Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition “click” reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)–(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation

Phenolics content, fruit flesh colour and browning in cultivated eggplant, wild relatives and interspecific hybrids and implications for fruit quality breeding

[EN] Increasing the content in bioactive phenolics in the eggplant (Satanum melongena) fruit is of interest, but may result in enhanced browning. We evaluated six varieties of S. melongena, 22 accessions of wild related species and 42 interspecific hybrids between cultivated eggplant and wild relatives for phenolics content, fruit flesh colour, polyphenol oxidase (PPO) activity, and fruit flesh browning. Wild relatives generally had a higher content in phenolics and a broader range of variation than cultivated eggplant. Chlorogenic acid was the predominant ( > 65.0%) phenolic acid in cultivated eggplant and its primary genepool wild ancestor S. insanum, while for the other wild species on average represented < 50% of the chromatogram peak area. Fruit flesh colour was lighter in S. melongena than in the wild species, while PPO activity and browning was much higher in wild species of the secondary and tertiary genepools. Interspecific hybrids between S. melongena and S. insanum were intermediate in their characteristics, while those with secondary and tertiary genepool species were more similar to the wild species. No significant correlations were found between total phenolics or chlorogenic acid contents and fruit flesh browning, but PPO activity was correlated to both the degree of browning (r = 0.404) and colour difference (r = 0.458). The results indicate that wild species can contribute to improving the bioactive properties of eggplant without affecting negatively fruit flesh colour and browning.This work has been funded in part by the initiative "Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives", which is supported by the Government of Norway (GS13044 and GS17011). This project is managed by the Global Crop Diversity Trust with the Millennium Seed Bank of the Royal Botanic Gardens, Kew and implemented in partnership with national and international gene banks and plant breeding institutes around the world. For further information see the project website: http://www.cwrdiversity.org/. Funding has also been received from the European Union's Horizon 2020 - Research and Innovation Framework Programme under grant agreement No 677379 (G2P-SOL project: Linking genetic resources, genomes and phenotypes of Solanaceous crops) and from Spanish Ministerio de Economia y Competitividad and Fondo Europeo de Desarrollo Regional (grant AGL2015-64755-R from MINECO/FEDER). Prashant Kaushik is grateful to ICAR for a pre-doctoral grant. Pietro Gramazio is grateful to Universitat Politecnica de Valencia for a pre-doctoral (Programa FPI de la UPV-Subprograma 1/2013 call) contract.Kaushik, P.; Gramazio, P.; Vilanova Navarro, S.; Raigón Jiménez, MD.; Prohens Tomás, J.; Plazas Ávila, MDLO. (2017). Phenolics content, fruit flesh colour and browning in cultivated eggplant, wild relatives and interspecific hybrids and implications for fruit quality breeding. Food Research International. 102:392-401. https://doi.org/10.1016/j.foodres.2017.09.028S39240110

Chemoproteomics reveals Toll-like receptor fatty acylation

Partial funding for Open Access provided by The Ohio State University Open Access Fund.Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.This work was supported by funding from the NIH/NIAID (grant R00AI095348 to J.S.Y.), the NIH/NIGMS (R01GM087544 to HCH), and the Ohio State University Public Health Preparedness for Infectious Diseases (PHPID) program. NMC is supported by the Ohio State University Systems and Integrative Biology Training Program (NIH/NIGMS grant T32GM068412). BWZ is a fellow of the National Science Foundation Graduate Research Fellowship Program (DGE-0937362)

Cell penetration, herbicidal activity, and in-vivo-toxicity of oligo-arginine derivatives and of novel guanidinium-rich compounds derived from the biopolymer cyanophycin Grogg, Marcel and Hilvert, Donald and Ebert, Marc-Olivier and Beck, Albert K. and Seebach, Dieter and Kurth, Felix and Dittrich, Petra S. and Sparr, C. and Wittlin, Sergio and Rottmann, Matthias and Mäser, Pascal

Oligo‐arginines are thoroughly studied cell‐penetrating peptides (CPPs, Figures 1 and 2). Previous in‐vitro investigations with the octaarginine salt of the phosphonate fosmidomycin (herbicide and anti‐malaria drug) have shown a 40‐fold parasitaemia inhibition with P. falciparum, compared to fosmidomycin alone (Figure 3). We have now tested this salt, as well as the corresponding phosphinate salt of the herbicide glufosinate, for herbicidal activity with whole plants by spray application, hoping for increased activities, i.e. decreased doses. However, both salts showed low herbicidal activity, indicating poor foliar uptake (Table 1). Another pronounced difference between in‐vitro and in‐vivo activity was demonstrated with various cell‐penetrating octaarginine salts of fosmidomycin: intravenous injection to mice caused exitus of the animals within minutes, even at doses as low as 1.4 μmol/kg (Table 2). The results show that use of CPPs for drug delivery, for instance to cancer cells and tissues, must be considered with due care. The biopolymer cyanophycin is a poly‐aspartic acid containing argininylated side chains (Figure 4); its building block is the dipeptide H‐βAsp‐αArg‐OH (H‐Adp‐OH). To test and compare the biological properties with those of octaarginines we synthesized Adp8‐derivatives (Figure 5). Intravenouse injection of H‐Adp8‐NH2 into the tail vein of mice with doses as high as 45 μmol/kg causes no symptoms whatsoever (Table 3), but H‐Adp8‐NH2 is not cell penetrating (HEK293 and MCF‐7 cells, Figure 6). On the other hand, the fluorescently labeled octamers FAM‐(Adp(OMe))8‐NH2 and FAM‐(Adp(NMe2))8‐NH2 with ester and amide groups in the side chains exhibit mediocre to high cell‐wall permeability (Figure 6), and are toxic (Table 3). Possible reasons for this behavior are discussed (Figure 7) and corresponding NMR spectra are presented (Figure 8)

Standard Model Physics at the HL-LHC and HE-LHC

The successful operation of the Large Hadron Collider (LHC) and the excellent performance of the ATLAS, CMS, LHCb and ALICE detectors in Run-1 and Run-2 with $pp$ collisions at center-of-mass energies of 7, 8 and 13 TeV as well as the giant leap in precision calculations and modeling of fundamental interactions at hadron colliders have allowed an extraordinary breadth of physics studies including precision measurements of a variety physics processes. The LHC results have so far confirmed the validity of the Standard Model of particle physics up to unprecedented energy scales and with great precision in the sectors of strong and electroweak interactions as well as flavour physics, for instance in top quark physics. The upgrade of the LHC to a High Luminosity phase (HL-LHC) at 14 TeV center-of-mass energy with 3 ab$^{-1}$ of integrated luminosity will probe the Standard Model with even greater precision and will extend the sensitivity to possible anomalies in the Standard Model, thanks to a ten-fold larger data set, upgraded detectors and expected improvements in the theoretical understanding. This document summarises the physics reach of the HL-LHC in the realm of strong and electroweak interactions and top quark physics, and provides a glimpse of the potential of a possible further upgrade of the LHC to a 27 TeV $pp$ collider, the High-Energy LHC (HE-LHC), assumed to accumulate an integrated luminosity of 15 ab$^{-1}$