The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.

Sirt1 activation by resveratrol is substrate sequence-selective.

Sirtuins are protein deacetylases used as therapeutic targets. Pharmacological Sirt1 activation has been questioned since the in vitro activator resveratrol failed to stimulate deacetylation of several physiological substrates. We tested the influence of substrate sequence by analyzing resveratrol effects on Sirt1-dependent deacetylation of 6802 physiological acetylation sites using peptide microarrays. Resveratrol stimulated deacetylation of a small set of sites and inhibited deacetylation of another set, whereas most substrates were hardly affected. Solution assays confirmed these substrate categories, and statistical analysis revealed their sequence features. Our results reveal substrate sequence dependence for Sirt1 modulation and suggest substrates contributing to resveratrol effects.

An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms.

Sirtuin enzymes regulate metabolism and aging processes through deacetylation of acetyl-lysines in target proteins. More than 6,800 mammalian acetylation sites are known, but few targets have been assigned to most sirtuin isoforms, hampering our understanding of sirtuin function. Here we describe a peptide microarray system displaying 6,802 human acetylation sites for the parallel characterisation of their modification by deacetylases. Deacetylation data for all seven human sirtuins obtained with this system reveal isoform-specific substrate preferences and deacetylation substrate candidates for all sirtuin isoforms, including Sirt4. We confirm malate dehydrogenase protein as a Sirt3 substrate and show that peroxiredoxin 1 and high-mobility group B1 protein are deacetylated by Sirt5 and Sirt1, respectively, at the identified sites, rendering them likely new in vivo substrates. Our microarray platform enables parallel studies on physiological acetylation sites and the deacetylation data presented provide an exciting resource for the identification of novel substrates for all human sirtuins.

Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity.

The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimer's disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay. The microtubule-assembly-promoting properties of parvulin 17 seem to depend on its PPIase activity. Thus, catalytic deficient variants of parvulin 17 were not able to promote microtubule formation. Accordingly, inhibitors of parvulin 17 activity also prevent parvulin-catalyzed tubulin polymerization. The analysis of tubulin interaction sites on parvulin using peptide microarrays revealed that tubulin interacts with the substrate binding pocket of parvulin. Additionally, ß-tubulin peptide scan on microarrays demonstrates interaction of parvulin 17 with an Arg-Pro-Asp motif corresponding to proline residue 87 of ß-tubulin. Confocal laser scanning microscopy points to a function of parvulin 17 in microtubule dynamics as well. Parvulin 17 is predominantly found in the cytosol and colocalizes with microtubules.

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Tungsten-containing aldehyde oxidoreductase of Eubacterium acidaminophilum.

Aldehyde oxidoreductase of Eubacterium acidaminophilum was purified to homogeneity under strict anaerobic conditions using a four-step procedure. The purified enzyme was present as a monomer with an apparent molecular mass of 67 kDa and contained 6.0 +/- 0.1 iron, 1.1 +/- 0.2 tungsten, about 0.6 mol pterin cofactor and zinc, but no molybdenum. The enzyme activity was induced if a molar excess of electron donors, such as serine and/or formate, were supplied in the growth medium compared to readily available electron acceptors such as glycine betaine. Many aldehydes served as good substrates, thus enzyme activity obtained with acetaldehyde, propionaldehyde, butyraldehyde, isovaleraldehyde and benzaldehyde differed by a factor of less than two. Kinetic parameters were determined for all substrates tested. Oligonucleotides deduced from the N-terminal amino acid sequence were used to isolate the encoding aorA gene and adjacent DNA regions. The deduced amino acid sequence of the aldehyde oxidoreductase exhibited high similarities to other tungsten-containing aldehyde oxidoreductases from archaea. Transcription of the aorA gene was monocistronic and started from a sigma 54-dependent promoter. Upstream of aorA, the gene aorR is localized whose product is similar to sigma 54-dependent transcriptional activator proteins and, thus, AorR is probably involved in the regulation of aorA expression.