Uncharged Reactivators of cholinesterases Inhibited by organophosphorus nerve agents

International audienc

Design and Development of New Uncharged Reactivators of OP-Inhibited Cholinesterases

International audienc

Uncharged Reactivators of cholinesterases Inhibited by organophosphorus nerve agents

International audienc

Extracellular endosulfatase Sulf-2 harbours a chondroitin/dermatan sulfate chain that modulates its enzyme activity

Sulfs represent a class of unconventional sulfatases, which differ from all other members of the sulfatase family by their structures, catalytic features and biological functions. Through their specific endosulfatase activity in extracellular milieu, Sulfs provide an original post-synthetic regulatory mechanism for heparan sulfate complex polysaccharides and have been involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) features a unique polysaccharide post-translational modification. We identified a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate binding domain. We found that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo using a mouse model of tumorigenesis and metastasis. In addition, we showed that mammalian hyaluronidase acted as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a unique proteoglycan enzyme and its newly-identified GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute in clarifying the conflicting data on the activities of the Sulfs and introduce a new paradigm into the study of these enzymes

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity

International audienceSulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human SuIf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs

Evidence for Post-Translational Processing of Vascular Endothelial (VE)-Cadherin in Brain Tumors: Towards a Candidate Biomarker

<div><p>Vessel abnormalities are among the most important features in malignant glioma. Vascular endothelial (VE)-cadherin is of major importance for vascular integrity. Upon cytokine challenge, VE-cadherin structural modifications have been described including tyrosine phosphorylation and cleavage. The goal of this study was to examine whether these events occurred in human glioma vessels. We demonstrated that VE-cadherin is highly expressed in human glioma tissue and tyrosine phosphorylated at site Y⁶⁸⁵, a site previously found phosphorylated upon VEGF challenge, via Src activation. <i>In vitro</i> experiments showed that VEGF-induced VE-cadherin phosphorylation, preceded the cleavage of its extracellular adhesive domain (sVE, 90 kDa). Interestingly, metalloproteases (MMPs) secreted by glioma cell lines were responsible for sVE release. Because VEGF and MMPs are important components of tumor microenvironment, we hypothesized that VE-cadherin proteolysis might occur in human brain tumors. Analysis of glioma patient sera prior treatment confirmed the presence of sVE in bloodstream. Furthermore, sVE levels studied in a cohort of 53 glioma patients were significantly predictive of the overall survival at three years (HR 0.13 [0.04; 0.40] p≤0.001), irrespective to histopathological grade of tumors. Altogether, these results suggest that VE-cadherin structural modifications should be examined as candidate biomarkers of tumor vessel abnormalities, with promising applications in oncology. </p> </div

Illustrative cases.

<p>All MRI examinations were performed using a 1.5 Tesla scanner (Philips Medical System). Standard MRI work-up systematically comprised at least one series of T2-weighted images (turbo spin echo, repetition time msec (RT)/echo time msec (ET=0.625/120; numbers of signals averaged ((NAS)=2; turbo factor=15) and T1-weigthed images (spin echo, RT/ET= 500/10; NAS=2) obtained prior to and after gadolinium injection. Representative contrast-enhanced images from low (a,b,c) and high (d,e,f) levels of sVE patients. (A,B,C) 36 years old man, oligodendroglioma grade III, in the left posterior temporal region (6 cm major axis): (A) Sagittal T1-weighted image after gadolinium injection shows diffuse and extensive contrast enhancement, (B) axial T2-weighted image shows heterogeneous aspect and few perilesional edema of the same lesion with (C) T2/fluid attenuated inversion recovery (FLAIR) shows infiltrative lesion with mass effect on ventricular junction. On all panels, the tumor area is indicated using a dotted white line. For this patient, sVE=296 ng/mL and overall survival was 12 months. (D,E,F) 60 years old women, glioblastoma in the left parietal region (3.5 cm major axis): (D) Sagittal T1-weighted image after gadolinium injection shows a ring of contrast enhancement around an area of hypointensity (necrosis). (E) axial T2-weighted image (T2) shows irregular contours and significative perilesional edema. (F) T2/fluid attenuated inversion recovery (FLAIR) shows few mass effect. For this patient, sVE=1.843 µg/mL and overall survival was 36 months.</p

Metalloproteinases are secreted by glioma cell line and induced VE-cadherin cleavage.

