Oxidative stress induces transient O‐GlcNAc elevation and tau dephosphorylation in SH‐SY5Y cells

O‐linked β‐N‐acetlyglucosamine or O‐GlcNAc modification is a dynamic post‐translational modification occurring on the Ser/Thr residues of many intracellular proteins. The chronic imbalance between phosphorylation and O‐GlcNAc on tau protein is considered as one of the main hallmarks of Alzheimer's disease. In recent years, many studies also showed that O‐GlcNAc levels can elevate upon acute stress and suggested that this might facilitate cell survival. However, many consider chronic stress, including oxidative damage as a major risk factor in the development of the disease. In this study, using the neuronal cell line SH‐SY5Y we investigated the dynamic nature of O‐GlcNAc after treatment with 0.5 mM H₂O₂ for 30 min. to induce oxidative stress. We found that overall O‐GlcNAc quickly increased and reached peak level at around 2 hrs post‐stress, then returned to baseline levels after about 24 hrs. Interestingly, we also found that tau protein phosphorylation at site S262 showed parallel, whereas at S199 and PHF1 sites showed inverse dynamic to O‐Glycosylation. In conclusion, our results show that temporary elevation in O‐GlcNAc modification after H₂O₂‐induced oxidative stress is detectable in cells of neuronal origin. Furthermore, oxidative stress changes the dynamic balance between O‐GlcNAc and phosphorylation on tau proteins.     

Disruption of O‐GlcNAc homeostasis during mammalian oocyte meiotic maturation impacts fertilization

Meiotic maturation and fertilization are metabolically demanding processes, and thus the mammalian oocyte is highly susceptible to changes in nutrient availability. O‐GlcNAcylation—the addition of a single sugar residue (O‐linked β‐N‐acetylglucosamine) on proteins—is a posttranslational modification that acts as a cellular nutrient sensor and likely modulates the function of oocyte proteins. O‐GlcNAcylation is mediated by O‐GlcNAc transferase (OGT), which adds O‐GlcNAc onto proteins, and O‐GlcNAcase (OGA), which removes it. Here we investigated O‐GlcNAcylation dynamics in bovine and human oocytes during meiosis and determined the developmental sequelae of its perturbation. OGA, OGT, and multiple O‐GlcNAcylated proteins were expressed in bovine cumulus oocyte complexes (COCs), and they were localized throughout the gamete but were also enriched at specific subcellular sites. O‐GlcNAcylated proteins were concentrated at the nuclear envelope at prophase I, OGA at the cortex throughout meiosis, and OGT at the meiotic spindles. These expression patterns were evolutionarily conserved in human oocytes. To examine O‐GlcNAc function, we disrupted O‐GlcNAc cycling during meiotic maturation in bovine COCs using Thiamet‐G (TMG), a highly selective OGA inhibitor. Although TMG resulted in a dramatic increase in O‐GlcNAcylated substrates in both cumulus cells and the oocyte, there was no effect on cumulus expansion or meiotic progression. However, zygote development was significantly compromised following in vitro fertilization of COCs matured in TMG due to the effects on sperm penetration, sperm head decondensation, and pronuclear formation. Thus, proper O‐GlcNAc homeostasis during meiotic maturation is important for fertilization and pronuclear stage development.     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

Mechanical stress alters protein O‐GlcNAc in human periodontal ligament cells

Protein O‐linked N‐acetylglucosamine (O‐GlcNAc) is a post‐translational modification of intracellular proteins that regulates several physiological and pathophysiological process, including response to various stressors. However, O‐GlcNAc's response to mechanical stress has not been investigated yet. As human periodontal ligament (PDL) cells are stimulated by compression force during orthodontic tooth movement that results in structural remodelling, in this study we investigated whether mechanical stress induces any alteration in protein O‐GlcNAc in PDL cells. In this study, PDL cells isolated from premolars extracted for orthodontic indications were exposed to 0, 1.5, 3, 7 and 14 g/cm² compression forces for 12 hours. Cell viability was measured by flow cytometry, and protein O‐GlcNAc was analysed by Western blot. Cellular structure and intracellular distribution of O‐GlcNAc was studied by immunofluorescence microscopy. We found that between 1.5 and 3 g/cm² mechanical compression, O‐GlcNAc significantly elevated; however, at higher forces O‐GlcNAc level was not increased. We also found that intracellular localization of O‐GlcNAc proteins became more centralized under 2 g/cm² compression force. Our results suggest that structural changes stimulated by compression forces have a significant effect on the regulation of O‐GlcNAc; thus, it might play a role in the mechanical stress adaptation of PDL cells.     

