The ubiquitin proteasome system classically selects its substrates for degradation by tagging them with ubiquitin. Here, we describe another means of controlling proteasome function in a global manner. The 26S proteasome can be inhibited by modification with the enzyme, O-GlcNAc transferase (OGT). This reversible modification of the proteasome inhibits the proteolysis of the transcription factor Sp1 and a hydrophobic peptide through inhibition of the ATPase activity of 26S proteasomes. The Rpt2 ATPase in the mammalian proteasome 19S cap is modified by O-GlcNAc in vitro and in vivo and as its modification increases, proteasome function decreases. This mechanism may couple proteasomes to the general metabolic state of the cell. The O-GlcNAc modification of proteasomes may allow the organism to respond to its metabolic needs by controlling the availability of amino acids and regulatory proteins.

The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. In this study, we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b+ myeloid precursors in the bone marrow (BM) in vivo, and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) in vitro. Addition of tumor exosomes at day 0 led to a significant block of differentiation into DC, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited the differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after the tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of BM cells isolated from IL-6 knockout mice. Addition of a rIL-6 to the IL-6 knockout BM cell culture restored the tumor exosome-mediated inhibition of DC differentiation. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking BM DC differentiation.

, an enzyme recently shown to be necessary for cell survival (2). UDP-GlcNAc, which is synthesized de novo from glucose via the hexosamine biosynthetic pathway serves as the substrate for protein O-GlcNAcylation. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins (3, 4) as well as with transcriptional levels of some genes (5-9). However, how glucose flux through the hexosamine pathway regulates gene transcription remains elusive. Based on the observation that many transcription factors are modified by O-GlcNAc (10, 11), most often in the transcriptional activation domain (10, 11), the intriguing hypothesis has been raised that O-GlcNAcylation of transcription factors could regulate gene transcription in response to glucose flux (8-10, 12). Because O-GlcNAcylation and phosphorylation often occur reciprocally in transcription factors (1, 9, 13), it has been difficult to elucidate the direct effects of O-GlcNAc on transcriptional regulation. Our finding that a region in the activation domain of the transcription factor Sp1 is exclusively subject to O-GlcNAcylation, but not to phosphorylation, allows us to assess the functional roles of O-GlcNAc in transcription directly (12).The first transcription factor shown to bear the O-GlcNAc modification was Sp1 (10), a ubiquitous transcription factor that plays a vital role in the control of TATA-less housekeeping gene transcription (14). The N-terminal portion of the molecule contains two glutamine-rich activation domains, each associated with a serine-͞threonine-rich region, whereas the C-terminal region contains the zinc-finger DNA-binding domain (15). The second glutamine-rich activation domain has been shown to be involved both in the homomultimerization of Sp1 (16) and in the interaction with the general transcription factor (TF II D) via TATA-binding protein-associated factor II 110 (TAF II 110) (17, 18). The homomultimerization is required for synergistic activation of transcription by Sp1 (19) whereas the interaction with TF II D via TAF II 110 is required for the effect of Sp1 on DNA polymerase II-dependent transcription. These protein-protein interactions appear to be dependent on the glutamine͞ hydrophobic patches within this domain of Sp1 (18), a motif that is conserved in the activation domains of other transcription factors such as CREB and VP16 (20,21). Furthermore, we have identified a dominant O-GlcNAcylation site in the C-terminal region of this domain (12). Based on these characteristics of this glutamine-rich activation domain of Sp1, we developed a model Sp1 peptide, termed SpE, which spans this domain and in which we could control the O-GlcNAc state either by mutagenesis or by use of different expression systems. Using an in vitro pulldown assay, we showed that the interaction of SpE with either the full-length Sp1 or TAF II 110 was inhibited dramatically by O-GlcNAcylation of the SpE peptide, suggesting that the OGlcNAc residue interrupted the hydrophobic interactions between the SpE peptide and its partners...

