Evidence for splice transcript variants of TMEM165, a gene involved in CDG.

BACKGROUND: Defects in TMEM165 gene cause a type-II Congenital Disorder of Glycosylation affecting Golgi glycosylation processes. TMEM165 patients exhibit psychomotor retardation, important osteoporosis, scoliosis, irregular epiphyses and thin bone cortex. TMEM165 protein is highly conserved in evolution and belongs to the family of UPF0016 membrane proteins which could be an unique group of Ca2+/H+ antiporters regulating Ca2+ and pH homeostasis and mainly localized in the Golgi apparatus. METHODS: RT-PCR from human brain tissues revealed TMEM165 splice-transcript variants. mRNA expression was analyzed by RT-Q-PCR. Expression plasmids allowed us to visualize isoform proteins and their subcellular localization. Their functions on glycosylation were achieved by looking at the gel mobility of highly glycosylated proteins in cells overexpressing isoforms. RESULTS: In this study, we highlight, as previously shown for other ion channels, the existence of TMEM165 splice-transcripts isoforms, in particular the Short-Form (SF) and the Long-Form (LF) transcripts, leading to a 129 aa and 259 aa protein isoform, respectively. These proteins both localize in the endoplasmic reticulum and have different effects on glycosylation compared to the wild-type protein (324 aa). We also point out that the SF is expressed at low levels in all human cells and tissues checked, excepted in brain, and forms homodimer. The LF was only expressed in the temporal lobe of human brain. GENERAL SIGNIFICANCE: The finding of numerous splice variants could lead to a family of TMEM165 isoforms. This family of TMEM165 splice transcripts could participate in the fine regulation of TMEM165 isoforms' functions and localizations.

Glucokinase expression is regulated by glucose through O-GlcNAc glycosylation.

Blood glucose fluctuates with the fasting-feeding cycle. One of the liver's functions is to maintain blood glucose concentrations within a physiological range. Glucokinase (GCK) or hexokinase IV, is the main enzyme that regulates the flux and the use of glucose in the liver leading to a compensation of hyperglycemia. In hepatocytes, GCK catalyzes the phosphorylation of glucose into glucose-6-phosphate. This critical enzymatic reaction is determinant for the metabolism of glucose in the liver which includes glycogen synthesis, glycolysis, lipogenesis and gluconeogenesis. In liver, simultaneous increase of glucose and insulin enhances GCK activity and gene expression, changes its subcellular location and interaction with regulatory proteins. The post-translational O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) acts as a glucose-sensitive modification and is believed to take part in hepatic glucose sensing by modifying key regulatory proteins. Therefore, we aimed to determine whether GCK is modified by O-GlcNAcylation in the liver of mice and investigated the role that this modification plays in regulating GCK protein expression. We demonstrated that endogenous GCK expression correlated with O-GlcNAc levels in the pathophysiological model ob/ob mice. More specifically, in response to the pharmacological inhibition of O-GlcNAcase (OGA) contents of GCK increased. Using the GlcNAc specific lectin succinylated-WGA and click chemistry labeling approaches, we demonstrated that GCK is modified by O-GlcNAcylation. Further, we demonstrated that siRNA-mediated Ogt knock-down not only decreases O-GlcNAc content but also GCK protein level. Altogether, our in vivo and in vitro results demonstrate that GCK expression is regulated by nutrient-sensing O-GlcNAc cycling in liver.

O-GlcNAcylation stabilizes ß-catenin through direct competition with phosphorylation at threonine 41.

Dysfunctions in Wnt signaling increase ß-catenin stability and are associated with cancers, including colorectal cancer. In addition, ß-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of ß-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of ß-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of ß-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of ß-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for ß-catenin stability. Analyses of ß-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the ß-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the ß-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of ß-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of ß-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.

The hexosamine biosynthetic pathway and O-GlcNAcylation drive the expression of ß-catenin and cell proliferation.

