Demystifying the O‑GlcNAc Code: A Systems View

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc), a process referred to as O-GlcNAcylation, occurs on a vast variety of proteins. Mounting evidence in the past several decades has clearly demonstrated that O-GlcNAcylation is a unique and ubiquitous modification. Reminiscent of a code, protein O-GlcNAcylation functions as a crucial regulator of nearly all cellular processes studied. The primary aim of this review is to summarize the developments in our understanding of myriad protein substrates modified by O-GlcNAcylation from a systems perspective. Specifically, we provide a comprehensive survey of O-GlcNAcylation in multiple species studied, including eukaryotes (e.g., protists, fungi, plants, Caenorhabditis elegans, Drosophila melanogaster, murine, and human), prokaryotes, and some viruses. We evaluate features (e.g., structural properties and sequence motifs) of O-GlcNAc modification on proteins across species. Given that O-GlcNAcylation functions in a species-, tissue-/cell-, protein-, and site-specific manner, we discuss the functional roles of O-GlcNAcylation on human proteins. We focus particularly on several classes of relatively well-characterized human proteins (including transcription factors, protein kinases, protein phosphatases, and E3 ubiquitin-ligases), with representative O-GlcNAc site-specific functions presented. We hope the systems view of the great endeavor in the past 35 years will help demystify the O-GlcNAc code and lead to more fascinating studies in the years to come

Demystifying the O‑GlcNAc Code: A Systems View

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc), a process referred to as O-GlcNAcylation, occurs on a vast variety of proteins. Mounting evidence in the past several decades has clearly demonstrated that O-GlcNAcylation is a unique and ubiquitous modification. Reminiscent of a code, protein O-GlcNAcylation functions as a crucial regulator of nearly all cellular processes studied. The primary aim of this review is to summarize the developments in our understanding of myriad protein substrates modified by O-GlcNAcylation from a systems perspective. Specifically, we provide a comprehensive survey of O-GlcNAcylation in multiple species studied, including eukaryotes (e.g., protists, fungi, plants, Caenorhabditis elegans, Drosophila melanogaster, murine, and human), prokaryotes, and some viruses. We evaluate features (e.g., structural properties and sequence motifs) of O-GlcNAc modification on proteins across species. Given that O-GlcNAcylation functions in a species-, tissue-/cell-, protein-, and site-specific manner, we discuss the functional roles of O-GlcNAcylation on human proteins. We focus particularly on several classes of relatively well-characterized human proteins (including transcription factors, protein kinases, protein phosphatases, and E3 ubiquitin-ligases), with representative O-GlcNAc site-specific functions presented. We hope the systems view of the great endeavor in the past 35 years will help demystify the O-GlcNAc code and lead to more fascinating studies in the years to come

High-Rate Solid Polymer Electrolyte Based Flexible All-Solid-State Lithium Metal Batteries

A flexible poly­(vinylidene fluoride)-polyetherimide@poly­(ethylene glycol) (PVDF-PEI@PEG) solid composite polymer electrolyte is prepared by an in situ thermal curing approach. The homogeneous PVDF-PEI composite porous membrane with an optimized PVDF and PEI weight ratio increases the amorphous phase, while the fast lithium ion transport channels are formed through the filled PEG electrolytes. The optimized polymer electrolyte exhibits high ionic conductivity of 2.36 × 10–4 S cm–1 at 60 °C and lithium ion transference number of 0.578 as well as excellent electrochemical stability window of 5.5 V. Moreover, the superior stability toward lithium metal anode enables over 3600 h cycling of the Li//Li symmetric cell at 0.1 mA cm–2. In particular, the LiFePO4//Li battery delivers high specific capacities of 132.4 and 111.5 mAh g–1 with a retention of 86.6% and 85.9% after 200 cycles at 2 C and 100 cycles at 3 C rate under 60 °C, respectively, demonstrating the feasibility as an energy storage device with high rate capability

