Evidence that only EWS among the FET proteins acquires a low partitioning property for the hyperosmotic stress response by O-GlcNAc glycosylation on its low-complexity domain.

FET proteins (FUS, EWS, and TAF15) share a common domain organization, bind RNA/DNA, and perform similarly multifunctional roles in the regulation of gene expression. Of the FET proteins, however, only EWS appears to have a distinct property in the cellular stress response. Therefore, we focused on the relationship between hyperosmotic stress response and post-translational modifications of the FET proteins. We confirmed that the hyperosmotic stress-dependent translocation from the nucleus to the cytoplasm and the cellular granule formation of FET proteins, and that EWS is less likely to partition into cellular granules in the cytoplasm than FUS or TAF15. The domain involved in the less partitioning property of EWS was found to be its low-complexity domain (LCD). Chemoenzymatic labeling analysis of O-linked ß-N-acetylglucosamine (O-GlcNAc) residues revealed that O-GlcNAc glycosylation occurs frequently in the LCD of EWS. A correlation was observed between the glycosylation of EWS and the less partitioning property under the hyperosmotic stress. These results suggest that among the FET proteins, only EWS has acquired the unique property through O-GlcNAc glycosylation. The glycosylation may play an essential role in regulating physiological functions of EWS, such as transcriptional activity, in addition to the property in cellular stress response.

Physiological levels of poly(ADP-ribose) during the cell cycle regulate HeLa cell proliferation.

Protein targets of polyADP-ribosylation undergo covalent modification with high-molecular-weight, branched poly(ADP-ribose) (PAR) of lengths up to 200 or more ADP-ribose residues derived from NAD+. PAR polymerase 1 (PARP1) is the most abundant and well-characterized enzyme involved in PAR biosynthesis. Extensive studies have been carried out to determine how polyADP-ribosylation (PARylation) regulates cell proliferation during cell cycle, with conflicting conclusions. Since significant activation of PARP1 occurs during cell lysis in vitro, we changed the standard method for cell lysis, and using our sensitive ELISA system, quantified without addition of a PAR glycohydrolase inhibitor and clarified that the PAR level is significantly higher in S phase than that in G1. Under normal condition in the absence of exogenous DNA-damaging agent, PAR turns over with a half-life of <40 s; consistent with significant decrease of NAD+ levels in S phase, which is rescued by PARP inhibitors, in line with the observed rapid turnover of PAR. PARP inhibitors delayed cell cycle in S phase and decreased cell proliferation. Our results underscore the importance of a suitable assay system to measure rapid PAR chain dynamics in living cells and aid our understanding of the function of PARylation during the cell cycle.

O-GlcNAc glycosylation stoichiometry of the FET protein family: only EWS is glycosylated with a high stoichiometry.

Of the FET (fused in sarcoma [FUS]/Ewing sarcoma protein [EWS]/TATA binding protein-associated factor 15 [TAF15]) family of heterogeneous nuclear ribonucleoprotein particle proteins, FUS and TAF15 are consistently and EWS variably found in inclusion bodies in neurodegenerative diseases such as frontotemporal lobar degeneration associated with FUS. It is speculated that dysregulation of FET proteins at the post-translational level is involved in their cytoplasmic deposition. Here, the O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation stoichiometry of the FET proteins was chemoenzymatically analyzed, and it was found that only EWS is dynamically glycosylated with a high stoichiometry in the neural cell lines tested and in mouse brain. It was also confirmed that EWS, but not FUS and TAF15, is glycosylated with a high stoichiometry not only in the neural cells but also in the non-neural cell lines tested. These results indicate that O-GlcNAc glycosylation imparts a physicochemical property on EWS that is distinct from that of the other FET proteins in most of cell lineages or tissues.

The glycosylation stoichiometry of EWS species in neuronal cells.

Although Ewing sarcoma protein (EWS) is known to be glycosylated by O-linked ß-N-acetylglucosamine (O-GlcNAc), the dynamics and stoichiometry of its glycosylation remain obscure. Here, we report a dynamic change in the glycosylation stoichiometry of EWS species during neuronal differentiation of embryonic carcinoma P19 cells. Our findings suggest that O-GlcNAc glycosylation participates in the regulation of EWS functions in neuronal cells.

Global increase in O-linked N-acetylglucosamine modification promotes osteoblast differentiation.

The balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts, and an imbalance in this bone metabolism leads to osteoporosis. Here, we found that osteoblast differentiation in MC3T3-E1 cells is promoted by the inactivation of O-linked ß-N-acetylglucosaminidase (O-GlcNAcase) and suppressed by the inactivation of O-GlcNAc transferase, as indicated by extracellular matrix calcification. The expression of osteogenic genes such as alp, ocn, and bsp during osteoblast differentiation was positively regulated in a O-GlcNAc glycosylation-dependent manner. Because it was confirmed that Ets1 and Runx2 are the two key transcription factors responsible for the expression of these osteogenic genes, their transcriptional activity might therefore be regulated by O-GlcNAc glycosylation. However, osteoclast differentiation of RAW264 cells, as indicated by the expression and activity of tartrate-resistant acid phosphatase, was unaffected by the inactivation of either O-GlcNAcase or O-GlcNAc transferase. Our findings suggest that an approach to manipulate O-GlcNAc glycosylation could be useful for developing the therapeutics for osteoporosis.

Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells.

Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughout adipogenesis. Instead, an adipogenesis-dependent increase in O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.

Pyrobacteriopheophorbide-a derivatives possessing various hydrophilic esterifying groups at the C17-propionate residues for photodynamic therapy.

Aiming at the application to photodynamic therapy, natural bacteriochlorophyll-a was converted to chemically stable free-base derivatives possessing different kinds of hydrophilic C17-propionate residues. These semi-synthetic bacteriochlorins were found to have self-assembling ability in an aqueous environment and formed stable J-type aggregates in a cell culture medium containing 0.2% DMSO. The electronic absorption spectra of all the sensitizers showed Qy absorption maxima at 754 nm in DMSO as their monomeric states, while a drastic shift of the red-most bands to ca. 880 nm was observed in the aqueous medium. The circular dichroism spectra in the medium showed much intense signals compared to those measured in DMSO, supporting the formation of well-ordered supramolecular structures. By introducing hydrophilic side chains, the bacteriochlorin sensitizers could be dispersed in the aqueous medium as their J-aggregates without the use of any surfactants. Cellular uptake efficiencies as well as photodynamic activities were evaluated using human cervical adenocarcinoma HeLa cells. Among the 11 photosensitizers investigated, the best result was obtained for a charged derivative possessing trimethylammonium terminal (17-CH2CH2COOCH2CH2N+(CH3)3I-) and photocytotoxicity of EC50 = 0.09 µM was achieved by far-red light illumination of 35 J/cm2 from an LED panel (730 nm).

Terminal differentiation program of skeletal myogenesis is negatively regulated by O-GlcNAc glycosylation.

BACKGROUND: O-Linked ß-N-acetylglucosaminylation (O-GlcNAcylation) on the Ser/Thr residue of nucleocytoplasmic proteins is a dynamic post-translational modification found in multicellular organisms. More than 500 proteins involved in a wide range of cellular functions, including cell cycle, transcription, epigenesis, and glucose sensing, are modified with O-GlcNAc. Although it has been suggested that O-GlcNAcylation is involved in the differentiation of cells in a lineage-specific manner, its role in skeletal myogenesis is unknown. METHODS AND RESULTS: A myogenesis-dependent drastic decrease in the levels of O-GlcNAcylation was found in mouse C2C12 myoblasts. The global decrease in O-GlcNAcylation was observed at the earlier stage of myogenesis, prior to myoblast fusion. Genetic or pharmacological inactivation of O-GlcNAcase blocked both the myogenesis-dependent global decrease in O-GlcNAcylation and myoblast fusion. Although inactivation of O-GlcNAcase affected neither cell-cycle exit nor cell survival in response to myogenic stimulus, it perturbed the expression of myogenic regulatory factors. While the expression of myod and myf5 in response to myogenic induction was not affected, that of myogenin and mrf4 was severely inhibited by the inactivation of O-GlcNAcase. CONCLUSION: These results indicate that the terminal differentiation program of skeletal myogenesis is negatively regulated by O-GlcNAcylation. GENERAL SIGNIFICANCE: O-GlcNAcylation is involved in differentiation in a cell lineage-dependent manner, and a decrease in O-GlcNAcylation may have a common role in the differentiation of cells of muscle lineage.

Requirement of decreased O-GlcNAc glycosylation of Mef2D for its recruitment to the myogenin promoter.

Previously, we demonstrated that the expression of myogenin, a critical transcription factor for myogenesis, is negatively regulated by O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation in mouse C2C12 cells. In this study, we found that Mef2 family proteins, especially Mef2D which is a crucial transcriptional activator of myogenin, are O-GlcNAc glycosylated. Between the two splice variants of Mef2D, Mef2D1a rather than Mef2D1b appears to drive the initiation of myogenin expression in the early stage of myogenesis. A deletion mutant analysis showed that Mef2D1a is glycosylated both in its DNA-binding and transactivation domains. A significant decrease in the glycosylation of Mef2D was observed in response to myogenic stimulus in C2C12 cells. Inhibition of the myogenesis-dependent decrease in the glycosylation of Mef2D suppressed its recruitment to the myogenin promoter. These results indicate that the expression of myogenin is regulated, at least in part, by the decreased glycosylation-dependent recruitment of Mef2D to the promoter region, and this is one of the negative regulatory mechanisms of skeletal myogenesis by O-GlcNAc glycosylation.