The short half-life protooncogene ß-catenin acquires a remarkable stability in a large subset of cancers, mainly from mutations affecting its proteasomal degradation. In this sense, colorectal cancers (CRC) form a group of pathologies in which early steps of development are characterized by an aberrant expression of ß-catenin and an uncontrolled proliferation of epithelial cells. Diet has long been described as an influence in the emergence of CRC, but the molecular events that link metabolic disorders and CRC remain elusive. Part of the explanation may reside in hexosamine biosynthetic pathway (HBP) flux. We found that fasted mice being force-fed with glucose or glucosamine leads to an increase of ß-catenin and O-GlcNAcylation levels in the colon. MCF7 cells possessing intact Wnt/ß-catenin signaling heavily expressed ß-catenin when cultured in high glucose; this was reversed by the HBP inhibitor azaserine. HBP inhibition also decreased the expression of ß-catenin in HT29 and, to a lesser extent, HCT116 cells. The same observation was made with regard to the transcriptional activity of ß-catenin in HEK293 cells. Inhibition of HBP also blocked the glucose-mediated proliferation capacity of MCF7 cells, demonstrating that glucose affects both ß-catenin expression and cell proliferation through the HBP. The ultimate element conducting these events is the dynamic posttranslational modification O-GlcNAcylation, which is intimately linked to HBP; the modulation of its level affected the expression of ß-catenin and cell proliferation. In accordance with our findings, we propose that metabolic disorders correlate to CRC via an upregulation of HBP that reverberates on high O-GlcNAcylation levels including modification of ß-catenin.

[Signaling and metabolic predispositions linked to the colorectal cancer]. / Signalisation et prédispositions métaboliques liées au cancer colorectal.

The setting up and the progression of the colorectal cancer (CCR) follow a sequence of events that are spatio-temporally rigorously orchestrated. The failures that specifically target the signaling pathways responsible for the cancerization of the colorectal mucosa have been well described and among these it seems that a dysregulation of the Wnt/ß-catenin pathway is involved in the triggering of near 90 % of the cases. It has been also described that several risk factors linked to metabolic disorders (feeding, insulin resistance, metabolic syndrome, etc.) predispose individuals to CCR but no rational explanations were given. We propose that, since it is implicated in the control of the insulin pathway among other actions, the nutritional sensor O-GlcNAcylation may be the element linking these metabolic disorders to CCR.

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of {beta}1,4-galactosyltransferase 1 and {alpha}2,6-sialyltransferase 1.

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged ß1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.

Dysregulation of the nutrient/stress sensor O-GlcNAcylation is involved in the etiology of cardiovascular disorders, type-2 diabetes and Alzheimer's disease.

O-GlcNAcylation is widespread within the cytosolic and nuclear compartments of cells. This post-translational modification is likely an indicator of good health since its intracellular level correlates with the availability of extracellular glucose. Apart from its status as a nutrient sensor, O-GlcNAcylation may also act as a stress sensor since it exerts its fundamental effects in response to stress. Several studies report that the cell quickly responds to an insult by elevating O-GlcNAcylation levels and by unmasking a newly described Hsp70-GlcNAc binding property. From a more practical point of view, it has been shown that O-GlcNAcylation impairments contribute to the etiology of cardiovascular diseases, type-2 diabetes and Alzheimer's disease (AD), three illnesses common in occidental societies. Many studies have demonstrated that O-GlcNAcylation operates as a powerful cardioprotector and that by raising O-GlcNAcylation levels, the organism more successfully resists trauma-hemorrhage and ischemia/reperfusion injury. Recent data have also shown that insulin resistance and, more broadly, type-2 diabetes can be controlled by O-GlcNAcylation of the insulin pathway and O-GlcNAcylation of the gluconeogenesis transcription factors FoxO1 and CRCT2. Lastly, the finding that AD may correspond to a type-3 diabetes offers new perspectives into the knowledge of the neuropathology and into the search for new therapeutic avenues.