Inhibition of Sirtuin Deacylase Activity by Peroxynitrite.

Sirtuins are a class of enzymes that deacylate protein lysine residues using NAD+ as a cosubstrate. Sirtuin deacylase activity has been historically regarded as protective; loss of sirtuin deacylase activity potentially increases susceptibility to aging-related disease development. However, which factors may inhibit sirtuins during aging or disease is largely unknown. Increased oxidant and inflammatory byproduct production damages cellular proteins. Previously, we and others found that sirtuin deacylase activity is inhibited by the nitric oxide (NO)-derived cysteine post-translational modification S-nitrosation. However, the comparative ability of the NO-derived oxidant peroxynitrite (ONOO-) to affect human sirtuin activity had not yet been assessed under uniform conditions. Here, we compare the ability of ONOO- (donated from SIN-1) to post-translationally modify and inhibit SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity. In response to SIN-1 treatment, inhibition of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity correlated with increased tyrosine nitration. Mass spectrometry identified multiple novel tyrosine nitration sites in SIRT1, SIRT3, SIRT5, and SIRT6. As each sirtuin isoform has at least one tyrosine nitration site within the catalytic core, nitration may result in sirtuin inhibition. ONOO- can also react with cysteine residues, resulting in sulfenylation; however, only SIRT1 showed detectable peroxynitrite-mediated cysteine sulfenylation. While SIRT2, SIRT3, SIRT5, and SIRT6 showed no detectable sulfenylation, SIRT6 likely undergoes transient sulfenylation, quickly resolving into an intermolecular disulfide bond. These results suggest that the aging-related oxidant peroxynitrite can post-translationally modify and inhibit sirtuins, contributing to susceptibility to aging-related disease.

Zinc-chelating BET bromodomain inhibitors equally target islet endocrine cell types.

Inhibition of the bromodomain and extraterminal domain (BET) protein family is a potential strategy to prevent and treat diabetes; however, the clinical use of BET bromodomain inhibitors (BETis) is associated with adverse effects. Here, we explore a strategy for targeting BETis to ß cells by exploiting the high-zinc (Zn2+) concentration in ß cells relative to other cell types. We report the synthesis of a novel, Zn2+-chelating derivative of the pan-BETi (+)-JQ1, (+)-JQ1-DPA, in which (+)-JQ1 was conjugated to dipicolyl amine (DPA). As controls, we synthesized (+)-JQ1-DBA, a non-Zn2+-chelating derivative, and (-)-JQ1-DPA, an inactive enantiomer that chelates Zn2+. Molecular modeling and biophysical assays showed that (+)-JQ1-DPA and (+)-JQ1-DBA retain potent binding to BET bromodomains in vitro. Cellular assays demonstrated (+)-JQ1-DPA attenuated NF-ĸB target gene expression in ß cells stimulated with the proinflammatory cytokine interleukin 1ß. To assess ß-cell selectivity, we isolated islets from a mouse model that expresses green fluorescent protein in insulin-positive ß cells and mTomato in insulin-negative cells (non-ß cells). Surprisingly, Zn2+ chelation did not confer ß-cell selectivity as (+)-JQ1-DPA was equally effective in both ß and α cells; however, (+)-JQ1-DPA was less effective in macrophages, a nonendocrine islet cell type. Intriguingly, the non-Zn2+-chelating derivative (+)-JQ1-DBA displayed the opposite selectivity, with greater effect in macrophages compared with (+)-JQ1-DPA, suggesting potential as a macrophage-targeting molecule. These findings suggest that Zn2+-chelating small molecules confer endocrine cell selectivity rather than ß-cell selectivity in pancreatic islets and provide valuable insights and techniques to assess Zn2+ chelation as an approach to selectively target small molecules to pancreatic ß cells.NEW & NOTEWORTHY Inhibition of BET bromodomains is a novel potential strategy to prevent and treat diabetes mellitus. However, BET inhibitors have negative side effects. We synthesized a BET inhibitor expected to exploit the high zinc concentration in ß cells to accumulate in ß cells. We show our inhibitor targeted pancreatic endocrine cells; however, it was less effective in immune cells. A control inhibitor showed the opposite effect. These findings help us understand how to target specific cells in diabetes treatment.

SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates.

MOTIVATION: Cell-type-specific surface proteins can be exploited as valuable markers for a range of applications including immunophenotyping live cells, targeted drug delivery and in vivo imaging. Despite their utility and relevance, the unique combination of molecules present at the cell surface are not yet described for most cell types. A significant challenge in analyzing 'omic' discovery datasets is the selection of candidate markers that are most applicable for downstream applications. RESULTS: Here, we developed GenieScore, a prioritization metric that integrates a consensus-based prediction of cell surface localization with user-input data to rank-order candidate cell-type-specific surface markers. In this report, we demonstrate the utility of GenieScore for analyzing human and rodent data from proteomic and transcriptomic experiments in the areas of cancer, stem cell and islet biology. We also demonstrate that permutations of GenieScore, termed IsoGenieScore and OmniGenieScore, can efficiently prioritize co-expressed and intracellular cell-type-specific markers, respectively. AVAILABILITY AND IMPLEMENTATION: Calculation of GenieScores and lookup of SPC scores is made freely accessible via the SurfaceGenie web application: www.cellsurfer.net/surfacegenie. CONTACT: Rebekah.gundry@unmc.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses.

Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample preparation steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among laboratories. To overcome these challenges, the current study systematically evaluated key experimental parameters regarding the use of carboxylate-modified paramagnetic particles and determined those that are critical for maximum performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use standard operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concentrating the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides and that the approach is compatible with robotic liquid handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will especially benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.

N-terminal BET bromodomain inhibitors disrupt a BRD4-p65 interaction and reduce inducible nitric oxide synthase transcription in pancreatic ß-cells.

Chronic inflammation of pancreatic islets is a key driver of ß-cell damage that can lead to autoreactivity and the eventual onset of autoimmune diabetes (T1D). In the islet, elevated levels of proinflammatory cytokines induce the transcription of the inducible nitric oxide synthase (iNOS) gene, NOS2, ultimately resulting in increased nitric oxide (NO). Excessive or prolonged exposure to NO causes ß-cell dysfunction and failure associated with defects in mitochondrial respiration. Recent studies showed that inhibition of the bromodomain and extraterminal domain (BET) family of proteins, a druggable class of epigenetic reader proteins, prevents the onset and progression of T1D in the non-obese diabetic mouse model. We hypothesized that BET proteins co-activate transcription of cytokine-induced inflammatory gene targets in ß-cells and that selective, chemotherapeutic inhibition of BET bromodomains could reduce such transcription. Here, we investigated the ability of BET bromodomain small molecule inhibitors to reduce the ß-cell response to the proinflammatory cytokine interleukin 1 beta (IL-1ß). BET bromodomain inhibition attenuated IL-1ß-induced transcription of the inflammatory mediator NOS2 and consequent iNOS protein and NO production. Reduced NOS2 transcription is consistent with inhibition of NF-κB facilitated by disrupting the interaction of a single BET family member, BRD4, with the NF-κB subunit, p65. Using recently reported selective inhibitors of the first and second BET bromodomains, inhibition of only the first bromodomain was necessary to reduce the interaction of BRD4 with p65 in ß-cells. Moreover, inhibition of the first bromodomain was sufficient to mitigate IL-1ß-driven decreases in mitochondrial oxygen consumption rates and ß-cell viability. By identifying a role for the interaction between BRD4 and p65 in controlling the response of ß-cells to proinflammatory cytokines, we provide mechanistic information on how BET bromodomain inhibition can decrease inflammation. These studies also support the potential therapeutic application of more selective BET bromodomain inhibitors in attenuating ß-cell inflammation.