Regulated changes in the acetylation of α-tubulin on lys(40) during growth and organ development in fast plants, Brassica rapa L.

Recently, we confirmed the widespread occurrence of α-tubulin acetylation on Lys(40) in angiosperms. In the present study, we found that α-tubulin acetylation is regulated in a growth stage- and organ development-dependent manner in the rapid cycling Brassica rapa, also known as Fast Plants. Organ distribution analysis showed that the proportion of acetylated α-tubulin is high in the cotyledons of young plants and in the true leaves and flowers of mature plants. A correlation between the increase in the levels of α-tubulin acetylation and the maturation of true leaves was observed. In the mature leaves, the acetylated α-tubulin showed an uneven distribution pattern, and the cells in the region of the leaf margins contained a high proportion of acetylated α-tubulin. These results indicate that α-tubulin acetylation is dynamically regulated in plant organs during development, and that it might play an important role in microtubule functioning throughout the angiosperm's life cycle.

Acetylation of α-tubulin on Lys40 is a widespread post-translational modification in angiosperms.

Acetylation of α-tubulin on Lys(40) is thought to be a modification that regulates the dynamic instability of microtubules, but little is known about the occurrence of α-tubulin acetylation in plants. Here we report on a growth stage-dependent change in levels of α-tubulin acetylation and the organ distribution of acetylated α-tubulin in Arabidopsis thaliana plants. Widespread occurrence of α-tubulin acetylation in the leaves of 15 species (20 cultivars) of angiosperms was also confirmed. Our data indicate that acetylated α-tubulin is widespread in many angiosperms, but levels can differ, sometimes considerably, among different organs and developmental stages.

An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo.

PolyADP-ribosylation is a posttranslational modification of proteins that results from enzymatic synthesis of poly(ADP-ribose) with NAD+ as the substrate. A unique characteristic of polyADP-ribosylation is that the poly(ADP-ribose) chain can have 200 or more ADP-ribose residues in branched patterns, and the presence and variety of these chains can have substantive effects on protein function. To understand how polyADP-ribosylation affects biological processes, it is important to know the physiological level of poly(ADP-ribose) in cells. Under normal cell physiological conditions and in the absence of any exogenous DNA damaging agents, we found that the concentration of poly(ADP-ribose) in HeLa cells is approximately 0.04 pmol (25 pg)/106 cells, as measured with a double-antibody sandwich, enzyme-linked immunosorbent assay protocol that avoids artificial activation of PARP1 during cell lysis. Notably, this system demonstrated that the poly(ADP-ribose) level peaks in S phase and that the average cellular turnover of a single poly(ADP-ribose) is less than 40 s.

Characterization of O-GlcNAcylation in starfish (Asterina pectinifera) development from fertilization to bipinnaria larva.

Though O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins has been found in many multicellular organisms, its presence or absence in Echinodermata is unknown. Here we report the occurrence of O-GlcNAcylation in starfish (Asterina pectinifera) oocytes and the apparent O-GlcNAcylation pattern in starfish early development. O-GlcNAcylation might participate in the regulation of starfish development at the mid-blastula stage and thereafter.

Characteristic increase in nucleocytoplasmic protein glycosylation by O-GlcNAc in 3T3-L1 adipocyte differentiation.

O-Linked beta-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins is a ubiquitous post-translational modification in multicellular organisms studied so far. Since aberrant O-GlcNAcylation has a link with insulin resistance, it is important to establish the status of O-GlcNAcylation in differentiation of mesenchymal cells such as preadipocytes. In this study, we found a differentiation-dependent drastic increase in the level of O-GlcNAcylation in mouse 3T3-L1 preadipocytes. The occurrence of the increase in O-GlcNAcylation, which correlated with the expression of C/EBPalpha, was in part due to increased expression of O-GlcNAc transferase. In addition to the well-known O-GlcNAcylated proteins such as nucleoporins and vimentin, pyruvate carboxylase, long chain fatty acid-CoA ligase 1, and Ewing sarcoma protein were identified as the proteins which are heavily O-GlcNAcylated with the adipocyte differentiation. Both adipocyte differentiation and the differentiation-dependent increase in O-GlcNAcylation were blocked by 6-diazo-5-oxo-norleucine. These results suggest that O-GlcNAcylation particilates, at least in part, in adipogenesis.