The O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins is dynamic and abundant in the nucleus and cytosol. Several transcription factors, including Sp1, have been shown to contain this modification; however, the functional role of O-GlcNAc in these proteins has not been determined. In this paper we describe the use of the previously characterized glutamine-rich transactivation domain of Sp1 (B-c) as a model to investigate the role of O-GlcNAc in Sp1's transcriptionally relevant protein-to-protein interactions with the TATA-binding-protein-associated factor (TAF110) and holo-Sp1. When the model Sp1 peptide was overexpressed in primate cells, this 97-amino-acid domain of Sp1 was found to contain a dominant O-GlcNAc residue at high stoichiometry, which allowed the mapping and mutagenesis of this glycosylation site. In vitro interaction studies between this segment of Sp1 and Drosophila TAF110 or holo-Sp1 indicate that the O-GlcNAc modification functions to inhibit the largely hydrophobic interactions between these proteins. In HeLa cells, the mutation at the mapped glycosylation site was permissive for transcriptional activation. We propose the hypothesis that the removal of O-GlcNAc from an interaction domain can be a signal for protein association. O-GlcNAc may thereby prevent untimely and ectopic interactions.Many cytosolic and nuclear proteins (11,13,14,16,17) are covalently modified by the addition of monomeric O-linked N-acetylglucosamine (O-GlcNAc) groups. Furthermore, this modification has been found to undergo dynamic changes, often in a signal-dependent manner (21). Many proteins that form multimeric complexes have been shown to be glycosylated with O-GlcNAc (O-GlcNAcylated), and this modification has been compared to phosphorylation (13) with respect to controlling protein function. The aspects of proteins speculated to be altered by modification with O-GlcNAc (O-GlcNAcylation) include protein-protein interactions (13,14,17), protein stability (12, 29), and subcellular localization (29). The observation that many transcription factors are O-GlcNAcylated suggested that this modification may play a role in the control of transcription (19). To date, there has not been any direct evidence to implicate O-GlcNAcylation in any of these roles.We chose Sp1 as a model to better define the role of OGlcNAcylation. Sp1 is an ubiquitous transcription factor that plays a particularly vital role in the regulation of transcription from TATA-less promoters that commonly encode housekeeping genes (25). It is a well-characterized protein composed of 778 amino acids. The amino-terminal portion of the molecule contains two glutamine-rich domains, each of which is associated with serine-threonine-rich regions (20). These domains are involved in transcriptional activation. The carboxy-terminal region of the molecule contains the zinc-finger DNA-recognition domain. Sp1 is known to be phosphorylated upon binding DNA (18), and each molecule of Sp1 is thought to bear at least nine O-GlcNAc residues (19). More-detaile...

In common with many other cell types, synovial fibroblasts produce exosomes. In this study, we show that the exosomes produced by synovial fibroblasts obtained from individuals with rheumatoid arthritis (RASF), but not exosomes produced by synovial fibroblasts obtained from individuals with osteoarthritis, contain a membrane bound form of TNF-α as demonstrated by colloidal gold immunostaining of TNF-α and confirmed by both Western blot and mass spectrometry. The RASF-derived exosomes, but not exosomes derived from fibroblasts obtained from individuals with osteoarthritis, are cytotoxic for the L929 cell, a TNF-α-sensitive cell line, and stimulate activation of NF-κB and induction of collagenase-1 in RASF. These effects are blocked by addition of soluble TNFR1 (sTNFbp), suggesting that a TNF-α-signaling pathway mediates these biological activities. sTNFbp also reduced the production of exosomes by RASF, suggesting the interruption of a positive amplification loop. Exosomes can transmit signals between cells, and RASF exosomes, effectively taken up by anti-CD3-activated T cells, activated AKT and NF-κB and rendered these activated T cells resistant to apoptosis. Neutralization of exosomal membrane TNF-α by sTNFbp partially reversed this resistance, suggesting that not only TNF-α but also additional exosomal proteins may contribute to the development of apoptosis resistance.