CRB1-associated retinal dystrophies.To analyze the clinical characteristics, natural history, and genetics of Multicenter international retrospective cohort study.CRB1 variants. Macular optical coherence tomography (OCT) parameters, visual function, fundus characteristics, and associations between variables were the main outcome measures.Review of clinical notes, ophthalmic images, and genetic testing results of 104 patients (91 probands) with disease-causing The mean age of the cohort at the first visit was 19.8 ± 16.1 (median 15) years, with a mean follow-up of 9.6 ± 10 years. Based on history, imaging, and clinical examination, 26 individuals were diagnosed with retinitis pigmentosa , 54 with early-onset severe retinal dystrophy / Leber congenital amaurosis , and 24 with macular dystrophy . Severe visual impairment was most frequent after 40 years of age for patients with RP and after 20 years of age for EOSRD/LCA. Longitudinal analysis revealed a significant difference between baseline and follow-up best-corrected visual acuity in the 3 subcohorts. Macular thickness decreased in most patients with EOSRD/LCA and MD, whereas the majority of patients with RP had increased perifoveal thickness.CRB1 variants present with mild, adult-onset RP. EOSRD/LCA phenotype was significantly associated with null variants, and 167_169 deletion was exclusively present in the MD cohort. The poor OCT lamination may have a degenerative component, as well as being congenital. Disease symmetry and reasonable window for intervention highlight CRB1 retinal dystrophies as a promising target for trials of novel therapeutics.A subset of individuals with  CRB1, MIM 604210) have been associated with a wide and complex range of phenotypes. The most commonly reported is Leber congenital amaurosis (LCA) or early-onset severe retinal dystrophy (EOSRD), where CRB1 accounts for approximately 10% of all cases.,CRB1 represents up to 6.5%.,,Biallelic disease-causing variants in Crumbs homolog 1 ,,15,16—all of which can ultimately lead to retinal detachment and neovascular glaucoma.,,One of the distinctive features of CRB1 was first identified in several unrelated individuals with RP and PPRPE.,CRB1 encodes a transmembrane protein with multiple epidermal growth factor–like and laminin A globular–like domainsCRB1 is frequently reported as one of the most common causative genes for LCA/EOSRD.,CRB1 cohort as of this writing, report disease natural history, and explore potential endpoints for future interventional clinical trials.The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the ethics committees of the participating institutions.CRB1- associated retinopathy. This was defined as patients with an inherited retinal dystrophy (IRD) harboring 2 or more disease-causing CRB1 variants. The patients were identified by reviewing the genetics database of Moorfields Eye Hospital  and St John of Jerusalem Eye Hospital group , and their records were subsequently studied.The inclusion criterion for the current study was to have molecularly confirmed CRB1 variants (transcript reference: NM_201253.3: ENST00000367400.3), and the detailed description is provided in the supplemental material (Supplementary Methods). Pathogenicity of each variant was classified mainly according to the guidelines of the American College of Medical Genetics and Genomics (ACMG).,Genetic testing was performed with various available methods, such as direct Sanger sequencing, next-generation sequencing–based retinal dystrophy gene panels, whole exome sequencing, and whole genome sequencing. In silico molecular genetic analysis was performed for all detected ,All participants were seen by specialists in IRDs at referral sites. Clinical notes were reviewed, including family, medical, and ophthalmic history, best-corrected visual acuity (BCVA), refraction, slit lamp biomicroscopy findings, and funduscopy. BCVA was converted to logMAR for statistical analysis. Count fingers vision was given a value of 1.98 logMAR and hand motion, 2.28 logMAR; light perception and no light perception were 2.7 and 3 logMAR, respectively.Records of visual field were limited within our cohort; therefore, we only took into consideration central vision based on BCVA to classify patients. Low vision corresponds to patients with moderate and severe impairment. Asymmetric BCVA was defined as a difference ≥0.3 logMAR  between eyes. Refraction was undertaken by an optometrist for both adults and children, and spherical equivalent was calculated for refractive error.When available, we also assessed additional testing such as color and autofluorescence retinal imaging, near-infrared reflectance, and optical coherence tomography . Fovea-centered macular volume scans were performed in a 6-mm,Macular OCT scans were divided into 3 categories regarding their qualitative features, and associations were subsequently explored: group 1, characterized by normal lamination; group 2, where the retinal layers are generally discernible but appear ill-defined; and group 3, defined by a disorganized retina with coalescent layers (particularly within the inner retina) and a degree of increased reflectivity of the nuclear layers .36,37FIGTo allow direct comparison between normal and abnormal retinal architecture, we also compared normal (group 1) and abnormal OCT lamination (groups 2 and 3). Patients with macular cysts, edema, and/or only line scans due to poor fixation were excluded from quantitative assessments (details in Supplementary Methods). Normative data regarding OCT thickness in the general population was extracted from Grover and associates,Pattern and full-field electroretinogram  testing was performed in a subset of patients, incorporating the standards of the International Society for Clinical Electrophysiology of Vision (ISCEV) and including additional dark-adapted (DA) red flash ERGs.P <.05. Linear regressions and t test were used for parametric variables’ assessment. Welch t-test variation was employed when the sample sizes were significantly different. χ2 was undertaken to assess possible association between 2 categorical variables.Statistical analysis was carried out with GraphPad Prism 8.0.2 (GraphPad Software). The threshold of significance for all statistical tests was set at CRB1 variants were included in this cohort and ascertained for phenotyping, after a multidisciplinary team of ophthalmologists and clinical geneticists excluded other possible genotypes. Eighty-seven patients (84%) were seen at Moorfields Eye Hospital and 17 (16%) at St John of Jerusalem Eye Hospital. Seven additional patients had a typical phenotype for the disease ; however, a single disease-causing variant in CRB1 was identified. These 7 cases with presumed CRB1-associated disease were not included in the analysis. Supplementary Table S1 summarizes the genetics and the clinical phenotype of these patients.One hundred four patients (91 probands) with multiple Sixty-two individuals were male (60%) and 42 female (40%). The mean age ± SD at the first visit was 19.8 ± 16.1 years, with a median of 15 years. Fifty-three participants had their first visit as children (age <16 years). The mean follow-up time of the cohort was 9.6 ± 10 years, and the overall age at their latest visit was 29.6 ± 17.2 years. The clinical findings from the cohort are summarized in Based on ophthalmic history, imaging, and clinical examination, 54 individuals presented with EOSRD/LCA (52%), 26 were diagnosed with RP (25%), and 24 with MD (23%). Within the EOSRD/LCA subcohort, the mean age of onset was 2 ± 2.2 years (median 1), with poor central vision and secondly nystagmus as the most prevalent initial symptoms and signs. Among the RP group, the mean age of onset was 13.7 ± 10.5 years (median 10), and the most common presenting symptom was nyctalopia, followed by constricted field. Lastly, the patients from the MD subcohort had a mean age of onset of 16.3 ± 10.8 years (median 15) and primarily complained of decreased acuity.P < .0001), RP (P < .0001), and MD subcohorts (P = .047).Baseline BCVA was 1.6 ± 0.8 logMAR in those with EOSRD/LCA (mean age 16.2 ± 15.3 years), 0.9 ± 0.8 logMAR in patients with RP (mean age 23.9 ± 18.6 years), and 0.6 ± 0.4 logMAR in patients with MD (23.2 ± 13.7 years). Seven infants with EOSRD/LCA did not have accurate vision recorded , and 1 patient with RP did not have BCVA detailed in the medical records. The number of patients in each WHO category of visual impairment is displayed in The mean spherical equivalent in the RP cohort was +1.75 ± +1.75 (n=1 myopic patient); in the EOSRD/LCA, +5.75 ± +3.5 (n=2 myopic patients); and +0.75 ± +2.5 among the MD subcohort . High hyperopia  was found in 1 patient with RP, 1 with MD, and in 21 with EOSRD/LCA (39%).One individual with RP and 4 with EOSRD/LCA were diagnosed with keratoconus. Fifteen patients with RP had lens opacities (58%), diagnosed at 34 ± 13 years of age; 17 patients among the EOSRD/LCA group (31%) at age 34.5 ± 14; and only 2 patients in the MD cohort, at ages 54 and 71 years, respectively. Only 4 patients among the RP subcohort had glaucoma: 1 neovascular at age 41 years, 2 open angle at ages 32 and 35 years, and 1 acute angle closure at age 29 years.All EOSRD/LCA patients presented with diffuse, dense pigment in the retinal periphery, with both spicules and nummular pigment. They all had macular involvement, with a pigmented ring in the posterior pole in 16 patients, and coloboma-like severe atrophy in 4 , A and BSixteen patients with MD had normal retina outside the arcades, whereas 8 had peripheral areas of pigmented bone spicules or nummular lesions, and/or vessel thinning. The macular reflex was blunted in 12 patients, with mottled retinal pigment epithelium and signs of atrophy in the remaining 12—the latter often accompanied by pigmented bone spicules and affecting the nasal peripapillary area , E and FThirty-four individuals with EOSRD/LCA (mean age 24.1 ± 16.6 years), 11 with RP (23 ± 18 years), and 3 with MD (37.3 ± 11.1 years) had nummular pigment clumps. White or yellow dots were seen in 16 EOSRD/LCA patients (mean age 24 ± 9 years), in 3 patients with RP (19 ± 7.5 years), and in 2 MD patients, at the same time point where nummular pigment was present (25 and 35 years of age). Sixteen patients with EOSRD/LCA (mean age 22 ± 13.5 years) presented with PPRPE, 10 with RP (23 ± 12 years), and 3 with MD (38 ± 10.5 years). PPRPE was more readily identified with autofluorescence (AF) imaging, likely given the retinal pigment epithelial basis of AF imaging.Seven EOSRD/LCA patients had retinal telangiectasia (mean age 30 ± 10 years), 6 of which were associated with exudation, with 2 resulting in vitreous hemorrhage(s). Retinal telangiectasia was seen in 4 patients with RP, diagnosed at a mean age of 28 ± 8 years, and resulted in a range of complications including exudation, vitreous hemorrhage, and retinal detachment (RD). Optic disc drusen was the most common optic nerve abnormality, affecting 7 patients with LCA, 4 with RP, and 1 with MD. Two patients with EOSRD/LCA had gliosis on the optic disc.Ocular complications were seen in 12 individuals with EOSRD/LCA (21%); in 4 they were related to telangiectasia, whereas the remaining 8 corresponded to RD, iris nodules, anterior segment synechiae, retinal hamartoma, and corneal hydrops. Ocular complications were also reported in 6 patients with RP (22%): 4 were associated with retinal telangiectasia, and the remaining 2 were uveitis and asteroid hyalosis. Only 1 patient with MD was found to have unusual vessel sheathing at age 8 years, of unknown cause.Eighty-five patients from our cohort had macular OCT scans (82%): 38 (45%) with EOSRD/LCA, 25 (29%) with RP, and 22 (26%) with MD—all being included in the qualitative analysis. Quantitative assessment was possible in 15 patients with EOSRD/LCA, 8 with RP, and 10 with MD, because of image quality and sufficient scans being available. Eleven individuals from the EOSRD/LCA subcohort had follow-up scans (over 7 ± 3 years), 7 from the RP group (over 7.5 ± 1.52 years), and 10 with MD (7 ± 3 years), which enabled additional longitudinal analysis. Baseline and follow-up structural parameters are described in Supplementary Table S2.P = .002), outer ring thickness , inner ring volume (P = .002), and outer ring volume (P = .0004) compared with the normal population (P = .0008) and central macular volume (P = .0037); however, the outer ring volume was increased (P = .0004). Lastly, patients with MD showed significant thinning in all CMT (P = .003), IRT (P = .004), ORT (P = .01), central macular volume (P = .0017), and inner ring volume  and MD subgroups (P between .4 and .8). However, a significant association was found in patients with RP between age and ORT  and inner ring volume , 54% had decreased IRT (–1.5 µm/y), and 50% had decreased ORT (–0.6 µm/y). Within the RP subcohort, 67% had decreased CMT (–1.9 µm/y), 49% had decreased IRT , and 43% had decreased ORT (increase of 1.5 µm/y). Lastly, 59% patients with MD had decreased CMT over follow-up (–0.5 µm/y), 70% had decreased IRT (–1.1 µm/y), and 49% had decreased ORT (–1.8 µm/y).P = .007) and excluding the EOSRD/LCA group (P = .002), with a mean age of 22.3 ± 15.4 years for those having cysts and 34.6 ± 18.2 for those without. The cysts resolved during follow-up in 24% of eyes, without any treatment. The presence of outer layers at the fovea and perifovea was also recorded (Supplementary Results).Macular cystic spaces were present in 29% of our cohort as a whole, and in 52% and 42% of the RP and MD subgroups, respectively. The majority had only the internal nuclear layer (INL) affected (24 eyes), followed by both INL and outer nuclear layer (ONL) involved (19 eyes), and then ONL only (13 eyes). There was a significant difference in the age of patients with and without cysts, both analyzing the cohort as a whole (P = .03) and 2 and 3 (P = .0003), whereas no age difference was recorded between groups 1 and 2 (P = .49). Visual acuity was also significantly different between groups; 0.4 ± 0.32 logMAR in group 1, 1.25 ± 0.97 logMAR in group 2, and 3.27 ± 1.38 logMAR in group 3, with progressive VA decline as the OCT layers became more disorganized (P < .0001).In terms of qualitative assessment, overall, 8 patients had both eyes in OCT group 1 , 52 in group 2 , and 18 in group 3 , 16 in OCT group 3 , and 2 each eye in groups 2 and 3 (ages 15 and 27 years). The RP subcohort had 3 patients in group 1 , 17 in group 2 , 2 in group 3 (44 and 53 years old), 1 with one eye in group 1 and the other in group 2 (37 years old), and 2 with one eye in group 2 and the other in group 3 .P = .0005), whereas no age difference was found between groups 1 and 2 of the RP (P = .28) and MD subcohorts (P = .07).Lastly, the MD group had 5 patients in group 1 , 15 in group 2 , 1 with one eye in group 1 and the other in group 2 (15 years old), and 1 with one eye in group 2 and the other in group 3 (23 years old). Findings are summarized in P = .81). The visual acuity of the abnormally laminated group was 1.75 ± 1.39 logMAR, which was significantly different from group 1 (P < .0001).In addition, given image quality variation, potential challenges to robustly distinguish between OCT groups 2 and 3, and the desire to compare definitively normal with abnormal architecture, we also compared group 1  vs groups 2 and 3 combined . The latter (group 2+3) had a mean age of 26.9 ± 16.8 years, and there was no significant age difference when compared to the normal lamination group  with EOSRD/LCA changed from group 2 to 3, whereas 5 (3 patients) with RP and 5 (3 patients) with MD went from group 1 to group 2 during follow-up (further details in Supplementary Results).r2 = 0.98), and on the peak times of the DA 10 ERG a- and b-waves and LA 30-Hz ERGs . PERG N95 data were available for analysis in 12 of 14 patients with a detectable P50 component. The N95:P50 ratio had a mean value of 1.7 (range 1.4-3.0), which is similar to the reference group for the lab (mean = 1.5)Twenty-nine patients (28%) underwent ff-ERG and PERG. There was a high degree of inter-ocular ERG symmetry based on amplitudes of the DA 0.01, DA 3, and DA 10 ERG a- and b-waves, LA 30-Hz ERG and LA 3 (single flash) ERG b-waves , RP (P < .0001), and MD (P = .016) subcohorts.Follow-up BCVA was obtained in 41 (76%) individuals with EOSRD/LCA, 20 (77%) with RP and 20 (83%) with MD. Mean follow-up time was 9.9 ± 12.1 years for patients with EOSRD/LCA, 9.9 ± 9.8 for the RP subcohort, and 7.6 ± 5.7 for MD patients. Follow-up BCVA was 1.9 ± 0.7 logMAR in patients with EOSRD/LCA (mean age 27 ± 17 years), 1.3 ± 1 logMAR in patients with RP (mean age 38 ± 13 years), and 0.8 ± 0.5 logMAR in MD patients (mean age 30 ± 16). There was a significant difference between baseline and follow-up BCVA in the EOSRD/LCA (The rate of BCVA decline was 0.06 logMAR (3 letters)/year in patients with EOSRD/LCA, 0.07 logMAR (3.5 letters)/year in patients with RP, and 0.04 logMAR (2 letters)/year in patients with MD. Fifteen patients (37%) with EOSRD/LCA dropped 15 ETDRS letters or more over follow-up in at least 1 eye, 10 (50%) with RP, and 7 (35%) with MD. Fourteen patients (34%) with EOSRD/LCA progressed to more advanced WHO categories of visual impairment, 7 (35%) with RP, and 6 (30%) with MD. Overall, 20 patients (25%) became severely sight impaired and/or blind over follow-up: 12 with EOSRD/LCA, 5 with RP, and 3 with MD.CRB1-RP patients in our sample experienced loss of thickness at the fovea and parafovea, while increasing in the perifovea; however, the small sample size available for longitudinal assessment should be borne in mind. MD patients had progressive thinning of the posterior pole—associated with poor retinal lamination—and discontinuous/narrower outer layers.Macular OCT longitudinal evaluation demonstrated that EOSRD/LCA patients may experience slowly decreasing retinal thickness (despite their baseline thickening), progressive loss of lamination, and degeneration of both central and pericentral outer retinal layers over follow-up. CRB1 variants were found in our cohort, 46 of which were missense (59%), 11 frameshift (14%), 10 nonsense (13%), 5 canonical splice site alteration (6%), 2 missense with significant splice site alteration (3%), 2 intronic variants (3%), 1 in-frame deletion (1%), and 1 deletion of exon 12 (1%) (Supplementary Table S3). Schematic representation of these detected variants and the status of evolutionary conservation are presented (Seventy-eight different P = .001) and MD (P = .003) phenotypes, rather than with RP  were significantly more associated with both EOSRD/LCA  and developed EOSRD/LCA, and 1 nephew harbored p.(Ser478ProfsTer24) and the in-frame p.(IIe167_Gly169del) and presented with MD.Intrafamilial variability was present in 2 families. In one family, a sibling had mild, asymptomatic MD and the other had RP with decreased vision since her 20s (homozygous p.Ser638Leu). In the other family, 3 different CRB1 retinopathy as of this writing, with 23 novel CRB1 variants identified. Deep phenotyping data are presented, including detailed analyses of demographic features, fundus imaging, morphologic characteristics, and quantitative data of electrophysiological assessment, with the aim of establishing clear phenotypes, potential outcome measures, genotype-phenotype correlations, and laying the foundations for future studies that aim to develop therapies and treatment strategies.This study characterizes the largest cohort of patients with molecularly confirmed CRB1-RP patients from the fifth decade of life. Patients with MD appeared to have an overall good prognosis, with relatively preserved visual function until adult age in at least 1 eye. The findings highlight the window of opportunity for potential treatments such as gene therapy.Low vision was the most common WHO category for individuals with EOSRD/LCA in the first 2 decades of life, followed by blindness from 20 years on. In the RP group, there was severe visual impairment from age 40 years, whereas in the MD group it was only seen in a minority of cases from age 28 years. This is similar to the observations from Talib and associates,CRB1-RP by Mathijssen and associates,Individuals with EOSRD/LCA may need relatively early intervention in childhood and adolescence because of early macular structural and functional involvement, whereas patients with RP or MD would potentially benefit up until their late 30s. The latter is in agreement with Talib and associatesP = .001). Regarding BCVA asymmetry, we report a smaller proportion than previous cohorts (19% in this report vs 33% and 31% in other groups).,Employing their statistical approach of age vs BCVA of the best-seeing eye (instead of age vs BCVA of both eyes as we have herein), we still obtained a significant association .CRB1 phenotypes, with 39% of the EOSRD/LCA patients having high hyperopia (>+5.00 D). Wang and associatesCRB1-LCA cohort to be highly far-sighted, and Mathijssen and associatesCRB1-RP cohort. This was not, however, a pathognomonic feature in CRB1-MD (29% of our patients were myopic and 16% were emmetropic), corroborating a previous study.Hyperopia was the prevalent refractive error in all ,,CRB1 on one occassion,The most prevalent posterior segment feature within our cohort was maculopathy, which was present in 97% of our cohort, with variable severity, and consistent with smaller studies.,Macular OCT revealed a range of features; 65% of the eyes from our cohort had ill-defined lamination group 2; , and 24%CRB1 may also involve a degenerative process and not only a congenital one. This is consistent with previous suggestions, although far larger studies with greater serial data are required.,To mitigate this potential limitation and thereby minimize possible bias, we added the comparison of normal and abnormal lamination groups. Patients in group 3 were older and our longitudinal data (available in 7% of eyes) showed progressive loss of defined lamination on qualitative review; therefore, the poorly laminated and disorganized OCT in ,,,CRB1-MD seemed to be exempt from the retinal thickening characteristic.Quantitative analyses of OCT have revealed a strong association between CRB1 and a thickened retina, reported in animal models,CRB1-related poor adhesion between cells, which leads to poor lamination and abnormal anatomy, consequently occupying volume in a less efficient, broader way.,Some groups have also reported age-dependent posterior pole thinning.,,,CRB1-RP and -MD, affecting mostly younger individuals, and it can be self-limiting. This has caused CRB1 to be included within the differential diagnoses considered in cystic maculopathies, such as those secondary to variants in RS1 and NR2E3.Macular cystic spaces were present in a lower percentage in our cohort when compared to other groups, and this may be related to the higher proportion of EOSRD/LCA patients, who are rarely affected by macular fluid accumulation.CRB1 retinopathy. CRB1 transcript CRB1-A has been located within Müller glia cells in mouse and human retina,,CRB1 phenotypes.Although macular fluid accumulation is also seen in other RP genotypes,CRB1-related disease, having a carrier frequency of approximately 2:10 000.,The variant c.2843G>A has been reported the most commonly occurring in individuals with CRB1 variant in non-Asians,,The in-frame deletion p.(Ile167_Gly169del) was reported to be the most prevalent disease-causing Another frequent variant among the MD group was p.(Pro836Thr), present in 7 Black African individuals (4 families). This variant was first described by den Hollander and associates, seen in a patient with RP and PPRPE, without a known second variant, and with no details regarding race.,,Lastly, we were able to test the association of null variants and different phenotypes. We not only found them significantly associated to EOSRD/LCA, as previously suggested by other groups,CRB1, occupying nearly all the AAV packing capacity, has made the development of a therapy for these retinopathies challenging.CRB1 macular OCT features will likely be helpful toward its inclusion among the inherited retinal dystrophy genotypes that are currently used to train and validate deep learning systems for automated diagnosis and classification.The large size of Inherent limitations of our study are its retrospective design, the differences in follow-up time between patients, not all data being available for every individual, and different types of accessible data, acquired with diverse methods and protocols. The above-mentioned limitations were ameliorated by the size of our genetically confirmed cohort, the wide age range of our patients, the international representation, and their detailed medical records.CRB1 retinopathy as of this writing, identifies novel CRB1 variants, and delineates genotype-phenotype correlations. Clinical and functional phenotypes are detailed, establishing the range and combinations of features that will aid diagnosis and inform patient management. Potential endpoints for future natural history and interventional clinical trials are highlighted, laying the foundations for future studies that aim to develop therapies and treatment strategies.In conclusion, this multinational investigation characterizes the largest cohort of patients with molecularly confirmed


