Sumoylation regulates FMRP-mediated dendritic spine elimination and maturation.

Fragile X syndrome (FXS) is the most frequent inherited cause of intellectual disability and the best-studied monogenic cause of autism. FXS results from the functional absence of the fragile X mental retardation protein (FMRP) leading to abnormal pruning and consequently to synaptic communication defects. Here we show that FMRP is a substrate of the small ubiquitin-like modifier (SUMO) pathway in the brain and identify its active SUMO sites. We unravel the functional consequences of FMRP sumoylation in neurons by combining molecular replacement strategy, biochemical reconstitution assays with advanced live-cell imaging. We first demonstrate that FMRP sumoylation is promoted by activation of metabotropic glutamate receptors. We then show that this increase in sumoylation controls the homomerization of FMRP within dendritic mRNA granules which, in turn, regulates spine elimination and maturation. Altogether, our findings reveal the sumoylation of FMRP as a critical activity-dependent regulatory mechanism of FMRP-mediated neuronal function.

mGlu5 receptors regulate synaptic sumoylation via a transient PKC-dependent diffusional trapping of Ubc9 into spines.

Sumoylation plays important roles in the modulation of protein function, neurotransmission and plasticity, but the mechanisms regulating this post-translational system in neurons remain largely unknown. Here we demonstrate that the synaptic diffusion of Ubc9, the sole conjugating enzyme of the sumoylation pathway, is regulated by synaptic activity. We use restricted photobleaching/photoconversion of individual hippocampal spines to measure the diffusion properties of Ubc9 and show that it is regulated through an mGlu5R-dependent signalling pathway. Increasing synaptic activity with a GABAA receptor antagonist or directly activating mGlu5R increases the synaptic residency time of Ubc9 via a Gαq/PLC/Ca(2+)/PKC cascade. This activation promotes a transient synaptic trapping of Ubc9 through a PKC phosphorylation-dependent increase of Ubc9 recognition to phosphorylated substrates and consequently leads to the modulation of synaptic sumoylation. Our data demonstrate that Ubc9 diffusion is subject to activity-dependent regulatory processes and provide a mechanism for the dynamic changes in sumoylation occurring during synaptic transmission.

Activity-dependent regulation of the sumoylation machinery in rat hippocampal neurons.

BACKGROUND INFORMATION: Sumoylation is a key post-translational modification by which the Small Ubiquitin-like MOdifier (SUMO) polypeptide is covalently attached to specific lysine residues of substrate proteins through a specific enzymatic pathway. Although sumoylation participates in the regulation of nuclear homeostasis, the sumoylation machinery is also expressed outside of the nucleus where little is still known regarding its non-nuclear functions, particularly in the Central Nervous System (CNS). We recently reported that the sumoylation process is developmentally regulated in the rat CNS. RESULTS: Here, we demonstrate that there is an activity-dependent redistribution of endogenous sumoylation enzymes in hippocampal neurons. By performing biochemical and immunocytochemical experiments on primary cultures of rat hippocampal neurons, we show that sumoylation and desumoylation enzymes are differentially redistributed in and out of synapses upon neuronal stimulation. This enzymatic redistribution in response to a neuronal depolarisation results in the transient decrease of sumoylated protein substrates at synapses. CONCLUSIONS: Taken together, our data identify an activity-dependent regulation of the sumoylation machinery in neurons that directly impacts on synaptic sumoylation levels. This process may provide a mechanism for neurons to adapt their physiological responses to changes occurring during neuronal activation.

Developmental regulation and spatiotemporal redistribution of the sumoylation machinery in the rat central nervous system.

BACKGROUND: Small Ubiquitin-like MOdifier protein (SUMO) is a key regulator of nuclear functions but little is known regarding the role of the post-translational modification sumoylation outside of the nucleus, particularly in the Central Nervous System (CNS). METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the expression levels of SUMO-modified substrates as well as the components of the sumoylation machinery are temporally and spatially regulated in the developing rat brain. Interestingly, while the overall sumoylation is decreasing during brain development, there are progressively more SUMO substrates localized at synapses. This increase is correlated with a differential redistribution of the sumoylation machinery into dendritic spines during neuronal maturation. CONCLUSIONS/SIGNIFICANCE: Overall, our data clearly demonstrate that the sumoylation process is developmentally regulated in the brain with high levels of nuclear sumoylation early in the development suggesting a role for this post-translational modification during the synaptogenesis period and a redistribution of the SUMO system towards dendritic spines at a later developmental stage to modulate synaptic protein function.

Sig1R protein regulates hERG channel expression through a post-translational mechanism in leukemic cells.

Sig1R (Sigma-1receptor) is a 25-kDa protein structurally unrelated to other mammalian proteins. Sig1R is present in brain, liver, and heart and is overexpressed in cancer cells. Studies using exogenous sigma ligands have shown that Sig1R interacts with a variety of ion channels, but its intrinsic function and mechanism of action remain unclear. The human ether-à-gogo related gene (hERG) encodes a cardiac channel that is also abnormally expressed in many primary human cancers, potentiating tumor progression through the modulation of extracellular matrix adhesive interactions. We show herein that sigma ligands inhibit hERG current density and cell adhesion to fibronectin in K562 myeloid leukemia cells. Heterologous expression in Xenopus oocytes demonstrates that Sig1R potentiates hERG current by stimulating channel subunit biosynthesis. Silencing Sig1R in leukemic K562 cells depresses hERG current density and cell adhesion to fibronectin by reducing hERG membrane expression. In K562 cells, Sig1R silencing does not modify hERG mRNA contents but reduces hERG mature form densities. In HEK cells expressing hERG and Sig1R, both proteins co-immunoprecipitate, demonstrating a physical association. Finally, Sig1R expression enhances both channel protein maturation and stability. Altogether, these results demonstrate for the first time that Sig1R controls ion channel expression through the regulation of subunit trafficking activity.