Molecular docking-guided synthesis of NSAID-glucosamine bioconjugates and their evaluation as COX-1/COX-2 inhibitors with potentially reduced gastric toxicity.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a powerful class of inhibitors targeting two isoforms of the family of cyclooxygenase enzymes (COX-1 and COX-2). While NSAIDs are widely used in the management of pain, in particular as a treatment for osteo- and rheumatoid arthritis, their long-term use has been associated with numerous on- and off-target effects. As the carboxylic acid moiety present in common NSAIDs is responsible for some of their adverse effects, but is not required for their anti-inflammatory activity, we sought to mask this group through direct coupling to glucosamine, which is thought to prevent cartilage degradation. We report herein the conjugation of commonly prescribed NSAIDs to glucosamine hydrochloride and the use of molecular docking to show that addition of the carbohydrate moiety to the parent NSAID can enhance binding in the active site of COX-2. In a preliminary, in vitro screening assay, the diclofenac-glucosamine bioconjugate exhibited 10-fold greater activity toward COX-2, making it an ideal candidate for future in vivo studies. Furthermore, in an intriguing result, we observed that the mefenamic acid-glucosamine bioconjugate displayed enhanced activity toward COX-1 rather than COX-2.

Acyl-CoA Oxidases Fine-Tune the Production of Ascaroside Pheromones with Specific Side Chain Lengths.

Caenorhabditis elegans produces a complex mixture of ascaroside pheromones to control its development and behavior. Acyl-CoA oxidases, which participate in ß-oxidation cycles that shorten the side chains of the ascarosides, regulate the mixture of pheromones produced. Here, we use CRISPR-Cas9 to make specific nonsense and missense mutations in acox genes and determine the effect of these mutations on ascaroside production in vivo. Ascaroside production in acox-1.1 deletion and nonsense strains, as well as a strain with a missense mutation in a catalytic residue, confirms the central importance of ACOX-1.1 in ascaroside biosynthesis and suggests that ACOX-1.1 functions in part by facilitating the activity of other acyl-CoA oxidases. Ascaroside production in an acox-1.1 strain with a missense mutation in an ATP-binding site at the ACOX-1.1 dimer interface suggests that ATP binding is important for the enzyme to function in ascaroside biosynthesis in vivo. Ascaroside production in strains with deletion, nonsense, and missense mutations in other acox genes demonstrates that ACOX-1.1 works with ACOX-1.3 in processing ascarosides with 7-carbon side chains, ACOX-1.4 in processing ascarosides with 9- and 11-carbon side chains, and ACOX-3 in processing ascarosides with 13- and 15-carbon side chains. It also shows that ACOX-1.2, but not ACOX-1.1, processes ascarosides with 5-carbon ω-side chains. By modeling the ACOX structures, we uncover characteristics of the enzyme active sites that govern substrate preferences. Our work demonstrates the role of specific acyl-CoA oxidases in controlling the length of ascaroside side chains and thus in determining the mixture of pheromones produced by C. elegans.

Biosynthetic tailoring of existing ascaroside pheromones alters their biological function in C. elegans.

Caenorhabditis elegans produces ascaroside pheromones to control its development and behavior. Even minor structural differences in the ascarosides have dramatic consequences for their biological activities. Here, we identify a mechanism that enables C. elegans to dynamically tailor the fatty-acid side chains of the indole-3-carbonyl (IC)-modified ascarosides it has produced. In response to starvation, C. elegans uses the peroxisomal acyl-CoA synthetase ACS-7 to activate the side chains of medium-chain IC-ascarosides for ß-oxidation involving the acyl-CoA oxidases ACOX-1.1 and ACOX-3. This pathway rapidly converts a favorable ascaroside pheromone that induces aggregation to an unfavorable one that induces the stress-resistant dauer larval stage. Thus, the pathway allows the worm to respond to changing environmental conditions and alter its chemical message without having to synthesize new ascarosides de novo. We establish a new model for biosynthesis of the IC-ascarosides in which side-chain ß-oxidation is critical for controlling the type of IC-ascarosides produced.