Effects of downregulated HDAC6 expression on the proliferation of lung cancer cells.

Histone deacetylase 6 (HDAC6) is a multifunctional, cytosolic protein deacetylase that primarily acts on alpha-tubulin. Here we report that stable knockdown of HDAC6 expression causes a decrease in the steady-state level of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor alpha, in A549 lung cancer cells. The decreased levels of in EGFR in HDAC6-knockdown cells, which correlated with increased acetylation of microtubules, were due to increased turnover of EGFR protein. Despite the decrease in EGFR levels, A549 cells lacking functional HDAC6 appeared to grow normally, probably due to increased expression of extracellular signal-regulated kinases 1 and 2. Indeed, HDAC6-knockdown cells were more sensitive than control cells to the MEK inhibitor U0126. These results suggest that HDAC6 inhibitors combined with inhibitors of growth factor signaling may be useful as cancer therapy.

Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: a new paradigm for metabolic control of signal transduction and transcription.

The glycosylation of serine and threonine residues with beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of nuclear and cytoplasmic proteins in multicellular eukaryotes. This highly dynamic glycosylation/deglycosylation of protein is catalyzed by the nucleocytoplasmic enzymes, UDP-G1cNAc: polypeptide O-beta-N-acetylglucosaminyltransferase (OGT)/O-beta-N-acetylglucosaminidase. OGT is required for embryonic stem cell viability and mouse ontogeny, thus O-GlcNAc is essential for the life of eukaryotes. The gene encoding O-GlcNAcase maps to a locus important to late-onset Alzheimer's disease. All known O-GlcNAc-modified proteins are also phosphoproteins that form reversible multimeric protein complexes. There is both a global and often site-specific reciprocal relationship between O-GlcNAc and O-phosphate in many cellular responses to stimuli. Thus, regulation of the protein-protein interaction(s) and/or protein function by dynamic glycosylation/phosphorylation has been hypothesized. In this chapter, we will review the current status of dynamic glycosylation/phosphorylation of several important regulatory proteins including c-Myc, estrogen receptors, Sp1, endothelial nitric oxide synthase, and beta-catenin. Various aspects of subcellular localization, association with binding partners, activity, and/or turnover of these proteins appear to be regulated by dynamic glycosylation/ phosphorylation in response to cellular signals or stages.

Intracellular and extracellular O-linked N-acetylglucosamine in the nervous system.

Addition of O-linked N-acetylglucosamine (O-GlcNAc) to the hydroxyl group of serine and threonine residues (O-GlcNAcylation) is a post-translational modification common to multicellular eukaryotes. To date, O-GlcNAcylations have been divided into two categories: the first involves nucleocytoplasmic and mitochondrial (intracellular) O-GlcNAcylation catalyzed by O-GlcNAc transferase (OGT), and the second involves O-GlcNAcylation in the secretory pathways (extracellular) catalyzed by epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT). Intracellular O-GlcNAcylation is involved in essential cellular and physiological processes such as synaptic activity, neuronal morphogenesis, and learning and memory. Moreover, intracellular O-GlcNAc might have a neuroprotective effect, protecting against neurodegenerative diseases such as Alzheimer's disease. EGF repeats on extracellular matrix proteins and the extracellular region of transmembrane proteins have recently been found to be modified by O-GlcNAc in the mouse cerebral cortex. EOGT is responsible for Adams-Oliver syndrome, a rare congenital disorder characterized by aplasia cutis congenita and terminal transverse limb defects, often accompanied by cardiovascular and neurological defects. Thus, a mechanistic understanding of O-GlcNAc in the regulation of its target proteins is of importance from both a basic science and a clinical-translational perspective. In this review, we summarize the current understanding of the physiological and pathological significances of both types of O-GlcNAcylations found in the nervous system.

Analysis of new biomarkers for cholangiocarcinoma.

Cholangiocarcinoma is one of the most serious diseases in northeast Thailand, where its incidence is reported to be the highest in the world. We tried to develop a new method to detect cholangiocarcinoma in the early stages using serum proteins. We found that after fluorescent labeling of the sugar moiety of serum proteins, a new peak was identified, which might be a promising marker for cholangiocarcinoma.

Petroacetylene, a new polyacetylene from the marine sponge Petrosiasolida that inhibits blastulation of starfish embryos.

A new C30 linear polyacetylene compound designated petroacetylene (1) has been isolated from the marine sponge Petrosia solida Hoshino 1981, collected off the coast of Amami-Oshima, Kagoshima Prefecture, Japan. The structure was elucidated on the basis of spectroscopic data and chemical means. Petroacetylene (1) inhibited blastulation of starfish embryos at a concentration of 3.1 µg mL(- 1) or greater.