslational modifications including phosphorylation, acetylation, and now N-methylation. There are several C-terminal lysines on p53 that increase its stability. The addition of the methylation modifications adds complexity to p5 3 and fine-tunes its activity and ultimately suppresses tumor formation. ... 

The phenylselenocysteine has also been used successfully to chemically append analogues of methyl- or acetyl-lysine, important histone modifications that can contribute to chromatin structure and accessibility of transcriptional machinery in eukaryotes. By introducing phenylselenocysteine into the Xenopus histone H3, both acetyl-lysine and mono-, di-, and trimethyl-lysine analogues were appended to the purified unnatural amino acid-containing FI 3 protein (Figure 10). " Additionally, the H3 protein with a modification mimicking acetylation of lysine 9 can be deactylated by a histone deacetylation complex and is also a substrate for phosphorylation by Aurora B kinase. Such purified and chemically labeled histones are likely functional in nucleosomes, and preparation of specifically modified histones for comprehensive analysis of chromatin structure and accessibility is particularly suited to this chemical labeling technique. 

Fig. 1. Histone modifications on the nucleosome core particle. The nucleosome core particle showing 6 of the 8 core histone N-terminal tail domains and 2 C-terminal tails. Sites of post-translational modification are indicated by coloured symbols that are defined in the key (lower left) acK = acetyl lysine, meR = methyl arginine, mcK = methyl lysine, PS = phosphoryl serine, and uK = ubiquitinated lysine. Residue numbers are shown for each modification. Note that H3 lysine 9 can be either acetylated or methylated. The C-terminal tail domains of one H2A molecule and one H2B molecule are shown (dashed lines) with sites of ubiquitination at H2A lysine 119 (most common in mammals) and H2B lysine 123 (most common in yeast). Modifications are shown on only one of the two copies of histones H3 and H4 and only one tail is shown for H2A and H2B. Sites marked by green arrows are susceptible to cutting by trypsin in intact nucleosomes. Note that the cartoon is a compendium of data from various organisms, some of which may lack particular modifications e.g., there is no H3meK9 in S. cerevisiae. (From Ref [7].)...

The co-repressor KAP-1 functionally links the DNA-binding Kruppel-associated box zinc finger proteins to the NURD complex by recruiting the Mi-2a subunit. [251]. This interaction requires a tandem PHD/bromodomain motif in which the individual domains appear to act together as a functional unit. The nature of any possible acetylated lysine targets of the bromodomain remains unclear but it is not excluded that this domain could bind to an acetylated lysine in Mi-2a rather than to an acetylated histone tail. 

Figure 1.3 Structure of double bromodomain of TAFl [56] (PDB entry lEQF). Because the TAFl bromodomains are separated by 2SA, they are proposed to simultaneously bind acetylated lysines 5 and 12 or 8 and 16 of histone H4.

Figure 2.3 Structures of mammalian classic histone deacetylases. Ribbon representation of the conserved catalytic domain of (a) class I human HDAC8 in complex with trichostatin A (TSA PDB code lt64), (b) human HDAC8 Tyr306Phe inactive mutant in complex with a peptidic acetyl-lysine substrate (PDB code 2v5w), (c) class I la human...

The secondary structure of the proteins are shown as dark gray helices and the beta strands and coil regions are in light gray. The zinc ions are shown as spheres, (b) The NAD molecule bound to the enzyme and the acetylated peptide of p53 are shown as ball and sticks. The acetylated lysine is labeled. 

Non-isotopic assays have been developed using synthetic acetylated forms of fluorescein-labeled substrates. A number of artificial substrates containing acetylated lysines based on some proteins, such as histones, p53 and p300/CBP, are available for HDAC enzyme activities [11-14]. These include the generic peptides (Fluor-de-Lys), Ac-GA-(NH-Ac-K)-amino-4-methylcoumarin (AMC), Boc-K(Ac)-7-AMC, Ac-NH-GGK(Ac)-AMC [12] and Ac-NH-RHK(Ac)K(Ac)-AMC [14]. The generic peptide... 

The action of HDACIs attributes to cell growth arrest and apoptosis. HDACIs have shown to disrupt cell cycle progression, arresting cells mainly in G1 phase. In parallel, quantification of acetylated lysines of histones and G1 checkpoint proteins, such as p53 and p21, has been used to determine the effect of HDACIs on cell cycle progression. Cell cycle arrest following HDACI treatment correlates with the induction of p21 expression in a p53-independent manner. As shown in Figure 6.2,... 

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