Sirtuins 1 and 2 Are Universal Histone Deacetylases

Lysine acetylation serves as an epigenetic marker for myriad cellular processes, such as signaling, differentiation, DNA repair, angiogenesis, and the like. Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD⁺-dependent histone deacetylases that operate as post-translational regulators for the deacetylation of acetyllysine. Here, we discuss the ability for SIRT1 and SIRT2 to deacetylate monoacetylated histone H3 on two separate architecturesthe peptide and the nucleosome. In addition, we analyze the site-specificity of SIRT1 and SIRT2 on 10 different monoacetylated histone H3 nucleosomes. By utilizing a rapid screening array, SIRT1 and SIRT2 were found to demonstrate heightened enzymatic activity when incubated with nucleosomal substrates over their peptide counterparts. These two enzymes displayed little site-specificity among the acetyl-nucleosomes screened, contrary to previous expectations, as well. The implication of the overall nonspecificity of SIRT1 and SIRT2 on the nucleosome suggests that these sirtuin enzymes have an adaptive nature, harnessing an ability to respond to various cellular situations, rather than an enzyme specifically designed for a particular task or function

Probing the Catalytic Charge-Relay System in Alanine Racemase with Genetically Encoded Histidine Mimetics

Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme’s catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme

Banca electrónica en el Ecuador

El sector bancario en el Ecuador realizó un avance tecnológico. Su conexión electrónica con el sistema financiero internacional promueve, a mediano plazo, cambios significativos en el sector. Mediante el enlace electrónico los bancos se adelantan a los gobiernos en la integración regional

Suppression of an AGG mutation at S2 of sfGFP by.

<p>(<b>A</b>) Expression of sfGFP in cells transformed with pETtrio-pylT(CCU)-sfGFP2AGG and grown in the absence or presence of 5 mM BocK. (<b>B</b>) The ESI-MS analysis of sfGFP expressed in the presence of 5 mM BocK.</p

Suppression of an opal mutation at S2 of sfGFP by the PylRS- pair.

<p>(<b>A</b>) Expression of sfGFP with an opal mutation. Lanes 1 and 2 were transformed with pETtrio-pylT(UCA)-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 3 and 4 were transformed with pETtrio-pylT(UCA)-sfGFP2TGA and grown in the absence or presence of 5 mM BocK. Each protein shown in the gel represents their real relative expression levels. ESI-MS of sfGFP expressed in cells transformed with pETtrio-pylT(UCA)-sfGFP2TGA and grown in the (<b>B1</b>) absence or (<b>B2</b>) presence of 5 mM BocK.</p

Suppression of an opal mutation at N134 of sfGFP by different variants.

<p>(<b>A</b>) Proteins shown in the gel represent their real relative expression levels. Lanes 1 and 2 were transformed with pETtrio-pylT(UCA)G73C-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 3 and 4 were transformed with pETtrio-pylT(UCA)G73A-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 5 and 6 were transformed with pETtrio-pylT(UCA)G73U-sfGFP134TGA and grown in the absence or presence of 5 mM BocK; lanes 7 and 8 were transformed with pETtrio-pylT(UCA)-sfGFP134TGA and grown in the absence or presence of 5 mM BocK. The ESI-MS analysis of sfGFP expressed in cells transformed with pETtrio-pylT(UCA)G73U-sfGFP134TGA and grown in the (<b>B1</b>) absence or (<b>B2</b>) presence of 5 mM BocK.</p

The structures of Pyl, BocK and AzF.

<p>The structures of Pyl, BocK and AzF.</p

A Chemical Biology Approach to Reveal Sirt6-targeted Histone H3 Sites in Nucleosomes

As a member of a highly conserved family of NAD⁺-dependent histone deacetylases, Sirt6 is a key regulator of mammalian genome stability, metabolism, and life span. Previous studies indicated that Sirt6 is hardwired to remove histone acetylation at H3K9 and H3K56. However, how Sirt6 recognizes its nucleosome substrates has been elusive due to the difficulty of accessing homogeneous acetyl-nucleosomes and the low activity of Sirt6 toward peptide substrates. Based on the fact that Sirt6 has an enhanced activity to remove long chain fatty acylation from lysine, we developed an approach to recombinantly synthesize histone H3 with a fatty acylated lysine, <i>N</i>^ε<i>-</i>(7-octenoyl)-lysine (OcK), installed at a number of lysine sites and used these acyl-H3 proteins to assemble acyl-nucleosomes as active Sirt6 substrates. A chemical biology approach that visualizes OcK in nucleosomes and therefore allows direct sensitization of Sirt6 activities on its acyl-nucleosome substrates was also formulated. By combining these two approaches, we showed that Sirt6 actively removes acylation from H3K9, H3K18, and H3K27; has relatively low activities toward H3K4 and K3K23; but sluggishly removes acylation at H3K14, H3K36, H3K56, and H3K79. Overexpressing Sirt6 in 293T cells led to downregulated acetylation at H3K18 and K3K27, confirming these two novel Sirt6-targeted nucleosome lysine sites in cells. Given that downregulation of H3K18 acetylation is correlated with a poor prognosis of several cancer types and H3K27 acetylation antagonizes repressive gene regulation by di- and trimethylation at H3K27, our current study implies that Sirt6 may serve as a target for cancer intervention and regulatory pathway investigation in cells

Cross recognitions between different anticodons of tRNA^Pyl and nonsense mutations at N134 of sfGFP.

<p>Cells were transformed with pETtrio-PylT(NNN)-PylRS-sfGFP134N’N’N’ and grown in the presence of 5 mM BocK (NNN and N’N’N’ denote anticodons and codons specified in the figure). Proteins shown in the gel represent their real relative expression levels.</p

Facile Removal of Leader Peptides from Lanthipeptides by Incorporation of a Hydroxy Acid

The biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products typically involves a precursor peptide which contains a leader peptide that is important for the modification process, and that is removed in the final step by a protease. Genome mining efforts for new RiPPs are often hampered by the lack of a general method to remove the leader peptides. We describe here the incorporation of hydroxy acids into the precursor peptides in <i>E. coli</i> which results in connection of the leader peptide via an ester linkage that is readily cleaved by simple hydrolysis. We demonstrate the method for two lantibiotics, lacticin 481 and nukacin ISK-1