HexNAcQuest: A Tool to Distinguish O‑GlcNAc and O‑GalNAc

Protein glycosylation plays crucial roles in the regulation of diverse biological processes. As a critical step, mass spectrometry-based site-specific analysis of protein glycosylation is important to better understand these events. Despite the great progress, characterization of structural isomers of glycans and glycopeptides remains challenging. In typical glycoproteomic analysis, collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) fragmentation produces abundant saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues. However, it has been difficult to distinguish isobaric GalNAc and GlcNAc modifications by using mass spectrometry only. By using intensities of oxonium ions of standard O-GlcNAc/O-GalNAc peptides, we systematically investigated the fragmentation patterns of different ions. Then a binary logistic regression model was established by training comprehensive data sets from glycoproteomics studies reported. The model was then tested with independent O-glycoproteomics data sets, with reliable classification achieved (>87% accuracy). In comparison to empirical observations and criteria used previously, our model is accurate and generalized. Based on this model, a corresponding Web server HexNAcQuest has been constructed, which is freely accessible to users. The model can also be easily integrated in MS-based glycoproteomics workflows to distinguish the isobaric HexNAc modifications

Design and Preparation of Novel Nitro-Oxide-Grafted Nanospheres with Enhanced Hydrogen Bonding Interaction for <i>O</i>‑GlcNAc Analysis

As an essential modification, O-linked β-N-acetylglucosamine (O-GlcNAc) modulates the functions of many proteins. However, site-specific characterization of O-GlcNAcylated proteins remains challenging. Herein, an innovative material grafted with nitro-oxide (N→O) groups was designed for high affinity enrichment for O-GlcNAc peptides from native proteins. By testing with synthetic O-GlcNAc peptides and standard proteins, the synthesized material exhibited high affinity and selectivity. Based on the material prepared, we developed a workflow for site-specific analysis of O-GlcNAcylated proteins in complex samples. We performed O-GlcNAc proteomics with the PANC-1 cell line, a representative model for pancreatic ductal adenocarcinoma. In total 364 O-GlcNAc peptides from 267 proteins were identified from PANC-1 cells. Among them, 183 proteins were newly found to be O-GlcNAcylated in humans (with 197 O-GlcNAc sites newly reported). The materials and methods can be facilely applied for site-specific O-GlcNAc proteomics in other complex samples

Image_2_SIRT1 Mediates Effects of FGF21 to Ameliorate Cisplatin-Induced Acute Kidney Injury.tif

Acute kidney injury (AKI) is a common complication in cancer patients. Kidney function is closely related to patients’ quality of life and tumor prognosis. Cisplatin is a highly effective anti-tumor drug. However, the use of cisplatin is limited by its nephrotoxicity. It has been reported that FGF21 has a renal-protective function, but the mechanisms by which it does so remain unclear. In this study, we show that the expression of FGF21 is significantly upregulated in both in vitro and in vivo cisplatin-induced AKI models. Administration of recombinant FGF21 to cisplatin-induced AKI mice resulted in significantly decreased blood urea nitrogen (BUN) and serum creatinine levels, as well as significantly reduced protein levels of kidney injury molecule-1 (TIM-1), C-caspase 3, and Bax. H&E-stained kidney sections from cisplatin-induced AKI mice treated with recombinant FGF21 showed a relatively normal renal tissue structure, a reduced number of necrotic sites and vacuolar changes, and decreased casts, suggesting alleviated renal tubular injury. Experiments with an AKI cell model (cisplatin-treated HK-2 cells) yielded similar results as the mouse model; recombinant FGF21 significantly downregulated protein expression levels of TIM-1, C-caspase 3, and Bax. Furthermore, administration of recombinant FGF21 to cisplatin-treated AKI models significantly increased SIRT1 expression, and the beneficial effects of FGF21 on kidney injury were reversed by SIRT1 knockdown. Collectively, our results suggest that SIRT1 mediates the protective effect of FGF21 on cisplatin-induced kidney injury.</p

O‑GlcNAcPRED-DL: Prediction of Protein O‑GlcNAcylation Sites Based on an Ensemble Model of Deep Learning