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion: Analysis of N‐ and O‐linked glycans and identification and elimination of a xylose‐based O‐linked tetrasaccharide core in the linker region

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion (huLCAT‐Fc) is a chimeric protein produced by fusing human Fc to the C‐terminus of the human enzyme via a linker sequence. The huLCAT‐Fc homodimer contains five N‐linked glycosylation sites per monomer. The heterogeneity and site‐specific distribution of the various glycans were examined using enzymatic digestion and LC‐MS/MS, followed by automatic processing. Almost all of the N‐linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N‐linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N‐linked site exclusively contain typical asialobiantennary structures. HuLCAT‐Fc was also confirmed to have mucin‐type glycans attached at T₄₀₇ and S₄₀₉. When LCAT‐Fc fusions were constructed using a G‐S‐G‐G‐G‐G linker, an unexpected +632 Da xylose‐based glycosaminoglycan (GAG) tetrasaccharide core of Xyl‐Gal‐Gal‐GlcA was attached to S₄₁₈. Several minor intermediate species including Xyl, Xyl‐Gal, Xyl‐Gal‐Gal, and a phosphorylated GAG core were also present. The mucin‐type O‐linked glycans can be effectively released by sialidase and O‐glycanase; however, the GAG could only be removed and localized using chemical alkaline β‐elimination and targeted LC‐MS/MS. E₄₁₆ (the C‐terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O‐xylosyltransferase. HuLCAT‐Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPP E₄₁₆GS₄₁₈G G G GDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.     

O‐Acyl isopeptide method: development of an O‐acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aβ1‐42 isopeptide

The O‐acyl isopeptide method was developed for the efficient preparation of difficult sequence‐containing peptide. Furthermore, development of the O‐acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid‐phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid β peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Osteoporosis in neurodegeneration

Osteoporosis affects bone microarchitecture and reduces bone mass. There are more than 200 million people with osteoporosis worldwide, and the prevalence is slowly increasing. The highest prevalences are found in Scandinavia and USA, also slowly increasing. A parallel increase in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and multiple sclerosis has been noted since the middle of this century. Osteoporosis is more common in patients with each of these neurodegenerative conditions than in the general population. Several metals with neurotoxic properties accumulate in bone and can substitute for calcium in hydroxyapatite, the main mineral component of bone. Especially cadmium, but also lead, aluminum and arsenic affect bone mineral density negatively. Metals with neurotoxic properties have also been found in brain and cerebrospinal fluid from patients with Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and multiple sclerosis, and markers for neurodegeneration such as amyloid beta peptide and amyloid precursor protein have been detected in bone tissue from patients with osteoporosis. A common mechanism contributing to the pathogenesis of both neurodegeneration and osteoporosis can be suspected. The hypothesis that neurodegenerative disorders are associated with osteoporosis is presented and discussed.     

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l ‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.     

Investigation of the synthetic route to pepstatin analogues by SPPS using O‐protected and O‐unprotected statine as building blocks

The synthetic route to pepstatin derivatives by a solid phase peptide synthesis using either O‐protected or O‐unprotected statine as a building block has been investigated. Statine was prepared according to a modified literature procedure, whereas protection of its 3‐hydroxyl moiety using tert‐butyldimethylsilylchloride (TBSCl) provided the novel O‐TBS‐protected statine building block. The O‐tert‐butyldimethylsilyl (TBS)‐protected statine approach provides an improved synthetic strategy for the preparation of statine‐containing peptides as demonstrated by the synthesis of the pepstatin analogue iva‐Val‐Leu‐Sta‐Ala‐Sta. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

BRI2 as a central protein involved in neurodegeneration

BRI2 is a protein that when mutated causes familial British and familial Danish dementias. Upon cleavage, the mutated BRI2 proteins release the peptides ABri and ADan, which are amyloidogenic and accumulate in the brains of patients. Although BRI2 has an unknown function, several reports indicate that it could play multiple roles. For example, the fact that it exists at the cell surface as a homodimer indicates that it could be involved in cell signaling events by acting as a receptor. BRI2 also interacts with amyloid precursor protein (APP), involved in Alzheimer's disease (AD). In cell cultures and mouse models of AD, BRI2 inhibits APP processing and reduces amyloid β peptide deposition. The interaction between the two proteins could be responsible for the neuropathological similarities between familial British/Danish dementias and AD. The study of BRI2, which is central in familial British and Danish dementia, could unravel underlying molecular mechanisms of neurodegeneration.     

Regulation of autophagy by polyphenols: Paving the road for treatment of neurodegeneration

In the present paper, we will discuss on the importance of autophagy in the central nervous system, and outline the relation between autophagic pathways and the pathogenesis of neurodegenerative disorders. The potential therapeutic benefits of naturally occurring phytochemicals as pharmacological modulators of autophagy will also be addressed. Our findings provide renewed insight on the molecular modes of protection by polyphenols, which is likely to be at least in part mediated not only by their potent antioxidant and anti-inflammatory effects, but also through modulation of autophagic processes to remove the aberrant protein aggregates.     