Ras-GTPase-activating protein binding protein 1 (G3BP1) is an oncogenic factor, which highly expressed in a variety of cancers. In recent years, G3BP1 has been reported to promote the development of prostate cancer by inhibiting the degradation of AR through inhibiting SPOP. However, whether G3BP1 contributes in a similar manner to the abnormal accumulation of ERα, which is also an important target for hormone therapy, remains unknown. This article addresses this issue and explores potential mechanisms.Bioinformatics tools were used for G3BP1 expression analysis, survival analysis, and clinical association analysis. Immunohistochemical staining was used to examine the correlation between G3BP1 and ERα in EC patients. In addition, western blot and co-immunoprecipitation were used to detect the half-life of G3BP1 and mutant, and the effect of G3BP1 and mutant on the ubiquitination and degradation of ERα mediated by SPOP. Then, the oncogenic functions of G3BP1 dependent on the SPOP/ERα axis were determined by CCK8 cell proliferation assay, colony formation assay and cell migration assay. Finally, we established the EC cells treated or untreated with fulvestrant, exploring the possibility of fulvestrant combined with the reduction of G3BP1 to improve the efficacy of fulvestrant.G3BP1 is abnormally high expressed and characterized by high-frequency mutation in EC. In addition, there is a positive correlation between G3BP1 protein and ERα protein. Mechanistically, both G3BP1 and mutant, the latter is displaying the longer half-life, competitively bind SPOP with ERα, thereby inhibiting SPOP-mediated ubiquitination and degradation of ERα. Functionally, G3BP1 and mutant promote the proliferation and migration of EC cells by regulating the G3BP1/SPOP/ERα axis. However, fulvestrant can reverse the cancer-promoting effects of G3BP1 and mutant.G3BP1 and its mutant positively regulate ERα signaling pathway by inhibiting SPOP-mediated ubiquitination and degradation of ERα, indicating the promising effect of fulvestrant on the suppression the occurrence and development of EC with high expressed G3BP1 and G3BP1 mutants.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01342-7. Endometrial cancer (EC) is one of the most common gynecological malignancies and the sixth most common malignant disease in the world . At presERα is an important nuclear transcription factor that mediates estrogen-responsive cancers, including EC . Upon biRas-GTPase-activating protein binding protein 1 (G3BP1) is a protein of 466 amino acid whose primary biological function is to promote the assembly of stress granules in the cytoplasm of eukaryotic cells in response to certain environmental stresses . As a suHerein, we demonstrated that G3BP1 protein is highly expressed and is positively correlated with ERα in EC tissues. Mechanistically, we found that G3BP1 and its EC-associated mutant dramatically inhibit SPOP-mediated ubiquitination and degradation of ERα. Functionally, we found that G3BP1 and its mutant promote EC cell proliferation and metastasis, and this effect could be reversed by the fulvestrant, an ERα antagonist commonly used in clinical, indicating a promising idea for improving the efficacy of EC treatment.HEK 293T, HEC-1-A and AN3-CA cells were purchased from the American Type Culture Collection. HEK 293T and HEC-1-A cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin solution. AN3-CA cells were cultured in MEM supplemented with 10% FBS and 1% penicillin–streptomycin solution. All cells were grown at 37 °C with 5% CO2.Cells were transfected with siRNA  using Lipo6000™ or Lipo8000™ Transfection Reagent  as described by the manufacturer's instructions. The siRNA oligonucleotide sequences of G3BP1 and SPOP are shown in Table SThe following antibodies and reagents were used: G3BP1 , ERα , SPOP , GAPDH , FLAG , Myc , HA , Anti-Rabbit , Goat Anti-Rabbit , anti-Flag M2 agarose beads , MG-132 , CHX .https://portal.gdc.cancer.gov/) and the GSE17025 of GEO cohort (https://www.ncbi.nlm.nih.gov/gds/). The protein expression and clinicopathological features were derived from Clinical Proteomic Tumor Analysis Consortium . The FPKM was transformed to TPM [Log2 (FPKM + 1)]. And the analysis was conducted by the “BiocManager”, “heatmap”, “limma”, “ggplot2” “survival” and “survminer” packages in R software (version 4.2.1). Pearson correlation analysis was used to screen out the genes significantly related to G3BP1 expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzed the gene sets and their biological functions based on CPTAC. The mutation frequency and mutation site of G3BP1 were derived from TCGA and cBioPortal cohort .The mRNA expression (FPKM) and clinicopathological features were derived from The Cancer Genome Atlas . Next, anti-Flag M2 agarose beads (Sigma) were added to the cell lysates overnight. Subsequently, the bound beads are then washed 3 times with BC100 buffer. The bound proteins were eluted using FLAG-peptide (Sigma)/BC100, and added with SDS-PAGE solution to boil for Western blot.The GST fusion protein was fixed on glutathione-Sepharose beads (Amersham Biosciences). After washing with a pull-down buffer, the beads were incubated with recombinant FLAG-labeled proteins for 2 h. The beads are then washed 5 times with a binding buffer and re-suspended in the sample buffer. The binding proteins were added with SDS-PAGE solution to boil for Western blot.Cell lysates or immunoprecipitations were subjected to SDS-PAGE and protein transfer to nitrocellulose membranes . The membrane was blocked in Tris-buffered saline (pH 7.4) with 5% skim milk and 0.1% Tween-20, incubated overnight with primary antibody at 4°, then incubated with secondary antibody for 1 h. Proteins of interest were visualized using the ECL Chemiluminescence System . WB was performed for 2–3 times from at least two independent experiments and representative pictures were shown.Total RNA was isolated from cells using Trizol reagent . cDNA synthesis was performed using the HiScript® II 1st Strand cDNA Synthesis Kit  according to manufacturer's instructions. PCR amplification was performed using SYBR Green PCR Master Mix Kit . All quantization were normalized to the level of endogenous control GAPDH. The primer sequences for qRT-PCR are shown in Table SOne hundred twenty pairs of paraffin-embedded EC tissues were cut to 4 μm thickness and roasted at 65℃ for 3 h. Then, roasted tissue sections were dewaxed with xylene and rehydrated with ethanol. Antigen repair is performed by heating tissue sections in a pressure cooker in Ethylene Diamine Tetra acetic Acid (EDTA) antigen repair buffer, PH9.0  for 10 min and then cooling them naturally to room temperature. Then, the endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide for 20 min, permeated with 1% Triton X-100  for 30 min, blocked with 10% donkey serum for 15 min, and incubated at 4℃ overnight with G3BP1 antibody  (1:400) or ERα antibody  (1:200). The next day, sections were incubated with secondary antibody  (1:500) at room temperature for 1 h, stained with DAB assay kit  for 5–7 min, and stained with hematoxylin  for 10s.All samples were reviewed by two independent pathologists experienced in IHC evaluation who had no knowledge of clinical outcomes in these patients. They assessed the percentage of positively stained cells and staining intensity to semi-quantitatively determine G3BP1 and ERα expression. The percentage score of positive staining cells was as follows: 0, < 10%; 1, 10%-50%; 2, > 50%. The staining intensity is graded as follows: 0 (no or weak staining = light yellow), 1 (moderate staining = yellow brown), and 2 (strong staining = brown). The total score of G3BP1 and ERα expression was the sum of the percentage of cells and the staining intensity score for positive staining, with the total score ranging from 0 to 4. For statistical analysis, the final score was a combination of independent scores assigned by the two pathologists reported in this study. Any differences in scores were resolved through discussion between the two pathologists.The cell proliferation rate was determined using Cell Counting Kit-8 (CCK-8)  according to the manufacturer's instructions. AN3-CA and HEC-1-A cells were inoculated into the 96-well plate with a density of 1500 cells per well (three wells in each group). During 0–5 d culture, 10 μl CCK-8 solution was added at the same time every day and cultured for 2 h. After these, OD values of each well were measured at 450 nm using a microplate absorbance instrument . Each assay was repeated three times.AN3-CA and HEC-1-A cells were inoculated in 6-well plates with 2000 cells per well (three wells in each group). After 10–15 days of culture, the cells were fixed in 4% polymethanol at 4° for 30 min and then stained with crystal violet solution  for 15–20 min. The images of cell colonies were captured by smartphone and analyzed by Image J software.Transwell chambers  were also used to measure cell migration. 40,000 cells were seeded in each upper chamber with 200 μl fresh medium without FBS, and 500 μl medium with 10% FBS in each lower chamber. Three duplicate wells were set up for each group. After 24 h, the cells were fixed in 4% polymethanol at 4° for 30 min and then stained with crystal violet solution  for 15–20 min. Clean the upper chamber and count the number of cells on the lower surface of the membrane.The expression vectors for SPOP were described previously . The G3BP < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001.Statistical analysis data were analyzed with student's t-test and presented as mean value ± standard deviation of triplicate measurements in three separate experiments. Statistical calculations were performed using GraphPad Prism 8 software. The images were analyzed and quantified using ImageJ software. * indicates http://ualcan.path.uab.edu/index.html), we found that the level of G3BP1 protein is correlated with the clinical stage and grade of patients with EC , including missense mutation, synonymous mutation, and nonsense mutation  approved by the Food and Drug Administration (FDA), which exhibits its antitumor effect through two mechanisms . On the To test this notion, we utilize DMSO and fulvestrant (100 nM) to treat the AN3-CA and HEC-1-A cells with G3BP1 overexpression, G3BP1 Q392* overexpression, SPOP overexpression, G3BP1 and SPOP co-overexpression, and G3BP1 Q392* and SPOP co-overexpression, respectively Fig. A, S2A. TFurthermore, we utilize DMSO and fulvestrant (100 nM) to treat the AN3-CA and HEC-1-A cells with SPOP knockdown, G3BP1 knockdown and SPOP and G3BP1 co-knockdown Fig. A. CCK8 cTherefore, it can be inferred that in EC patients with high G3BP1, fulvestrant can be applied to reverse the occurrence and development of EC. In addition, ERα expression is often higher in patients with high G3BP1, so G3BP1 can improve the sensitivity of patients with EC to fulvestrant. Since G3BP1 Q392* has a stronger oncogenic effect than G3BP1, fulvestrant may be more effective in EC patients with G3BP1 Q392*.In this article, we identified a carcinogenic protein G3BP1 in EC that is positively associated with ERα protein expression. It promotes ERα accumulation by inhibiting the SPOP-mediated ERα ubiquitination and degradation, which leads to the proliferation and metastasis of EC. In addition, G3BP1 Q392* displays more suppressive effect than wildtype G3BP1, due to its longer half-life than G3BP1, but these effects could be reversed by fulvestrant, providing a promising idea for improving the efficacy of fulvestrant in EC patients with G3BP1-positive plus ERα-positive.Notably, the expression of G3BP1 differs only at the protein level, but not at the mRNA level, suggesting that some post-translational modifications of G3BP1may be disrupted, resulting in the high expression of G3BP1 protein in EC. However, there are few studies on the post-translational modification of G3BP1, which mainly focus on the methylation, phosphorylation, and acetylation. Moreover, these post-translational modifications only affected the G3BP1-mediated SGs assembly but did not affect the expression of G3BP1. For further study, KEGG analysis and GO analysis were performed on genes associated with overexpression of G3BP1 protein based on CPTAC cohort  Kaplan-Meier survival curves of EC based on the TCGA cohort. (B) Heat map of clinical characteristics of EC based on TCGA cohort. (C) Heat map of clinical characteristics of EC based on CPTAC cohort. (D) Gene co-expression circle of G3BP1 based on TCGA cohort. (E) KEGG analysis was performed based on CPTAC cohort. (F) Based on CPTAC cohort, R package was used to enrich gene ontology, including BP, molecular MF and CC. Data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Supplementary Fig. 2. The promoting effects of G3BP1 and G3BP1 Q392* on the proliferation and metastasis of endometrial carcinoma partly depend on SPOP/ERα axis. (A) AN3-CA and HEC-1-A cells were transfected with indicator plasmids. 48h after transfection, cell lysates were prepared and the protein levels of ERα, SPOP and G3BP1 were determined by western blot. (B) CCK8 cell proliferation analysis was used to detect the proliferation ability of AN3-CA and HEC-1-A cells. (C) Colony formation assay was used to detect the colony formation ability of AN3-CA and HEC-1-A cells. (D) Cell migration assay was used to detect the metastasis ability of AN3-CA and HEC-1-A cells. Data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Supplementary Fig. 3. Fulvestrant can reverse the promoting effects of G3BP1 and G3BP1 Q392* on endometrial carcinoma. (A) HEC-1-A cells were transfected with indicator plasmids. The cells were cultured with DMSO and fulvestrant (100nM) in complete medium. 48h after transfection, cell lysates were prepared and the protein levels of ERα, SPOP and G3BP1 were determined by western blot. (B) CCK8 cell proliferation analysis was used to detect the proliferation ability of HEC-1-A cells. (C) Colony formation assay was used to detect the colony formation ability of HEC-1-A cells. (D) Cell migration assay was used to detect the metastasis ability of HEC-1-A cells. Data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001Additional file 2.


Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. The functional relevance of these interactions was confirmed by in vitro conjugation and in vivo cellular assays. Therefore, highly curved phagophoric rims not only serve as a geometric cue for hAtg3 recruitment, but also their interaction with hAtg3 promotes LC3-PE conjugation by targeting its catalytic center to the membrane surface and bringing substrates into proximity. Our studies advance the notion that autophagosome biogenesis is directly guided by the spatial interactions of Atg3 with highly curved phagophoric rims. Here, Ye et al use high-resolution NMR in conjunction with in vitro and in vivo assays to show the catalytically important C-terminal regions of human Atg3 are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. In eukaryotes, autophagy is essential for maintaining cellular homeostasis and basic functions; its dysfunction leads and/or contributes to many disease processes including neurodegeneration, infection, cardiovascular impairment, and tumorigenesis10.Macroautophagy (autophagy) is a major intracellular degradation and recycling process initiated by forming a cup-like membrane structure known as the phagophore. As the phagophore expands, cytosolic components that are targeted for degradation, such as aggregated proteins, damaged organelles, and foreign organisms including viruses and bacteria, are engulfed. Following the phagophore elongation, it is sealed to create a double-membrane vesicle called the autophagosome. The autophagosome is then delivered to the lysosome to degrade and recycle its contents11. This LC3-PE conjugate regulates phagophore expansion and serves as a docking site for autophagic cargos14. This conjugate reaction is catalyzed by three ubiquitin-like conjugation enzymes: the E1-like Atg7, the E2-like Atg3, and the E3-like Atg12-Atg5/Atg16 complex. Despite their conceptual similarities, Atg7, Atg3, and Atg12-Atg5/Atg16 are structurally and functionally distinct from canonical E1, E2, and E3 enzymes22. For instance, unlike canonical E2 enzymes, in vitro Atg3 alone selects substrate PE lipids and can catalyze the transfer of LC3 from an LC3-Atg3 intermediate to PEs without the Atg12-Atg5/Atg16 complex25. As this reaction occurs at the membrane surface with PEs in a lipid bilayer phase, the protein-lipid interaction of Atg3 plays an essential role in its function. In fact, Atg3’s function requires the presence of an N-terminal amphipathic helix (NAH) that selectively interacts with membranes that contain lipid-packing defects, such as the leading edge of the growing phagophore where membranes are highly curved29. Our recent studies have further revealed that the membrane association of the NAH of human Atg3 (hAtg3) is necessary but insufficient to catalyze LC3-PE conjugation30. We have discovered a conserved region following the hAtg3 NAH that tightly coordinates its membrane geometry-sensitive interaction with its catalytic activity. In addition, enhancing the interaction of Atg3 with membranes by acetylation of its NAH reportedly extends the duration of autophagy31. However, how the C-terminal located catalytic center is directed to the membrane surface for effective LC3-PE conjugation remains obscure.A key step in autophagosome biogenesis is the conjugation of LC3 family proteins to phosphatidylethanolamine (PE)38 to position the catalytic residue Cys264 at the membrane surface and bring substrates, LC3 and PE lipids, into proximity to promote LC3-PE conjugation. In addition, NMR hydrogen/deuterium exchange studies revealed that the catalytic loop and following α-helix of hAtg3 are conformationally dynamic in aqueous solution and are conducive to interaction with the membrane, even though the general hAtg3 core structure is similar to those previously reported for yeast and Arabidopsis thaliana39. Together, our studies advance an emerging concept that the interactions of Atg3 with the highly curved membrane rims of the phagophore spatially regulate autophagosome biogenesis40.In this study, we have used NMR spectroscopy to determine the solution structure of hAtg3 and discovered that both its NAH and catalytically important C-terminal regions directly interact with the membrane. The functional significance of this interaction was confirmed by in vitro and in vivo loss-of-function mutational analyses. Our results suggest that hAtg3 exploits a multifaceted membrane-association mechanism41. In an earlier work, we showed that residues 90 to 190 form an unstructured loop and that deletion of these amino acids (hAtg3Δ90–190) minimally perturbed hAtg3’s core structure and did not affect its function in vitro . We have also shown that residues 1 to 25 are unstructured in aqueous solution but undergo a conformational change to form an amphipathic α-helix upon interaction with the membrane42. Deletion of both regions  causes a minimal perturbation in the core structure as evidenced by an overlay of the TROSY spectra of hAtg3Δ90–190 and hAtg3ΔN25, Δ90–190 shown in Supplementary Fig. ΔN25, Δ90–190 construct produced high-resolution spectra, it was used for NMR structure determination. This construct is very similar to a yeast Atg3 (yAtg3) version that was recently selected for X-ray structure determination43.hAtg3 is designated as an intrinsically disordered protein since more than 1/3 of its 314 residues are in unstructured regionsΔN25, Δ90–190  is shown in Fig. ΔN25, Δ90–190 onto the structures of yeast and Arabidopsis thaliana Atg3 (yAtg3 and AtAtg3) demonstrate a conserved E2-like fold. The reported Cα RMSDs of hAtg3 to yAtg3 and AtAtg3 structures by Chimera are 1.1 Å and 1.0 Å, respectively. The most notable deviations are observed in helix F (according to the secondary structure definition in the yAtg318), which immediately follows the catalytic loop containing Cys264  exchange experiments. Instead of quantitatively determining an exchange rate constant, for the purpose of this discussion, we classified the peaks observed in TROSY spectra into three groups according to their intensities in H/D exchange experiments. Group I amides include those that are in fast exchange  relative to a reference spectrum collected in an H2O buffer. Group II amides exchanged slowly  remain unassigned42. Resonances from these residues are either missing or display pronounced exchange broadening in the TROSY spectrum. Thus, these regions are likely involved in conformational exchanges in bicelle-bound hAtg3Δ90–190. During sample optimization, we noticed that hAtg3Δ90–190 in bicelles is stable only for one or two days at pH 6.5, but at pH 7.5, it remains stable for several weeks. We hypothesized that the membrane interaction of a protonated His residue in hAtg3Δ90–190 contributes to its instability at lower pH. This observation led us to substitute His266, one of two fully conserved His residues  shows similar conjugase activity in an in vitro LC3B-PE conjugation assay  that produced a high-quality TROSY spectrum  around the catalytic site of hAtg3 upon the binding of its NAH to bicelles 47. On a sample of perdeuterated 15N, 2H- hAtg3Δ90–190, 4M mixed with 12% (w/v) bicelles  in an 80% D2O and 20% H2O buffer, irradiation at 1.28 ppm (hydrophobic tail residues of lipids) with a 40 ms Gaussian pulse for 1.6 s resulted in the effective saturations of all lipid resonances, except the peak from choline methyl groups, due to spin diffusion . By comparison, hAtg3 mutants that disrupt this C-terminal membrane interaction still bind to the membrane, as described below.To probe the insertion depth of these membrane-interacting regions, we performed NMR paramagnetic relaxation enhancement (PRE) experiments using 5-doxylstearic acid (5-DSA)13Cα and 13Cβ shifts in bicelle-bound hAtg3Δ90–190, 4M indicate that residues 265 to 277 are in a helical conformation  that are fully conserved and experience large cross-saturation effects , a polar Ser (F296S), or a small hydrophobic Ala (F296A) residue nearly abolished its activity. Again, these mutations form the LC3B-hAtg3 intermediate and bind to the membrane normally  in yAtg3 was reported to dramatically increase its conjugase activity. This result is inconsistent with our finding that hAtg3F296S is impaired in LC3B-PE conjugation, suggesting that yAtg3 and hAtg3 may have subtly different catalytic mechanisms. To further investigate this, we introduced wild-type hAtg3 and the hAtg3F296S or hAtg3F296L variant into Atg3−/− mouse embryonic fibroblasts (MEFs) by lentiviral transduction , the expression of hAtg3 rescued the LC3B-PE conjugation and autophagic flux , directly interact with the membrane. The functional relevance of these interactions is demonstrated by in vitro conjugation and in vivo cellular assays. Intriguingly, the catalytic loop and helix F of hAtg3 are conformationally flexible in aqueous solution as evidenced by resonance exchange broadenings and fast H/D exchanges. This observation further supports the model that they are conformationally plastic and susceptible to structural rearrangements that are critical for their conjugase activity53.During autophagy the Atg3 enzyme catalyzes a covalent conjugation of Atg8 family proteins to the amino group of PE lipids. The reaction product Atg8-PE is required for the successful formation of an autophagosome. Since this reaction is carried out at the membrane surface with PE serving as one of two reaction substrates, the functional importance of the interaction between Atg3 and membrane has been long recognized. In an early study of yAtg3, PE lipids were reportedly required to anchor in a bilayer structure for Atg8-PE conjugation to occur, and the conjugation reaction required the presence of negatively charged lipids and proceeded more efficiently with a higher percentage of PEs in the membrane (up to 75% percent)30. However, in our previous study, we could only obtain ~78% of backbone resonance assignments of bicelle-bound hAtg3Δ90–190 because of exchange broadenings42. Here, we overcame this challenge by introducing mutations (e.g. H266L mutation) that stabilize hAtg3’s interaction with the membrane and achieved near-complete (~93%) backbone resonance assignments for bicelle-bound hAtg3Δ90–190, 4M. Consistent with our previous study, perturbed residues, induced by NAH binding to the membrane, map to a continuous surface around residue Cys264 and are located on one side of the protein. Moreover, NMR cross-saturation experiments provided evidence that, besides the NAH, the catalytic loop and two following helices (F and G) also directly interact with the membrane. These C-terminal/membrane interactions are functionally indispensable; mutations that are designed to disrupt these interactions abolish or dramatically reduce LC3-PE conjugation in vitro and in vivo. We propose a model that summarizes the key findings of this study and delineates the process of hAtg3/membrane interaction  induced PIP clustering55. In addition, during the activation of phospholipase A2 (PLA2), the membrane may function as an allosteric ligand56.We recently reported substantial chemical shift