Deep,Quantitative Coverage of the Lysine Acetylome Using Novel Anti-acetyl-lysine Antibodies and an Optimized Proteomic Workflow

Introduction of antibodies specific for acetylated lysine has significantly improved the detection of endogenous acetylation sites by mass spectrometry. Here, we describe a new, commercially available mixture of anti-lysine acetylation (Kac) antibodies and show its utility for in-depth profiling of the acetylome. Specifically, seven complementary monoclones with high specificity for Kac were combined into a final anti-Kac reagent which results in at least a twofold increase in identification of Kac peptides over a commonly used Kac antibody. We outline optimal antibody usage conditions, effective offline basic reversed phase separation, and use of state-of-the-art LC-MS technology for achieving unprecedented coverage of the acetylome. The methods were applied to quantify acetylation sites in suberoylanilide hydroxamic acid-treated Jurkat cells. Over 10,000 Kac peptides from over 3000 Kac proteins were quantified from a single stable isotope labeling by amino acids in cell culture labeled sample using 7.5 mg of peptide input per state. This constitutes the deepest coverage of acetylation sites in quantitative experiments obtained to-date. The approach was also applied to breast tumor xenograft samples using isobaric mass tag labeling of peptides (iTRAQ4, TMT6 and TMT10-plex reagents) for quantification. Greater than 6700 Kac peptides from over 2300 Kac proteins were quantified using 1 mg of tumor protein per iTRAQ 4-plex channel. The novel reagents and methods we describe here enable quantitative, global acetylome analyses with depth and sensitivity approaching that obtained for other well-studied post-translational modifications such as phosphorylation and ubiquitylation, and should have widespread application in biological and clinical studies employing mass spectrometry-based proteomics.Lysine acetylation (Kac)¹ is a well conserved, reversible post-translational modification (PTM) involved in multiple cellular processes (1). Acetylation is regulated by two classes of enzymes: lysine acetyltransferases (KATs) and histone deacetylases (HDACs) (2–4). This modification was originally identified as a nuclear event on histone proteins and has been long appreciated for its role in epigenetic and DNA-dependent processes. With the help of a growing number of large-scale acetylation studies, it has become evident that lysine acetylation is ubiquitous, also occurring on cytoplasmic and mitochondrial proteins and has a role in signaling, metabolism, and immunity (1, 4–6). Therefore, the examination of lysine acetylation on nonhistone proteins has gained a prominent role in PTM analysis.To date, the identification of large numbers of acetylation sites has been challenging because of the substoichiometric nature of this modification (7, 8). Additionally, global acetylation is generally less abundant than phosphorylation and ubiquitylation (1). The introduction of antibodies specific for lysine acetylation has significantly improved the ability to enrich and identify thousands of sites (9–14). A landmark study by Choudhary et al. used anti-Kac antibodies to globally map 3600 lysine acetylation sites on 1750 proteins, thereby demonstrating the feasibility of profiling the acetylome (10). A more recent study by Lundby et al. investigated the function and distribution of acetylation sites in 16 different rat tissues, and identified, in aggregate, 15,474 acetylation sites from 4541 proteins (12).Although anti-acetyl lysine antibodies have been a breakthrough for globally mapping acetylation sites (9–12), it remains a challenge to identify large numbers of lysine acetylation sites from a single sample, as is now routinely possible for phosphorylation and ubiquitylation (13, 15–18). To improve the depth-of-coverage in acetylation profiling experiments there is a clear need for (1) alternative anti-acetyl lysine antibodies with higher specificity, (2) optimized antibody usage parameters, and (3) robust proteomic workflows that permit low to moderate protein input. In this study, we describe a newly commercialized mixture of anti-Kac antibodies and detail a complete proteomic workflow for achieving unprecedented coverage of the acetylome from a single stable isotope labeling by amino acids in cell culture (SILAC) labeled sample as well as isobaric tags for relative and absolute quantitation (iTRAQ)- and tandem mass tag (TMT)-labeled samples.     

Structural basis of pyrrole polymerization in human porphobilinogen deaminase

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.     