SLC26A9 stimulates CFTR expression and function in human bronchial cell lines.

We investigated the possible functional- and physical protein-interactions between two airway Cl(-) channels, SLC26A9 and CFTR. Bronchial CFBE41o- cell lines expressing CFTR(WT) or CFTR(ΔF508) were transduced with SLC26A9. Immunoblots identified a migrating band corresponding to SLC26A9 present in whole-cell lysates as on apical membrane of cells grown on polarized filters. CFTR levels were increased by the presence of SLC26A9 in both CFTR(WT) and CFTR(ΔF508) cell lines. In CFBE41o- cells and CFBE41o-/CFTR(WT) cells transduced with SLC26A9, currents associated to the protein expression were not detected. However, the forskolin (FK)-stimulated currents were enhanced in SLC26A9-transduced cells compared to control cells. Therefore, the presence of SLC26A9 resulted in an increase in CFTR activity (same % of CFTR((inh)-172) or GlyH-101 inhibition in both groups). In CFBE41o-/CFTR(ΔF508) cells transduced with SLC26A9 (at 27°C), a current associated to the protein expression was also lacking. FK-stimulated currents and level of CFTR((inh)-172) inhibition were not different in both groups. The presence of SLC26A9 in Xenopus oocytes expressing CFTR also enhanced the FK-stimulated currents as compared to oocytes expressing CFTR alone. This stimulation was mostly linked to CFTR. An enhancement of FK-stimulated currents was not found in oocytes co-expressing SLC26A9 and CFTR(ΔF508). In conclusion, in both protein expression systems used, SLC26A9 stimulates CFTR activity but not that of CFTR(ΔF508). Our co-immunoprecipitation studies demonstrate a physical interaction between both anion channels. We propose as an alternative hypothesis (not exclusive) to the known SLC26A9-STAS domain/CFTR interaction, that SLC26A9 favors the biogenesis and/or stabilization of CFTR, leading to stimulated currents.

Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates.

BACKGROUND: Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes. RESULTS: Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries), providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation. CONCLUSION: For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis.

Characterization of SLC26A9, facilitation of Cl(-) transport by bicarbonate.

SLC26 family members are anionic transporters involved in Cl(-) and HCO(3)(-) absorption or secretion in epithelia. SLC26A9, preferentially expressed in the lung, is a poorly characterized member of this family. In this study, we investigated the transport properties of human SLC26A9 to determine its functional and pharmacological characteristics. SLC26A9 protein expression results in the appearance of an anionic current exhibiting an apparently linear current/voltage relationship and increases in (36)Cl influxes and effluxes. The sequences of conductivity, Cl(-) >I(-) > NO(3)(-) >/= gluconate > SO(4) (2-) and selectivity (P(x)/P(CI)), I(-) > NO(3)(-) > Cl(-) > gluconate > SO(4)(2-) are found. Cl(-) channel inhibitors DIDS and NS 3623 inhibit SLC26A9 associated currents while the specific CFTR inhibitor (CFTR(inh)-172) or glybenclamide has little effect. Elevation of intracellular cAMP (a CFTR activator) is also ineffective whereas increasing intracellular calcium blocks the SLC26A9 associated currents. The HCO(3)(-) conductance mediated by the SLC26A9 protein expression is low and no intracellular pHi changes are detectable under conditions favoring a Cl(-)/HCO(3)(-) exchange. However, the presence of HCO(3)(-)/CO(2) stimulates the Cl(-)-transporting activity of SLC26A9 in Xenopus laevis oocytes or SLC26A9-transduced COS-7 cells. As an important initial step in characterizing SLC26A9 function, we conclude that SLC26A9 is a Cl(-) channel and we suggest that HCO(3)(-) acts as a modulator of the channel. SLC26A9 physiological role in airway epithelia and its potential interaction with CFTR remain to be elucidated.

The two N-glycans present on bovine Pofut1 are differently involved in its solubility and activity.

O-Fucosylation is a post-translational glycosylation in which an O-fucose is covalently attached to the hydroxyl group of a specific serine or threonine residue. This modification occurs within the consensus sequence C2X(4-5)(S/T)C3 present on epidermal growth factor-like repeats of several proteins, including the Notch receptors and their ligands. The enzyme responsible for the addition of O-fucose to epidermal growth factor-like repeats is protein O-fucosyltransferase 1. Protein O-fucosyltransferase 1-mediated O-fucosylation is essential in Notch signaling, folding and targeting to the cell surface. Here, we studied the expression pattern of protein O-fucosyltransferase 1 in cattle and showed that the active enzyme is present in all tissues examined from embryo and adult as a glycoprotein with two N-glycans. By comparing protein O-fucosyltransferase 1 sequences available in databases, we observed that mammalian protein O-fucosyltransferase 1 enzymes possess two putative N-glycosylation sites, and that only the first is conserved among bilaterians. To gain more insight regarding the significance of N-glycans on protein O-fucosyltransferase 1, we substituted, by site-directed mutagenesis, bovine protein O-fucosyltransferase 1 N65, N163 or both, with L or Q. We demonstrated that the loss of N-glycan on N163 caused a slight decrease in protein O-fucosyltransferase 1 activity. In contrast, glycosylation of N65 was crucial for protein O-fucosyltransferase 1 functionality. Loss of glycosylation at N65 resulted in aggregation of protein O-fucosyltransferase 1, suggesting that N-glycosylation at this site is essential for proper folding of the enzyme.