<p>(A) Conditioned media from Astrocytoma grade IV (LN229, LN) and Astrocytoma grade III (U87, U) cells lines were tested for protease activities using a zymography assay. Inhibition of protease activities by EDTA identified MMPs. (B) U87 (U) cell line media induced VE-cadherin cleavage from HUVECs (90 kDa fragment). Glioma cell line conditioned media was added to HUVEC confluent monolayer during two hours and HUVEC (H) conditioned media was analyzed for sVE content by western blot. The effect was impaired by broad spectrum MMPs inhibitor (GM6001, I). (C) Western blot analysis of glioma patient sera at dilution 1:50, 1:100, 1:500 revealed the presence of the 90 kDa fragment of VE-cadherin (sVE). (D) Deglycosylation Assay of sVE in serum shows, using two different antibodies to sVE that the soluble fragment is glycosylated. </p

VEGF induced-VE-cadherin extracellular domain cleavage is preceded by a Src-dependent VE-cadherin tyrosine phosphorylation.

<p>(A,B) HUVECs treated with VEGF (50 ng/mL) were analyzed for phosphotyrosinated-VE-cadherin in cell extracts (A) and VE-cadherin extracellular domain in conditioned medium (B): (A) VE-cadherin was immunoprecipitated from 200 µg of protein lysates and analyzed by SDS-PAGE and western blotting with the anti-phosphotyrosine antibody. VEGF induced a time-dependent tyrosine phosphorylation of VE-cadherin detectable after 2 min of stimulation. (B) Conditioned media from VEGF-stimulated HUVECs were concentrated and analyzed by SDS-PAGE and western blotting with human VE-cadherin antibody directed against VE-cadherin extracellular domain (BV9). A 90 kDa fragment corresponding to the full length VE-cadherin extracellular domain was already detectable after 10 min of VEGF stimulation. (C) HUVECs were pretreated with increasing concentrations of Src inhibitor PP2 (2.5 to 20 µM) for 15 min, prior to treatment with VEGF for 15 min. VE-cadherin was immunoprecipitated from 200 µg of protein lysates. PP2 concentrations higher than 2.5 µM completely inhibited VEGF-induced VE-cadherin tyrosine phosphorylation. (D) Analysis of conditioned media from an identical number of HUVECs pre-treated for 15 min with PP2 (5 µM) before VEGF stimulation for 15 min: the inhibitor decreased VEGF-induced VE-cadherin cleavage. (E,F) Src expression was inhibited by Src-siRNA in HUVECs 24 hours before VEGF stimulation (15min). Analysis of conditioned media showed that the knock-down of Src (controlled in F) decreased the level of soluble VE-cadherin in the media. (D,E,F) The signals were quantified using ImageJ software, error bars in graphs indicate S.D. and experiments were repeated at least three times in a similar configuration.</p

VE-cadherin expression and phosphorylation in human glioma tissues.

<p>(A,B) Representative (n=10 samples) micrographs of primary human glioma stained for VE-cadherin (in red; nuclei are stained in blue). Images were processed using Adobe Photoshop Scale bar: 25 μm. A capillary network positive for VE-cadherin was detected in all the tumors. (C) A 125 kDa fragment of VE-cadherin was highly detectable in glioblastoma (GBM) extract and not in non-tumor brain tissue (N). (D) Protein lysates from N and GBM were analyzed by SDS-PAGE and western blotting with the antiphophotyrosine antibody (clone 4G10). Several proteins with apparent molecular masses (indicated by filled arrowheads) ranging from 220 to 25 kDa displayed clearly strong tyrosine phosphorylation in GBM but not in non-tumor tissue. (E) 500 μg of GBM tissue lysate protein were immunoprecipitated with an anti-human VE-Cadherin antibody directed to C-term of the protein and blotted with the indicated antibodies (Ptyr or VE-cad). Immunoprecipitation of VE-cadherin from glioma extracts allowed to detect a tyrosine phosphorylated form of VE-cadherin. Sample loading was controlled using actin detection. (F) Active Src (phosphoY418) was highly detectable in GBM but not in non-tumor (N) brain extract. (G) Orthovanadate treated-HUVECs lysates (control: CTL) and GBM extracts (50 µg) were analyzed by western blot using antiphosphosite antibodies directed against Y⁶⁵⁸ and Y⁷³¹, and the antibody directed against pY⁶⁸⁵ VE-cadherin raised in our laboratories. Only pY⁶⁸⁵ was detected in GBM as in HUVECs upon VEGF stimulation (50 ng/mL). (H) Same experiment as described in (E), and immunoblotting with anti-Csk antibody and anti-pY⁶⁸⁵ VE-cadherin antibody. The association of Csk with VE-cadherin in GBM confirmed the phosphorylation at the site Y⁶⁸⁵ also detected with the antiphosphosite antibody. Filled arrowhead indicates the position of the IgG heavy chains of the crosslinking antibody. In all blots the position of size markers (in kDa) is indicated on the left. These experiments were repeated at least three times in a similar configuration.</p