The mechanism involved in the loss of PTEN expression in NSCLC tumor cells

Loss of PTEN expression is observed in most non-small cell lung cancers (NSCLC). However, the mechanism by which PTEN expression is regulated in NSCLC has not been fully elucidated. In this study, we investigated the role of DNA methyltransferases (Dnmts), microRNA-29b (miR-29b), and anti-miR-29b inhibitor in PTEN promoter methylation and PTEN gene expression in H358 NSCLC cells in vitro and in vivo. PTEN mRNA was measured by RT-PCR. PTEN and Dnmts protein levels were measured by Western blot. miR-29b expression was detected by Northern blot. A xenograft H358 tumor mouse model was established by subcutaneously inoculating H358 cells into the right hind limbs of nude mice. We found that radiation induced cell apoptosis and hypomethylation in PTEN promoter, PTEN and miR-29b expression, and downregulation of Dnmt1, 3a and 3b expression in H358 tumor cells. The effect of radiation on gene expression and apoptosis was blocked by anti-miR-29b inhibitor. In the xenograft H358 tumor model, anti-miR-29b inhibitor reversed radiation-induced tumor growth delay, PTEN reexpression and downregulation of Dnmts expression. Our study suggested that miR-29b is an upstream molecule of PTEN. miR-29b regulates PTEN gene expression through downregulating Dnmts expression and subsequently induces hypomethylation in PTEN promoter. Targeting therapy could be established in NSCLC by upregulating miR-29b expression.     

Spring colonisation of orchards by Anthonomus pomorum from adjacent forest borders

The early-season dispersal of the overwintered apple blossom weevil, Anthonomus pomorum (L.) (Coleoptera: Curculionidae), is a crucial stage in the colonisation of dwarf apple orchards adjacent to forests. We have conducted release-recapture studies with 1700 to 4000 marked weevils at two orchard sites in Switzerland over 2 years to characterise the spatial and temporal pattern of the dispersal process. The dispersal and colonisation of orchards in spring by overwintered weevils is dependent upon the prevailing temperature.An orientated dispersal from the forest border to the centre of the orchard was observed consistently, irrespective of the angle of the apple tree rows with respect to the forest border or of climatic conditions. The average dispersal distance of the weevils was 19 m. Approximately one third of the weevil population remained on the first tree encountered, the remainder of the population moved over short distances mainly along the tree rows. This dispersal pattern led to a strong edge effect with higher numbers of weevils occurring at the edges adjoining the forests as compared to the centre of orchards. The relevance of these findings to population dynamics and management of the pest is discussed.     

Flux-dependent increase in the stoichiometry of charge translocation by mitochondrial ATPase/ATP synthase induced by almitrine

After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.     

The role of microbial biofilms in deterioration of space station candidate materials

Formation of microbial biofilms on surfaces of a wide range of materials being considered as candidates for use on the International Space Station was investigated. The materials included a fibre-reinforced polymeric composite, an adhesive sealant, a polyimide insulation foam, teflon cable insulation, titanium, and an aliphatic polyurethane coating. They were exposed to a natural mixed population of bacteria under controlled conditions of temperature and relative humidity (RH). Biofilms formed on the surfaces of the materials at a wide range of temperatures and RHs. The biofilm population was dominated by Pseudomonas aeruginosa, Ochrobactrum anthropi, Alcaligenes denitrificans, Xanthomonas maltophila, and Vibrio harveyi. The biocide, diiodomethyl-p-tolyl sulfone, impregnated in the polyurethane coating, was ineffective against microbial colonization and growth. Degradation of the polyurethane coatings was monitored with electrochemical impedance spectroscopy (EIS). The impedance spectra indicated that microbial degradation of the coating occurred in several stages. The initial decreases in impedance were due to the transport of water and solutes into the polymeric matrices. Further decreases were a result of polymer degradation by microorganisms. Our data showed that these candidate materials for space application are susceptible to biofilm formation and subsequent degradation. Our study suggests that candidate materials for use in space missions need to be carefully evaluated for their susceptibility to microbial biofilm formation and biodegradation.     

Effects of habitat fragmentation on genetic diversity and gene flow among the Homidia socia (Collembola: Entomobryidae) populations in the Thousand Island Lake

In the present study, partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene of 22 island populations of the springtail Homidia socia in the Thousand Island Lake were sequenced. Across all sequences, 37 haplotypes were identified for the 510‐bp mitochondrial (mt) DNA COI gene. Haplotype 2 was the most common, and was distributed in the most of the 22 island populations. Haplotype diversity ranged from 0.065 to 0.733, and the total genetic diversity was 0.56216. The genetic characteristics of the 22 island populations were analyzed using the fixation index and gene flow, with values of 0.00043–0.94900 and 0.02703–703.72540, respectively. Comparison between (island area and isolations) with population genetic diversity revealed that there were no significant correlations between them, except for a significant correlation between the number of haplotypes and island area. Mantel tests showed that there was no significant correlation between geographic distance and genetic distance among various groups. All the results indicated that there were no obvious relationships between island characteristics and the genetic diversity of the springtails. We consider that the low dispersal capacity of springtails and the island patches surrounded by water in the Thousand Island Lake are the major factors affecting the genetic diversity of H. socia.