O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification (i.e., O-GlcNAcylation) on serine/threonine residues of proteins, regulating a plethora of physiological and pathological events. As a dynamic process, O-GlcNAc functions in a site-specific manner. However, the experimental identification of the O-GlcNAc sites remains challenging in many scenarios. Herein, by leveraging the recent progress in cataloguing experimentally identified O-GlcNAc sites and advanced deep learning approaches, we establish an ensemble model, O-GlcNAcPRED-DL, a deep learning-based tool, for the prediction of O-GlcNAc sites. In brief, to make a benchmark O-GlcNAc data set, we extracted the information on O-GlcNAc from the recently constructed database O-GlcNAcAtlas, which contains thousands of experimentally identified and curated O-GlcNAc sites on proteins from multiple species. To overcome the imbalance between positive and negative data sets, we selected five groups of negative data sets in humans and mice to construct an ensemble predictor based on connection of a convolutional neural network and bidirectional long short-term memory. By taking into account three types of sequence information, we constructed four network frameworks, with the systematically optimized parameters used for the models. The thorough comparison analysis on two independent data sets of humans and mice and six independent data sets from other species demonstrated remarkably increased sensitivity and accuracy of the O-GlcNAcPRED-DL models, outperforming other existing tools. Moreover, a user-friendly Web server for O-GlcNAcPRED-DL has been constructed, which is freely available at http://oglcnac.org/pred_dl

<i>O</i>‑GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry

As a dynamic post-translational modification, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP–GalNAz followed by copper-free azide–alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acryl­amido­propyl)­tri­methyl­ammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins

Online Integration of Multiple Sample Pretreatment Steps Involving Denaturation, Reduction, and Digestion with Microflow Reversed-Phase Liquid Chromatography−Electrospray Ionization Tandem Mass Spectrometry for High-Throughput Proteome Profiling

A facile integrated platform for proteome profiling was established, in which native proteins were online denatured and reduced within a heater, digested with an immobilized trypsin microreactor, and analyzed by microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (μRPLC-ESI-MS/MS). In comparison to the traditional off-line urea denaturation protocol, even more unique peptides were obtained by online heating in triplicate (14 ± 2 vs 11 ± 2 for myoglobin and 16 vs 12 ± 1 for BSA) within a significantly shortened pretreatment time of ∼3.5 min (including 1 min of thermal denaturation and reduction and ∼2.5 min of microreactor digestion). Moreover, proteins with concentrations ranging from 50 ng/mL (∼6 fmol) to 1 mg/mL (∼120 pmol) were positively identified by the online system. Such a platform was further successfully applied for analyzing the soluble fraction of mouse liver extract. Of all the 367 proteins identified from samples pretreated by the urea protocol and online heating, ∼40% were overlapped, showing the partial complementation of both approaches. All these results demonstrate that the online integrated platform is of great promise for high-throughput proteome profiling and improved identification capacity for low-abundance proteins with a minute sample amount

Design and Preparation of Novel Nitro-Oxide-Grafted Nanospheres with Enhanced Hydrogen Bonding Interaction for <i>O</i>‑GlcNAc Analysis

As an essential modification, O-linked β-N-acetylglucosamine (O-GlcNAc) modulates the functions of many proteins. However, site-specific characterization of O-GlcNAcylated proteins remains challenging. Herein, an innovative material grafted with nitro-oxide (N→O) groups was designed for high affinity enrichment for O-GlcNAc peptides from native proteins. By testing with synthetic O-GlcNAc peptides and standard proteins, the synthesized material exhibited high affinity and selectivity. Based on the material prepared, we developed a workflow for site-specific analysis of O-GlcNAcylated proteins in complex samples. We performed O-GlcNAc proteomics with the PANC-1 cell line, a representative model for pancreatic ductal adenocarcinoma. In total 364 O-GlcNAc peptides from 267 proteins were identified from PANC-1 cells. Among them, 183 proteins were newly found to be O-GlcNAcylated in humans (with 197 O-GlcNAc sites newly reported). The materials and methods can be facilely applied for site-specific O-GlcNAc proteomics in other complex samples