Sirtuins are NAD-dependent deacetylases that regulate important biological processes. Mammals have seven sirtuins, Sirt1-7. Four of them (Sirt4-7) have no detectable or very weak deacetylase activity. Here we found that Sirt5 is an efficient protein lysine desuccinylase and demalonylase in vitro. The preference for succinyl and malonyl groups was explained by the presence of an arginine residue (Arg105) and tyrosine residue (Tyr102) in the acyl pocket of Sirt5. Several mammalian proteins were identified to have succinyl or malonyl lysine modifications by mass spectrometry. Deletion of Sirt5 in mice appeared to increases the level of succinylation on carbamoyl phosphate synthase 1, a known target of Sirt5. Thus protein lysine succinylation may represent a posttranslational modification that can be reversed by Sirt5 in vivo.

The Sir2 family of enzymes or sirtuins are known as nicotinamide adenine dinucleotide (NAD)-dependent deacetylases1 and have been implicated in the regulation of transcription, genome stability, metabolism, and lifespan2, 3. However, four of the seven mammalian sirtuins have very weak deacetylase activity in vitro. Here we show that human Sirt6 efficiently removes long chain fatty acyl groups, such as myristoyl, from lysine residues. The crystal structure of Sirt6 reveals a large hydrophobic pocket that can accommodate long chain fatty acyl groups. We demonstrate further that Sirt6 promotes the secretion of tumor necrosis factor α (TNFα) by removing the fatty acyl modification on K19 and K20 of TNFα. Protein lysine fatty acylation has been known to occur in mammalian cells, but the function and regulatory mechanisms of this modification were unknown. Our data suggest that protein lysine fatty acylation is a novel mechanism that regulates protein secretion. The discovery of Sirt6 as an enzyme that controls protein lysine fatty acylation provides new opportunities to investigate the physiological function of the previously ignored protein posttranslational modification.

Tyrosine phosphorylation is a common protein posttranslational modification, which plays a critical role in signal transduction and the regulation of many cellular processes. Using a pro-peptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase/tRNA pair that allows the site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray crystal structure of the synthetase reveals a reconfigured substrate binding site formed by non-conservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site whose corresponding kinase is unknown and determined the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Protein ADP-ribosyltransferases catalyze the transfer of adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) onto specific target proteins. Sirtuins, a class of enzymes with NAD-dependent deacetylase activity, have been reported to possess ADP-ribosyltransferase activity, too. Here we used NAD analogues and 32P-NAD to study the ADP-ribosyltransferase activity of several different sirtuins, including yeast Sir2, human SirT1, mouse SirT4, and mouse SirT6. The results showed that an alkyne-tagged NAD is the substrate for deacetylation reactions but cannot detect the ADP-ribosylation activity. Furthermore, comparing with a bacterial ADP-ribosyltransferase diphtheria toxin, the observed rate constant of sirtuin-dependent ADP-ribosylation is >5000-fold lower. Compared with the kcat/Km values of the deacetylation activity of sirtuins, the observed rate constant of sirtuin-dependent ADP-ribosyltion is 500 times weaker. The weak ADP-ribosylation events can be explained by both enzymatic and nonenzymatic reaction mechanisms. Combined with recent reports on several other sirtuins, we propose that the reported ADP-ribosyltransferase activity of sirtuins is likely some inefficient side reactions of the deacetylase activity and may not be physiologically relevant.

Amyloid-beta peptide (Abeta) is the principal constituent of plaques associated with Alzheimer's disease (AD) and is thought to be responsible for the neurotoxicity associated with the disease. Copper binding to Abeta has been hypothesized to play an important role in the neruotoxicity of Abeta and free radical damage, and Cu2+ chelators represent a possible therapy for AD. However, many properties of copper binding to Abeta have not been elucidated clearly, and the location of copper binding sites on Abeta is also in controversy. Here we have used a range of spectroscopic techniques to characterize the coordination of Cu2+ to Abeta(1-16) in solution. Electrospray ionization mass spectrometry shows that copper binds to Abeta(1-16) at pH 6.0 and 7.0. The mode of copper binding is highly pH dependent. Circular dichroism results indicate that copper chelation causes a structural transition of Abeta(1-16). UV-visible absorption spectra suggest that three nitrogen donor ligands and one oxygen donor ligand (3N1O) in Abeta(1-16) may form a type II square-planar coordination geometry with Cu2+. By means of fluorescence spectroscopy, competition studies with glycine and L-histidine show that copper binds to Abeta(1-16) with an affinity of Ka approximately 10(7) M(-1) at pH 7.8. Besides His6, His13, and His14, Tyr10 is also involved in the coordination of Abeta(1-16) with Cu2+, which is supported by 1H NMR and UV-visible absorption spectra. Evidence for the link between Cu2+ and AD is growing, and this work has made a significant contribution to understanding the mode of copper binding to Abeta(1-16) in solution.