perturbations in the C-terminal regions surrounding the catalytic residue Cys264 of hAtg3 when its NAH binds to the membraneion Fig. . In the 43, a structural model of the Atg8-yAtg3 intermediate was proposed using molecular modeling in conjunction with mutagenesis and NMR CSPs. In this model, Atg8-yAtg3 adopts a closed-state, that is presumably more active for discharging Atg8 to PE lipids than an open-state, and a noncovalent interaction interface between yAtg3 and Atg8 is defined. Assuming that this interaction interface is conserved between yAtg3 and hAtg3, we have mapped the corresponding residues at the Atg8-yAtg3 interface onto the hAtg3 structure. As shown in Supplementary Fig. 13C-labeled CH3 groups of Ile, Leu, and Val residues in a perdeuterated protein background were explored to characterize protein/lipid interactions. Compared to backbone 15N and HN groups, 13C chemical shifts of sidechain CH3 groups are more specific to direct interactions. These results support our assumption that the multifaceted membrane interaction of hAtg3 uncovered in this study applies to the LC3B-hAtg3 intermediate, although details of the LC3B-hAtg3/lipid interactions may be additionally adjusted to accommodate the presence of LC3B. In contrast, little direct structural information is available for the LC3-Atg3 intermediate in complex with the Atg12-Atg5/Atg16. In yAtg3, a segment of fourteen residues (Ile129 to Lys142), referred to as Atg3E123IR , was shown to interact with residues of helices F and G and functionally inhibit its conjugase activity in the apo state. Removing this auto-inhibition by the Atg3E123IR/Atg12-Atg5 interaction was suggested to be a requirement for activating yAtg3. However, the corresponding residues of Atg3E123IR and its key interacting partners on helices F and G are not conserved in hAtg3. Additionally, yAtg3 has an active AIM/LIR motif, while higher organisms have evolved to lose the LIR from Atg3. Therefore, the Atg12-Atg5/Atg16 complex may use different molecular mechanisms to activate yAtg3 and hAtg3 for Atg8-PE and LC3-PE conjugations. Nonetheless, the plasticity of Atg3’s catalytically important C-terminal regions and their versatile interactions with the membrane, LC3, and the Atg12-Atg5/Atg6 complex (direct or indirect) are keys to the activation and regulation of its conjugase activity.Future studies, in particular structure determinations of the LC3-hAtg3 intermediate in the absence and presence of the Atg12-Atg5/Atg16 complex at the membrane surface, will illustrate how the multifaceted membrane association of NAH and C-terminus of hAtg3 tightly coordinates its curvature sensitivity and conjugase activity. In a recent study of yAtg3In summary, in this study we determined the solution structure of hAtg3 and demonstrated that its catalytic loop and adjacent helix F are conformationally dynamic in aqueous solution. Moreover, we identified direct interactions between its catalytically important C-terminal regions and the membrane. The functional implications of these hAtg3 C-terminal/membrane interactions were confirmed by mutagenesis and in vitro and in vivo assays. These interactions functionally link hAtg3 conjugase activity to NAH curvature sensitivity, thereby restricting LC3-PE conjugation to the highly curved rim of the phagophore. Thus conceptually, the membrane functions like an-E3 ligase for LC3-PE conjugation.−/− mouse embryonic fibroblasts (MEFs) provided by Dr. Shengkan (Victor) Jin  were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1× Antibiotic Antimycotic Solution (AA) .HEK293T cells  and Atg3F296S, and hAtg3F296L cDNAs were amplified by PCR . Purified PCR products were sub-cloned into pCDH1-CMV-mCherry-MCS-EF1-puro viral backbone using NheI and BamHI.hAtg3, hAtg3−/− MEFs and incubated for 24 h. Puromycin selection was started after 48 h post transduction and remained for 3 days. MEF cells were sorted with flow cytometry , v2) based on mCherry expression level. MEF cells with high expression of mCherry were used for in vivo experiments.Lentivirus was made by transfecting HEK293T cells with ViraPower packaging plasmids  from Invitrogen (K497500) and the viral vector using JetPrime . Four hours after transfection, the culture medium was replaced. The viral supernatant was collected at 24 and 48 h post-transfection, and filtered with 0.45 μM supor membrane . The viral supernatant was added to Atg3−/− MEFs expressing mCherry-hAtg3 in starvation medium with BafA1 was set to 1. The autophagic flux was calculated by subtracting the LC3-II value in the absence of BafA1 from that in the presence of BafA1 in starvation medium, and the values were then normalized with the value of Atg3−/− MEFs expressing mCherry-hAtg3 being set as 1. The data were analyzed by one-way ANOVA test followed by Turkey’s multiple comparisons test using GraphPad Prism7.0.Cells were incubated in complete or starvation media in the absence and presence of 100 nM bafilomycin A1 (BafA1)  for 3 h and subjected to immunoblotting using the mCherry antibody  with a ratio of 1:1000, the β-actin antibody  with a ratio of 1:10,000, and the LC3 antibody  with a ratio of 1:5000. To quantify the LC3-II expression level, western blot experiments were performed 4 times. The signal intensity of LC3-II was measured using Image Studio version 5 software (LI-COR Biotechnology), then normalized to the intensity of β-actin of the same sample. To calculate the relative LC3-II level, the LC3-II value of Atg330. All other hAtg3 mutants were generated using the Q5 Site-Directed Mutagenesis Kit  or QuikChange Multi Site-Directed Mutagenesis Kit  with corresponding primers  and D2O ) and 15NH4Cl  for 15N  labeled samples at 37 °C until OD600 reached 0.6. The temperature was then lowered to 25 °C and cells were induced with 0.5 mM IPTG for ~16 h and then harvested by centrifugation. For 13CH3-ILV-labeling, a single colony was cultured in 5 mL LB medium for 7 h. Cells from 0.2 mL of the culture were pelleted down, washed with 0.5 mL M9 D2O medium once, and then inoculated into 10 mL fresh M9 D2O medium at 37 °C for 15 h. The culture was then transferred into 400 mL fresh M9 D2O medium and incubated at 37 °C until OD600 reached 0.6. The temperature was then lowered to 25 °C, α-Ketobutyric acid-4-13C,3,3-D2  and 2-Keto-3-methyl-13C-butyric-4-13C,3-D acid  were added to the growth medium. After incubation for 1 h, cells were induced with 0.5 mM IPTG for ~16 h.Full-length human Atg3 (hAtg3), subcloned into the pET28a expression vector at the BamHI/XhoI site with His- and T7-tags at the N-termini, was kindly provided by Dr. Xuejun Jiang . For the studies here, a thrombin cleavage site was introduced at just before the start of hAtg3 N-terminus (with two amino acid residues Gly-Ser left with the rest of hAtg3 after thrombin cleavage), or hAtg3 without the N-terminal first 25 residues . These constructs as well as hAtg3 without residues 90–190 were obtained previously using primers listed in Supplementary Table 22 supplemented with benzonase nuclease  and complete protease inhibitor cocktail . The cells were lysed by sonication on ice with 2 s on and 7 s off intervals for 18 min total duration. Cell debris was removed by centrifugation  at 26,900 × g at 10 °C for 30 min. Supernatants were collected, and loaded onto a Ni-NTA column . The column was washed with PBS buffers  without and with 30 mM imidazole, and then eluted with PBS buffer containing 500 mM imidazole. The elute of hAtg3 and its mutants  were concentrated for biochemical assays. For NMR experiments, the elute of proteins from the Ni-NTA column was concentrated and exchanged to a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, and 2 mM BME, followed by the addition of 0.1% (v/v) TWEEN20  and 50 units thrombin  to remove the T7- and His-tags for an overnight agitation at 4 °C. The solution was then subjected to a Q-column  and further purified by size-exclusion chromatography using an S200 column  and a buffer of 50 mM HEPES, 1 M NaCl, and 1 mM DTT. LC3B and mouse Atg7 for in vitro assays were prepared as described previously30. Purified proteins were exchanged into a buffer of 50 mM HEPES, pH 7.5  or pH 6.5 (for NMR structure determination in aqueous solution), 150 mM NaCl, and 2 mM TCEP (tris(2-carboxyethyl)phosphine). Protein concentration was assayed using a Nanodrop .Cell pellets containing expressed proteins were suspended in a lysis buffer of 20 mM phosphate, pH 7.5, 300 mM NaCl, 2 mM beta-mercaptoethanol (BME), and 1 mM MgCl30. Typically, 5.0 μM hAtg3 or its mutant was mixed with 5 μM LC3B, 0.5 μM mouse Atg7, 1 mM MgCl2 in a reaction buffer  of 37 μL. 3.7 μL were removed for a control. Then 1 mM ATP (0.45 μL of 100 mM ATP) was added, and the reaction was allowed to proceed for 30 minutes at 37 °C for intermediate formation. 3.75 μL were removed to check for intermediate formations, and an additional 3.75 μL were incubated separately as a non-liposome containing control (CL). 1 mM sonicated liposomes  were then added to the reaction system . The reaction proceeded at 37 °C, with 5 μL aliquots removed at 0, 15, 30, 60, 120, and 180 minutes. Samples were mixed with 4 μL 4× protein loading buffer , and stored at −20 °C until analyzed by 18% polyacrylamide gel electrophoresis after heating for 3 min. 4× loading buffer without BME was used for the gel analysis of intermediates. Gels were imaged on a BioRad Chemidock MP imager, and analyzed using ImageJ. For the preparation of sonicated liposomes, POPC , DOPG , DOPE  in chloroform were added to a glass tube and dried to a thin film by spinning with heat for half an hour using a condenser rotor SpeedVac, followed by lyophilization overnight once the volatile organics were removed. Lipids were rehydrated with H2O for 1 h at 42 °C, with vortex and then freeze at −80 °C every 15 min. The mixture was then for bath sonication (BRANSON 3510R-MT Bransonic Ultrasonic Cleaner). The lipids were sonicated for 4 × 15-min intervals until clear, then stored at room temperature.In vitro LC3B-PE conjugation was conducted as previously described30. Typically, 2 μM hAtg3 (or its mutants) was incubated with 800 μM sonicated liposomes (POPC/DOPG/DOPE with a molar ratio of 3:2:5) or without liposomes as a control in a buffer A of 50 mM HEPES, 150 mM NaCl, and 2 mM TCEP at 37 °C for 1 h. After incubation, 12 μL from a total of 300 μL mixture were removed and mixed with 4 μL 4× protein loading buffer . The remaining mixture was centrifuged at 160,000 × g for 2 h at 4 °C (Optima MAX Ultracentrifuge). After removing the supernatant, the pellets were gently washed once with 288 μL buffer A and resuspended in 288 μL buffer A. Then, 12 μL of the supernatant (S) and the pellet resuspension (P) were mixed with 4 μL 4× protein loading buffer, respectively, for gel electrophoresis . Samples were heated at 95 °C for 3 min for gel electrophoresis.Liposome co-sedimentation assay was conducted as previously described57. Briefly, a stock solution of 40% neutral acrylamide and N, N′-methylenebisacrylamide in a 19:1 ratio  was mixed with a stock solution of 40% positively charged (3-acrylamidopropyl)-trimethylammonium chloride  or negatively charged 2-acrylamido-2-methyl-1-propanesulfonic acid  containing N,N’-methylenebisacrylamide  in a 19:1 ratio in equal volume. The mixtures or the stock solution containing only neutral acrylamide were diluted with 10× TBE buffer  to a final 5% acrylamide concentration for the negatively-charged or neutral gel, or a final 7% acrylamide concentration for the positively-charged gel. Polymerization was initiated by adding 0.1% ammonium peroxide sulfate and 1% tetramethylethylenediamine . The polymerization of 130 µL of the mixtures was carried out in a 3.2 mm diameter plastic tube. Polymerized gels were extensively washed in deionized water, and the water was changed at least 3 times within 2 days. The gels were dried over a 2-day period at 37 °C on a plastic support wrapped with polyvinylidene chloride (PVDC) foil. The dried gel was transferred to a 5 mm Shigemi tube for the NMR sample, and the protein solution was added. Vertical compression of the gel was achieved by inserting the Shigemi plunger into the tube and limiting the final length at around 14 mm. The sample was ready for NMR measurements after equilibration for at least 8 h at 4 °C. For a phage aligned NMR sample, 500 μM 15N, 2H-labeled protein was dissolved in 50 mM HEPES, pH 6.5, 150 mM NaCl and 2 mM TCEP containing 6.5 mg/mL phage Pf1 .For the RDC experiment, we prepared charged polyacrylamide gels as described previouslyG120C- hAtg3Δ90–190, H266L, C50A, C81A, C85A, C259A linked by a disulfide bond was prepared following a published protocol43. Briefly, 500 μM unlabeled LC3BG120C was first reduced by 10 mM DTT and desalted into buffer B . Then this LC3BG120C protein solution was mixed with 1:1 v/v buffer C ), incubated at room temperature (RT) for 20 min to form DPS-LC3BG120C, and desalted again into buffer B. Separately, 300 μM perdeuterated 13CH3-ILV-labeled hAtg3Δ90–190, H266L, C50A, C81A, C85A, C259A was reduced by 10 mM DTT and desalted into buffer B. Finally, this hAtg3 protein solution was mixed with DPS-LC3BG120C solution at a 1:1.5 molar ratio and incubated at RT for 1 h to form LC3BG120C- hAtg3Δ90–190, H266L, C50A, C81A, C85A, C259A. Cross-linking product was directly desalted into a buffer of 50 mM HEPES, pH 7.5, 150 mM NaCl and verified by SDS-PAGE gel with a purity of >90%.LC3B3. For H/D exchange experiments, 0.5 mM 15N-labeled proteins in 50 mM HEPES, pH 6.5, 150 mM NaCl, and 2 mM TCEP were lyophilized, and the powder was then dissolved in D2O  for immediate NMR collection. For bicelle samples, typically 0.5 mM 15N, 15N/13C, or 15N/13CH3-ILV 2H-labeled proteins were mixed with a final concentration of 12% (w/v) bicelles , or DMPC:DMPG:DHPC = 8:2:20 , or DMPC:DMPG:LPE:DHPC = 7:2:1:20 , or DMPC:DMPG:LPC:DHPC = 7:2:1:20  in 25 mM HEPES, pH 7.5, 75 mM NaCl, 2 mM TCEP. Bicelles were prepared as previously described30. Briefly, DMPC , DMPG , or LPE , or LPC  were mixed with double distilled H2O (ddH2O), frozen at −80 °C, and thawed at 42 °C for 3 freeze-thaw cycles. DHPC  in ddH2O was then added into the mixture and quickly vortexed to form a clear solution. The solution was frozen at −80 °C and slowly thawed at room temperature for use. For PRE experiments, 5-DSA  powder was dissolved in 24% (w/v) bicelles as a stock solution (100 mM), and titrated into NMR samples to reported concentrations; the mixture was vortexed, frozen at −80 °C, and thawed at room temperature for NMR data collections.Typical NMR samples contained 0.5 mM labeled proteins in a buffer of 50 mM HEPES, pH 6.5, 150 mM NaCl, 2 mM TCEP, and 0.02% NaN13C-edited HCCHTOCSY, 13C-edited NOESY, HCACO, 15N-edited NOESY and HBHA(CO)NH, 2D Hb(CbCgCd)Hd and Hb(CbCgCdCe)He spectra (Bruker Topspin3.2 pulse sequence library). The 3D 13C-NOESY and 15N-NOESY experiments (Bruker Topspin3.2 pulse sequence library) were collected with 150 ms mixing time. 1DNH and 2DC’H RDCs for resonances with little perturbation between an aqueous solution and alignment medium were measured using the ARTSY method58 and an IP-HSQC experiment59, respectively.All NMR data were acquired at 25 °C on a Bruker 600 MHz spectrometer except the NOESY data that were acquired on a Bruker 850 MHz spectrometer. Both instruments are equipped with cryoprobes. The data were processed using NMRPipe3.0 and analyzed using NMRViewJ (Version 9.1.0-b55 with Java 1.8.0). Backbone resonance assignments were carried out using triple resonance HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB experiments (Bruker Topspin3.2 pulse sequence library). Aliphatic and aromatic side-chain resonance assignments were obtained from the 3D α, Cβ, N, and C’ chemical shifts using TALOS+. NOESY assignments were obtained by a combined manual and automated analysis with CYANA 3.0. The structures were calculated and refined with XPLOR-NIH 3.3. PROCHECK, PSVS, and PDB validation servers were used to analyze the ten lowest-energy structures with acceptable covalent geometry. In the final structures, the percentage of residues that reside in the most favored, additionally allowed, and generally allowed regions of the Ramachandran diagram are 90.6%, 9.3%, and 0.1%, respectively, for ordered residues .Torsion angle restraints were derived from CP value < 0.05 was considered the threshold for statistical significance. The significance intervals (*) for P values are provided within the figure legend, along with the specific statistical test performed for each experiment: one-way ANOVA test followed by Turkey’s multiple comparisons test. The number of experimental replicates  is indicated within figure legends. The cell lines utilized were regularly authenticated through morphologic inspection and mycoplasma testing. To minimize clonal bias, the experiments were performed using pooled mCherry-positive cells sorted by flow cytometry.Data are presented as mean values ± SD (standard deviation), calculated using GraphPad Prism 7.0. Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Streptococcus than V3–V4 primers. In conclusion, primers targeting the V1–V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections.Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1–V2, V1–V3, V3–V4, V4–V5, V5–V7 and V6–V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1–V2, V3–V4, and V4–V5 regions generated more than 90% of the original input sequences. Primers targeting the V1–V2 and V1–V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1–V2 and V3–V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1–V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for  Microbiota are found in many habitats but are poorly characterized due to a high proportion of unnamed species-level taxa ,2. HowevThe use of targeted amplification and sequencing of the 16S ribosomal RNA gene to characterize the human oral bacterial community forms the basis of the Human Oral Microbiome Database . The 16SAlthough a small portion of the 16S rRNA gene has been considered sufficient to represent the full-length sequence in many community analyses , it is ialign_to_tree_mafft_fasttree implemented in QIIME2 (version 2020.6)  2020.6) .Plaque samples were obtained from all participants at the Department of Periodontics of Pusan National University Dental Hospital . Buccal swab samples were obtained from the mucosa of both cheeks using a sterile dental microbrush . Supragingival samples were collected by swabbing the surfaces of mesiobuccal molar sites. Participants were requested to refrain from food and oral hygiene (brushing or flossing the teeth) for 2 h before sampling, and all provided written informed consent. The study protocol was approved by the Institutional Review Board of Pusan National University Dental Hospital (PNUDH-2017-023). Samples from all subjects were stored at −80 °C until required.Total DNA was extracted from buccal and supragingival plaque using a Gram-positive DNA purification kit  following the manufacturer’s instructions. Final DNA concentrations were measured with a NanoDrop ND-1000 spectrophotometer . Samples were stored at −80 °C until required. The V1–V2 and V3–V4 regions of the 16S ribosomal RNA gene were subjected to PCR amplifications. The primer sequences used are listed in Basic microbiome analysis has been performed using QIIME2  and its Prevotella, Treponema, Capnocytophaga, Leptotrichia, Porphyromonas, and Fusobacterium. Taken together, primers targeting different sub-regions of 16S rRNA differed substantially in terms of the extent to which they confidently detected the oral microbiome.For in silico simulation, a set of full-length 16S sequences commonly used for oral microbiome studies was downloaded from a public database (eHOMD). Since these sequences incorporate PCR primer-binding sites, in silico amplicons for different sub-regions were generated using PCR primers commonly used in microbiome studies . First, Actinomyces, Staphylococcus, and Streptococcus genera than V1–V2 or V1–V3 primers  and the total 16S rRNA region. The phylogenetic trees constructed by primers targeting V1–V2 and V3–V4 had similar structures, whereas the V4–V5 primer pair produced a tree distinct from that produced by the total 16S rRNA primer pair . Periodoenticola . Phylogebspecies . OverallFollowing in silico simulations of oral microbiome detections, clinical samples were used to assess the performance of the V1–V2 and V3–V4 primers. In total, 70 samples from healthy adults  were used for the clinical study . First, After classifying taxa, species assigned by each primer and taxa predicted to be detected by the primers were compared. In terms of species detection, V1–V2 primers detected 335 species, with 108 taxa remaining unassigned at the species level. On the other hand, V3–V4 primers detected 269 species, with 102 taxa remained unassigned at the species level. Furthermore, V1–V2 primers exclusively assigned 98 species, whereas V3–V4 primers assigned only 32 species. Consequently, V1–V2 primers more effectively detected bacterial species B.Streptococcus, Haemophilus, Rothia, Gemella, Lautropia, Prevotella, Leptotrichia, Neisseria, and Fusobacterium, which accounted for over 60% of the total sequences for V1–V2 primers and over 70% for V3–V4 primers. As was observed for phylum analysis, the relative abundance of Streptococcus was significantly higher for V3–V4 primers (35.9%) than for V1–V2 primers (15.8%)  than V1–V2 primers (36.5%) C. At the (15.8%) D.Streptococcus on the relative abundances of other taxa, the correlation between V1–V2 and V3–V4 was analyzed after removing Streptococcus at the genus level. The correlation coefficient between V1–V2 and V3–V4 at the genus level was 0.72 when all taxa were evaluated . In contrast, V3–V4 primers identified 34.5% of taxa as Streptococcus, only assigning eight taxa to the species level (2.22%), and assigned the majority of taxa to the genus level (32.2%). Especially, V3–V4 primers failed to identify any S. oralis subspecies and S. mitis. For Fusobacterium analysis, both primers detected similar relative abundances at the genus level. V1–V2 primers identified nine species at the