The function of GluR1 and GluR2 in cerebellar and hippocampal LTP and LTD is regulated by interplay of phosphorylation and O‐GlcNAc modification

Long‐term potentiation (LTP) and long‐term depression (LTD) are the current models of synaptic plasticity and widely believed to explain how different kinds of memory are stored in different brain regions. Induction of LTP and LTD in different regions of brain undoubtedly involve trafficking of AMPA receptor to and from synapses. Hippocampal LTP involves phosphorylation of GluR1 subunit of AMPA receptor and its delivery to synapse whereas; LTD is the result of dephosphorylation and endocytosis of GluR1 containing AMPA receptor. Conversely the cerebellar LTD is maintained by the phosphorylation of GluR2 which promotes receptor endocytosis while dephosphorylation of GluR2 triggers receptor expression at the cell surface and results in LTP. The interplay of phosphorylation and O‐GlcNAc modification is known as functional switch in many neuronal proteins. In this study it is hypothesized that a same phenomenon underlies as LTD and LTP switching, by predicting the potential of different Ser/Thr residues for phosphorylation, O‐GlcNAc modification and their possible interplay. We suggest the involvement of O‐GlcNAc modification of dephosphorylated GluR1 in maintaining the hippocampal LTD and that of dephosphorylated GluR2 in cerebral LTP. J. Cell. Biochem. 109: 585–597, 2010. © 2010 Wiley‐Liss, Inc.     

Proteomic analysis and abrogated expression of O‐GlcNAcylated proteins associated with primary breast cancer

O‐GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, which catalyze the addition and removal of O‐GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor‐associated proteins are O‐GlcNAc modified, the total O‐GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O‐GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O‐GlcNAc immnoblotting and LC‐MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O‐GlcNAcylated or associated with O‐GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O‐GlcNAcylated or associated with O‐GlcNAcylation. Moreover, OGT knockdown showed that decreasing O‐GlcNAcylation was related to inhibition of the anchorage‐independent growth in vitro. These data indicate that aberrant protein O‐GlcNAcylation is associated with breast cancer. Abnormal modification of these O‐GlcNAc‐modified proteins might be one of the vital malignant characteristics of cancer.     

Highly Efficient Identification of O‐GalNAc Glycosylation by an Acid‐Assisted Glycoform Simplification Approach

Compared with N‐linked glycosylation, the analysis of O‐GalNAc glycosylation is extremely challenging due to the high structure diversity of glycans and lack of glycosidases to release O‐GalNAc glycans. In this work, a glycoform simplification strategy by combining HILIC enrichment with chemical de‐sialylation to characterize O‐GalNAc glycosylation of human serum is presented. This method is first validated by using the bovine fetuin as the test sample. It is found that more than 90% of the sialic acid residues can be removed from bovine fetuin by the acid‐assisted de‐sialylation method, which significantly simplifies the glycan structure and improves identification sensitivity. Indeed, the number of identified peptide backbones increases nearly one fold when this strategy is used. This method is further applied to analyze the human serum sample, where 185 O‐GalNAc modified peptide sequences corresponding to 94 proteins with high confidence (FDR (false detection rate) <1%) are identified. This straight forward strategy can significantly reduce the variations of glycan structures, and is applicable to analysis of other biological samples with high complexity.     

Arabidopsis O‐GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering

Post‐translational modification of proteins by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is catalyzed by O‐GlcNAc transferases (OGTs). O‐GlcNAc modification of proteins regulates multiple important biological processes in metazoans. However, whether protein O‐GlcNAcylation is involved in epigenetic processes during plant development is largely unknown. Here, we show that loss of function of SECRET AGENT (SEC), an OGT in Arabidopsis, leads to an early flowering phenotype. This results from reduced histone H3 lysine 4 trimethylation (H3K4me3) of FLOWERING LOCUS C (FLC) locus, which encodes a key negative regulator of flowering. SEC activates ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), a histone lysine methyltransferase (HKMT), through O‐GlcNAc modification to augment ATX1‐mediated H3K4me3 histone modification at FLC locus. SEC transfers an O‐GlcNAc group on Ser947 of ATX1, which resides in the SET domain, thereby activating ATX1. Taken together, these results uncover a novel post‐translational O‐GlcNAc modification‐mediated mechanism for regulation of HKMT activity and establish the function of O‐GlcNAc signaling in epigenetic processes in plants.     

Preventing N‐ and O‐formylation of proteins when incubated in concentrated formic acid

Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O‐formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20^oC) induces N‐formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N‐formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below –20^oC.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.