O-GlcNAc transferase associates with the MCM2-7 complex and its silencing destabilizes MCM-MCM interactions

International audienceO-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the Mini-Chromosome Maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2-7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2-7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilize MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2-7 complex and O-GlcNAcylation homeostasis might regulate MCM2-7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions

Exploring the Potential of β-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging

International audienceMonitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its O-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of β-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of β-catenin in HeLa cells. The changes in O-GlcNAcylation of β-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation

Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin

<div><p>Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the <i>O</i>-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability.</p></div

SUMO Interacting Motifs in human ΔLf and Lf from different species compared to the SIMr consensus (<i>D/E</i>)₃ V/C L/V I/V V—<i>E</i> [37].

<p>^aThe single-letter amino acid code is used; bold letters indicate the hydrophobic positions of the putative SUMO interacting motif, the acidic cluster is in italics, the SIM-associated serine residues are underlined.</p><p>^bThe numbering of the amino acid residues corresponds to human ΔLf.</p><p>^cThe numbering of the amino acid residues corresponds to Lfs.</p><p>SUMO Interacting Motifs in human ΔLf and Lf from different species compared to the SIMr consensus (<i>D/E</i>)₃ V/C L/V I/V V—<i>E</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129965#pone.0129965.ref037" target="_blank">37</a>].</p

SUMO predictive motifs in human ΔLf.

<p>^aSUMOsp (<a href="http://sumosp.biocuckoo.org/" target="_blank">http://sumosp.biocuckoo.org/</a>) and SUMOplot (<a href="http://www.abgent.com/tools/sumoplot/" target="_blank">htpp://www.abgent.com/tools/sumoplot/</a>) softwares were used.</p><p>^bThe single-letter amino acid code is used.</p><p>^cThe numbering of the amino acid residues corresponds to human ΔLf.</p><p>SUMO predictive motifs in human ΔLf.</p

ΔLf is modified by SUMOylation.

<p>A) Schematic overview of ΔLf showing the NLS and PEST sequences, the two putative DBD and the putative SIM domain. The amino acid residues targeted by post-translational modifications are shown, S10 as the main <i>O</i>-GlcNAc/P site, K379 and K391 as the two ubiquitinated lysines, K13 as a putative acetylation site. B) Mutation of K13, K308, K361 and K391 individual lysine residues did not abolish ΔLf SUMOylation. The first series of ΔLf mutant constructs (ΔLf^K13R, ΔLf^K308R, ΔLf^K361R, ΔLf^K391R and the M4S mutant constructs) were co-transfected with the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His antibodies and M2. The data presented correspond to one representative experiment of two conducted (n = 2). C) Expression of pCMV-3xFLAG-ΔLf^WT (WT) and the second series of SUMOylation mutant constructs. WT and the above constructs were transfected for 24 h prior to lysis. Whole cell extract was immunoblotted with either anti-FLAG M2 or anti-GAPDH antibodies. The data presented correspond to one representative experiment of at least seven conducted (n ≥ 7). NV: null vector (pCMV-3xFLAG). The level of expression of each mutant compared to WT is shown in the bar graph beneath the figure (n ≥ 7). D) ΔLf is SUMOylated and M5S is not. WT and the M5S mutant construct were co-transfected with or without the pSG5-His-SUMO-1 (His-SUMO-1) plasmid in HEK-293 cells for 24 h prior to lysis. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 antibodies and M2. Asterisks correspond to SUMO bands (mono-SUMO, 86 kDa; multi-SUMO, 97, 108, 119 kDa). Lysates from HEK-293 cells transfected with a null vector (NV) and from non-transfected (NT) cells were used as negative controls. The data presented correspond to one representative experiment of at least three conducted (n ≥ 3).</p

Competition between SUMOylation and ubiquitination at K379 controls ΔLf turnover.

<p>A) HEK-293 cells were co-transfected with K379 or NV constructs, His-SUMO-1 or/and HA-Ub-expression vectors for 24 h and then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. Total cell extracts were immunoprecipitated with M2 or used as input. Samples were immunoblotted with anti-HA (upper panel) or with anti-SUMO-1 (lower panel) antibodies. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control. NS: non-specific. The data presented correspond to one representative experiment of at least three conducted (n ≥ 3). Lane 6 corresponds to non-transfected cells. B) Cells were transfected with K379, either with the His-SUMO-1 or the HA-Ub expression vector and then incubated with fresh medium supplemented by 10 μg.mL^-1 CHX for the indicated time 24 h after transfection. K379 transfected cells were incubated without (left panel) or with (right panel) 10 μM MG132 for 2 h prior to lysis. Total protein extracts were immunoblotted with either M2 or anti-GAPDH antibodies. Detection was carried out using a Fusion SOLO camera (Vilbert Lourmat). The data presented (B) correspond to one representative experiment of at least five conducted. C-D) The M2 densitometric analyses are normalized for the matching GAPDH immunoblots and expressed as ratio D^K379/D^GAPDH as described in Materials and Methods. Data are shown as the means ± SD (n = 5).</p

SUMOylation of ΔLf represses its transcriptional activity.

<p>A) Cells were co-transfected with pGL3-S1^Skp1-Luc reporter vector and WT, SUMO mutant constructs or null vector in order to assay the relative transcriptional activity of WT and its SUMO mutants. Relative luciferase activities are expressed as described in Materials and Methods (n≥5; p < 0.05 (*)). B-E) Alteration of SUMOylation at K308 modulates ΔLf transcriptional activity. B) Knockdown of Ubc9 was performed using siUbc9/siCtrl as described in Materials and Methods and followed after 48 h of incubation by immunoblotting of the cell extracts with either anti-Ubc9 or anti-GAPDH antibodies. C) Knockdown of Ubc9 leads to a decrease in SUMOylation. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 or M2. Input was immunoblotted with M2, anti-SUMO-1 or anti-GAPDH antibodies and used as controls. D) Deconjugation of SUMO-1 from WT, K13 and K308 by SENP2. HEK-293 cells were co-transfected with WT or the K308 construct together with pSG5-His-SUMO-1 and pcDNA-SENP2-SV5 and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies. C-D) Cells were incubated with MG132 for 2 h prior to lysis and NEM added to lysis, IP and WB buffers. The data presented correspond to one representative experiment of at least six conducted (n ≥ 6) (B) and to one representative experiment of at least two conducted (n ≥ 2) (C, D). E) Cells were co-transfected with pGL3-S1^Skp1-Luc reporter vector, either WT or the K308 construct together with pSG5-His-SUMO-1 or pcDNA-SENP2-SV5. Relative luciferase activities were also assayed in Ubc9 invalidated cells. HEK-293 cells were reverse transfected for 24 h using siRNAs targeting Ubc9 (siUbc9) or a scrambled control sequence (siCtrl) before being transfected as described above to evaluate the relative transcriptional activities of ΔLf and the K308 mutant. Relative luciferase activities are expressed as described in Materials and Methods (n≥5; p < 0.05 (*), p < 0.01 (**)).</p