species level (2.67%), whereas V3–V4 primers assigned only three species at the species level (1.89%). V1–V2 primers distinguished four subspecies of F. nucleatum, whereas V3–V4 primers only detected F. nucleatum subvincetii. Taken together, V1–V2 primers outperformed V3–V4 primers by detecting a greater number of oral bacterial species and exhibiting higher resolutions for the identifications of Streptococcus and Fusobacterium at the species level  than for V1–V2 primers (15.8%). Since Streptococcus is one of the most prevalent bacteria in the oral cavity [Streptococcus was removed before comparing the abundance of other bacteria, the abundances of the other genera were similar, which suggests that primers exert a significant selective influence on particular species, and if these species are abundant within a community, it might impact the overall abundance of other bacteria.Analysis of average relative abundances revealed that the abundance of Bacillota was higher for V3–V4 primers (50%) than for V1–V2 primers (36.5%). Similarly, the relative abundance of l cavity , its abuStreptococcus and Fusobacterium. V1–V2 primers successfully assigned 58% of Streptococcus to 24 species, leaving 41% identified to the genus level, whereas V3–V4 primers only assigned 9.5% of Streptococcus to eight species and identified the majority of taxa at the genus level (93.5%). In silico, Streptococcus phylogenetic trees showed that V1–V2 primers could identify most streptococci to the species level, while V3–V4 primers failed to differentiate S. oralis and S. mitis , and V1–V2 primers identified nine species (2.67%), while V3–V4 primers identified three species (1.89%). Furthermore, V1–V2 primers were able to distinguish four subspecies of F. nucleatum, whereas V3–V4 primers only detected F. nucleatum subvincetii. Overall, these results suggest that the V1–V2 primers are more accurate than V3–V4 primers for identifying Streptococcus and Fusobacterium at the species level in oral microbiome studies.Analysis at the species level revealed differences in the relative abundances of S. mitis . Taken tStreptococcus and other species of interest were not quantified, and thus, we suggest that in future studies, this be undertaken by qPCR. Finally, since targeting specific regions of 16S rRNA can cause taxonomic classification ambiguities, we suggest that combining this with long-read sequencing should enable more accurate and sensitive identification of bacteria [The study results are conclusive, but there are several limitations. First, the number of clinical samples used in this study was relatively small. Thus well-designed, larger-scale studies are required to validate our results. In addition, various sampling sites, age groups, ethnic backgrounds and clinical diseases should be included in a future study. Second, the abundances of Streptococcus species. Since Streptococcus constitutes a significant proportion of oral microbial communities, our findings suggest that the V1–V2 primer set should be preferred when investigating microbial community dynamics in the oral environment.To conclude, our findings have significant implications for oral microbiome studies. In particular, they emphasize the importance of selecting PCR primers carefully. Furthermore, they show that the primary advantage of using the V1–V2 primer set is its superior ability to identify more taxa and its higher resolution for


Heparins, naturally occurring glycosaminoglycans, are widely used for thrombosis prevention. Upon application as anticoagulants in cancer patients, heparins were found to possess additional antitumor activities. Ectonucleotidases have recently been proposed as novel targets for cancer immunotherapy.In the present study, we discovered that heparin and its derivatives act as potent, selective, allosteric inhibitors of the poorly investigated ectonucleotidase NPP1 . Structure-activity relationships indicated that NPP1 inhibition could be separated from the compounds’ antithrombotic effect. Moreover, unfractionated heparin (UFH) and different low molecular weight heparins (LMWHs) inhibited extracellular adenosine production by the NPP1-expressing glioma cell line U87 at therapeutically relevant concentrations. As a consequence, heparins inhibited the ability of U87 cell supernatants to induce CD4+ T cell differentiation into immunosuppressive Treg cells.NPP1 inhibition likely contributes to the anti-cancer effects of heparins, and their specific optimization may lead to improved therapeutics for the immunotherapy of cancer. It prevents the formation of blood clots, and has therefore been established since decades as a subcutaneously applied anticoagulant drug. While other, orally available anticoagulants are becoming increasingly important, heparin has retained its outstanding role as a drug of choice in oncology for the prophylaxis and treatment of cancer-associated thrombosis –5. Severi) (+), diadenosine polyphosphates and cyclic dinucleotides such as cyclic guanosine--monophosphate-adenosine--monophosphate   and NTPDase1 (CD39) , enzymesi) , 14. CD7i) , 14. Thei) . In addiAMP) see Figure 1AMP) see –17. InhiAMP) see .In the present study, we investigated the potential of heparin and heparin-derived sulfopolysaccharides to inhibit ectonucleotidases. In fact, we discovered that heparin and its derivatives are potent and selective allosteric inhibitors of NPP1. This mechanism of action may contribute to the anti-cancer effects observed for heparins. The present results and the observed structure-activity relationships provide the basis for the future development of more potent NPP1 inhibitors lacking the associated anti-thrombotic activity.22.1®, Merck KGaA, Darmstadt, Germany. Sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogencarbonate, D-glucose, HEPES (N-[2-hydroxyethyl]-piperazine-N’-[2-ethanesulfonic acid]), calcium chloride, and magnesium sulfate were purchased from PAN Biotech GmbH . Dulbecco’s Modified Eagle Medium, fetal calf serum (FCS), penicillin/streptomycin (P/S) mixture, and L-glutamine were also obtained from PAN Biotech GmbH. The commercially available heparins were purchased from the following sources: unfractionated heparin-sodium : Heparin-5000-ratiopharm from Ratiopharm GmbH, Ulm, Germany; LMWHs: tinzaparin (2), Innohep® from LEO Pharma, Ballerup, Denmark, dalteparin (3), Fragmin® from Eurim Pharma GmbH, Saaldorf-Surheim, Germany, nadroparin (4), Fraxiparin®, Berenga Arzneimittel, Baden-Baden, Germany; enoxaparin (5), Clexane® from Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany; fondaparinux (6), Arixtra® from Aspen Pharma Trading Ltd, Dublin, Ireland. The non-commercial compounds 7-15 were synthetized as described , nucleosides , dipyridamole (DP), EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), disodium hydrogen phosphate, and sodium dodecyl sulfate were purchased from Sigma-Aldrichibed see .2.2N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2 mM CaCl2, 1 mM MgCl2, pH 9.00) and incubated with 20 ng of human recombinant soluble NPP1  expressed in murine myeloma NS0 cells obtained from R&D Systems GmbH . For initial screening and selectivity analysis, NPP1 prepared as previously described , and 7  versus four different substrate concentrations ranging from 50 to 500 μM of ATP. The assay procedure and operation conditions were the same as described above. The experiment was conducted twice, each in triplicates. A Lineweaver-Burk was calculated using GraphPad Prism 7.0 for predicting the inhibition type of the compound.Concentration-inhibition curves of the test compounds at human NPP1 were performed with ATP as substrate at a final concentration of 400 μM. Several different dilutions of the test compounds were prepared in assay buffer (10 mM 2.3p-nitrophenyl thymidine monophosphate (p-Nph-5´-TMP) as substrate to study potential inhibition of NPP3 and NPP5 using a colorimetric assay as previously described , and 0.09 µg of NPP3, or 0.4 µg of NPP5 were employed. The mixtures were incubated for 30 min at 37°C in case of NPP3, and for 100 min at 37°C in case of NPP5, and the reactions were subsequently terminated by adding 20 μL of 1.0 N aq. NaOH solution. The assay is based on the enzymatic ester hydrolysis of p-Nph-5’-TMP which results in the formation of the yellow p-nitrophenolate anion. The absorption was measured at 400 nm using a BMG PheraStar FS plate reader . Each analysis was repeated three times with triplicate measurements.Compounds 1, 2, 5, 6, and 7 were screened at different concentrations using 400 μM of escribed . Solubleescribed , and humescribed  were dil2.44A) as a substrate and human soluble NPP4 . The enzyme reaction was started by adding 0.14 μg of purified human NPP4 to the reaction mixture, which was incubated at 37°C for 90 min. The released product ATP was quantified using the luciferin-luciferase reaction. The firefly luciferase reacts with D-luciferin in the presence of the formed ATP and Mg2+. The resulting luminescence is a measure of the enzymatic activity, and the luminescence was read at 560 nm using a microplate reader (BMG PheraStar). Three independent experiments were performed, each in duplicate measurements; positive and negative controls were included.Selectivity studies on NPP4 were performed using a published luminescence-based assay versus 20 μM of diadenosine tetraphosphate  was 100 µM for all studied human NTPDases . Mixtures of enzymes with substrate and test compounds were incubated at 37°C for 30 min, and the enzymatic reaction was stopped by heating for 10 min at 95°C. The released products were separated by CE and quantified by their UV-absorption at 260 nm with ADP and AMP as external standards, as previously described  in the presence of test compounds, a CE-based assay system was used, according to a published procedure , 25. Theescribed ; positiv2.63H]AMP . Compound 7 was used at 800 µg/mL and compound 6 at 20 µg/mL for the selectivity test, while for initial screening compound 5 was tested at a concentration of 55 µM and 6 at 30 µM, and 1 and 2 were tested at 50 IU/mL (3H]-AMP. After the precipitation was completed (after at least 30 min on ice), the mixture was separated by filtration through GF/B glass fiber filters using a cell harvester . After washing each reaction vial three times with 400 µL of cold (4°C) demineralized water, 5 mL of the scintillation cocktail  was added and the radioactivity was measured by scintillation counting . Positive and negative controls were included.The assay was performed essentially as previously described . The enz2.7The mRNA isolated from several primary human glioblastoma cell specimens, namely 46Z, 78Z, 106Z, 138Z, from adults’ human brain-derived neural progenitors, 155Z and 167Z, and mRNA isolated from human glioblastoma cell lines T98G, LN229, U138, and U87, were studied with respect to gene expression of ectonucleotidases. A two-step RT-qPCR (quantitative reverse transcription polymerase chain reaction) technique was used. The reverse transcription and PCR amplification steps were performed in two separate reactions. mRNA samples