Insights into Lysine Deacetylation of Natively Folded Substrate Proteins by Sirtuins.

Sirtuins are NAD(+)-dependent lysine deacylases, regulating a variety of cellular processes. The nuclear Sirt1, the cytosolic Sirt2, and the mitochondrial Sirt3 are robust deacetylases, whereas the other sirtuins have preferences for longer acyl chains. Most previous studies investigated sirtuin-catalyzed deacylation on peptide substrates only. We used the genetic code expansion concept to produce natively folded, site-specific, and lysine-acetylated Sirt1-3 substrate proteins, namely Ras-related nuclear, p53, PEPCK1, superoxide dismutase, cyclophilin D, and Hsp10, and analyzed the deacetylation reaction. Some acetylated proteins such as Ras-related nuclear, p53, and Hsp10 were robustly deacetylated by Sirt1-3. However, other reported sirtuin substrate proteins such as cyclophilin D, superoxide dismutase, and PEPCK1 were not deacetylated. Using a structural and functional approach, we describe the ability of Sirt1-3 to deacetylate two adjacent acetylated lysine residues. The dynamics of this process have implications for the lifetime of acetyl modifications on di-lysine acetylation sites and thus constitute a new mechanism for the regulation of proteins by acetylation. Our studies support that, besides the primary sequence context, the protein structure is a major determinant of sirtuin substrate specificity.

Insights into K-Ras 4B regulation by post-translational lysine acetylation.

Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.

Identification of organ tissue types and skin from forensic samples by microRNA expression analysis.

The identification of organ tissues in traces recovered from scenes and objects with regard to violent crimes involving serious injuries can be of considerable relevance in forensic investigations. Molecular genetic approaches are provably superior to histological and immunological assays in characterizing organ tissues, and micro-RNAs (miRNAs), due to their cell type specific expression patterns and stability against degradation, emerged as a promising molecular species for forensic analyses, with a range of tried and tested indicative markers. Thus, herein we present the first miRNA based approach for the forensic identification of organ tissues. Using quantitative PCR employing an empirically derived strategy for data normalization and unbiased statistical decision making, we assessed the differential expression of 15 preselected miRNAs in tissues of brain, kidney, lung, liver, heart muscle, skeletal muscle and skin. We show that not only can miRNA expression profiling be used to reliably differentiate between organ tissues but also that this method, which is compatible with and complementary to forensic DNA analysis, is applicable to realistic forensic samples e.g. mixtures, aged and degraded material as well as traces generated by mock stabbings and experimental shootings at ballistic models.