from the cell lines (1 µg) were treated with 1 µL of DNase I  for the removal of contaminating genomic DNA, incubated at room temperature, followed by addition of 25 mM of EDTA (ethylenediaminetetraacetic acid) and incubated at 65°C for 10 min. Then, the reverse transcription was achieved with the iScript cDNA Synthesis Kit  using 0.5 µg of reaction mix available from the manufacturer, 1 µL of reverse transcriptase and RNA templates. Subsequently, 1 µL (5000 U/µL) of RNase H digest  was added to each cDNA sample and incubated for 20 min at 37°C. Finally, the cDNA samples were diluted to a final concentration of 2.5 ng/µL, and qPCR was performed using SYBR Green ; the cDNA (5 ng) was mixed with 250 nM of the respective sequence-specific primers of the different investigated ectonucleotidases, which have been checked for their efficiency . The isolated RNA was subsequently used for first strand cDNA synthesis, which was performed with the SuperScript Reverse Transcriptase IV kit . The reverse transcription was carried out according to the manufacturer’s instruction with 1 µg of total RNA and 50 pmol of oligo d(T)20 primer. PCR amplification of the NPP1 cDNA was achieved with the gene-specific forward primer 5’-GCCAAGGACCCCAACACCTATAAAG-3’ that binds downstream of the initial GC-rich region of the coding sequence (CDS) of NPP1 thereby omitting the first 204 bp of the CDS, and the following reverse primer 5’-CGACAGTTTCTGAACAATGCAATAG -3’. The PCR product was purified and extracted from a 1% agarose gel using the Zymoclean Gel DNA Recovery Kit . Afterwards, the purified cDNA was cloned into the pJet1.2 vector  following the manufacturer’s blunt-end cloning protocol. Then, the reaction product was transformed into competent DH5α cells. Multiple clones were sequenced using the standard pJet1.2for and pJet1.2rev primers, as well as the NPP1 gene specific primer 5’-GGATTGTATCCAGAATCTCATGGC-3’. The sequences were analyzed using SnapGene software (www.snapgene.com) and Clustal Omega  mixture and 1% L-glutamine. When confluent, the cell culture media was carefully removed and cells were frozen at -20°C overnight. The cells were then scraped off with a cell scraper using 2-3 mL of buffer , homogenized and centrifuged for 10 min at 1000 g. The supernatant was collected and centrifuged for 60 min at 48000 g and 4°C. The pellet was then resuspended twice with Tris buffer, pH 7.4, followed by vortexing and centrifugation. The whole procedure was performed on ice. Finally, the pellet was resuspended and homogenized. A protein concentration of 1.5 mg/mL was determined with the Bradford method  and detached with EDTA. Thereafter, they were counted by a Cell Counter CASY® 1 Model TT  to retrieve the number of viable cells, and washed three times with PBS before they were resuspended in Krebs-HEPES buffer at a concentration of 106 cells/mL (H-purine-9-ethanol hydrochloride)  measurements, the samples were centrifuged at 600 g to remove insoluble material like cell debris from the supernatant. Until measurement, samples were stored at -20°C. Nucleotides  and nucleosides , which were used as standard compounds to monitor efficient separation by CE, were dissolved in Krebs-HEPES buffer at a concentration of 10 µM and stored at -20°C (U87 human glioblastoma cells were grown in cell culture flasks until reaching a fully covered confluent cell layer. Cells were cultivated at 37°C and 5% COcells/mL . Adenosihloride) . Heparinhloride)  were addat -20°C . The capat -20°C .2.11+ T cells were extracted from buffy coats by positive selection using the StraightFrom™ Buffy Coat CD4 MicroBead Kit . CD4+ T cells were stimulated by ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator at day 1. At days 1 and 3, the indicated compounds or U87 cell supernatant, either of untreated or of heparin-treated cells were added to CD4+ T cells. Cells were harvested at day 5 and were investigated for CD25 and FoxP3 expression. Therefore, cells were washed once with staining buffer . To avoid nonspecific binding, cells were blocked for 15 min with 3% BSA in PBS. Anti-CD25 antibody  was added and the cells were incubated for 30 min in the dark. Subsequently, cells were washed once with staining buffer and fixed in 150 µL of fixation buffer , 0.1% saponin, and 0.5% Tween-20 in Dulbecco's phosphate-buffered saline) for 30 min at 4°C. Samples were washed once with permeabilization buffer . Next, cells were permeabilized in 75 µL of permeabilization buffer for 30 min at 4°C. Samples were spiked with anti-FoxP3 antibody, fluorescein isothiocyanate ((FITC-labeled), RRID : AB_439752, BioLegend Inc.) diluted in 25 µL of permeabilization buffer. After an incubation period of 30 min at 4°C, cells were washed twice with 150 µL permeabilization buffer and finally resuspended in 500 µL of staining buffer and transferred into a suitable plate. Samples were measured in triplicates using the Guava® easyCyte HT Flow Cytometer . Compensation and FMO (fluorescence minus one) controls were conducted. The gating strategy is displayed in Human CD43Heparin is a complex mixture of naturally occurring anionic, sulfated glycosaminoglycans extracted from pig gut mucosa. Unfractionated heparin  whose molecular weight is ranging from 8-25 kDa, is a linear polymer made up of 1→4 linked disaccharide repeating units, consisting of α-D-glucosamine, 2-deoxy-2-sulfamino-D-glucose, 2-acetamido-2-deoxy-D-glucose, α-L-iduronic or β-D-glucuronic acid with 2.7 (average number) sulfated groups per disaccharide repeating unit. Low molecular weight heparins (LMWHs) are obtained from UFH by chemical or enzymatic depolymerization resulting in polysaccharide fragments of smaller size. Three commercially available heparins, UFH (1), the LMWHs tinzaparin (2), and enoxaparin (5), and the synthetic pentasaccharide fondaparinux (6)  and tinzaparin (compound 2), dalteparin , and nadroparin  were applied as further representatives of LMWHs, in addition to enoxaparin (compound 5), displaying decreasing molecular weights in this order. Fondaparinux (compound 6) is a fully synthetic anticoagulant, which is based on the pentasaccharide sequence that represents the antithrombin binding site of heparin that is responsible for the anticoagulant effect. In addition to commercially available drugs, we tested the non-commercial heparin derivatives 7-15 see Figure 250 values ranged from 0.503 µM for UFH (compound 1) to 73 µM for G4271B (compound 11). Among the clinically used heparins, UFH (compound 1) and fondaparinux (6) appeared to be the most potent ones in inhibiting NPP1 activity with IC50 values of 8.29 µg/mL and 4.99 µg/mL, respectively, followed by tinzaparin (2) with an IC50 value of 18.4 µg/mL. These compounds have 2.7, 3.2 and 2.5 sulfate groups per disaccharide repeating unit on average. LMWHs possess a lower total number of sulfate groups compared to UFH; their IC50 values ranged from 20 to 190 µg/mL. Among the LMWHs, dalteparin (3) exhibits a MW of 6 kDa while nadroparin (4) and enoxaparin (5) have a lower MW of 4.5 kDa; compound 5 was found to be almost 5 times more potent than compounds 3 and 4.The IC50 values in a similar range, however a dodecasaccharide of 3.6 kDa (compound 10) showed stronger inhibition of NPP1 activity compared with 8 or 10 units . The non-anticoagulant heparin derivative (reduced oxyheparin) exhibited an IC50 value of 30 µM, thus, the ring-opening modification did not interfere with the compound’s inhibitory activity at NPP1. For direct comparison, the pIC50 values are depicted in Desulfation of position 6  instead of position 2  was advantageous, compound 9 being 35-fold more potent than 8. Compounds 10, 11, and 12, which differ by two or four units in length, displayed IC3.2Next, we studied the selectivity of some heparins versus other members of the ectonucleotidase family. UFH (1), fondaparinux (6), and G9694 (7) were selected representatives for anticoagulant and non-anticoagulant heparin derivatives Figure 43.3max values in m values (see compound 6) indicating preferred binding to the enzyme-substrate complex, or to increased Km values indicating preferred binding to the free enzyme (see inhibitor 7).Next, we investigated the mechanism of inhibition for compound 7 (G9694) and compound 6 (fondaparinux) as group representatives for semisynthetic non-anticoagulative heparin derivatives and for commercially available anticoagulants. ATP hydrolysis by NPP1 was determined in the absence and in the presence of the inhibitors. Four different concentrations of the substrate ATP were employed , each tested in the presence of 0, 0.4, and 4 µM of fondaparinux (6), or 0, 4, and 40 µM of G9694 (7). For the determination of the inhibition type, Lineweaver-Burk plots were computed Figure 53.4ENPP-1, -2 and -3), NTPDases  and 5’-ectonucleotidase/CD73 (NT5E) was determined by RT-qPCR (quantitative reverse transcription polymerase chain reaction).To test the compounds in a more complex system, we searched for a native cell line with high NPP1 expression. Thus, mRNA expression analysis of ectonucleotidases in several tumor cell lines was carried out. Altogether, 10 different cell lines were investigated with respect to 8 genes encoding members of the ectonucleotidase family. Expression of NPP1 was previously reported to be increased in human astrocytic brain tumors (glioblastoma), and NPP1 expression was observed to increase with tumor grade . NPP1 waIn the initially performed broader screening Table 2ATable 2APreliminary quantitative mRNA expression analysis of ectonucleotidases in human cell lines determined by qPCR.Table 2BQuantitative mRNA expression analysis of selected ectonucleotidases in specific human cell lines determined by qPCR.3.5Cancer cell lines typically show many mutations. To investigate whether NPP1 expressed in the human glioblastoma cell line U87 corresponds to the wildtype sequence or bears mutations, we performed a sequencing analysis based on extraction of RNA . Our data clearly confirmed that the sequence of NPP1 expressed in U87 cells was identical to the wildtype human NPP1 sequence , and of 38 µM (for AMP) were determined adenine (EHNA) was therefore added to prevent adenosine degradation by ADA  to block cellular adenosine uptake by the solute carrier transporters SLC29, better known as equilibrative nucleoside transporters (ENT) , 42. Aden by ADA . CombineHeparins were tested in a range of different concentrations. All investigated compounds led to a dose-dependent inhibition of adenosine formation. To be able to relate the tested concentrations to the clinically employed concentrations of these anticoagulants, we adapted them to the therapeutically applied amounts, given in international units (I.U.) for heparins 1, 2 and 3, and in µg for fondaparinux (6). To use a comparable scale and to select a mean therapeutic concentration, as well as a higher one that slightly exceeds the typical therapeutic doses, we selected 1 I.U./mL and 5 I.U./mL for UFH (1), tinzaparin (2) and enoxaparin (5), and 2 µg/mL and 10 µg/mL for fondaparinux (6). The non-commercial heparin derivatives G9694 (7), G5645 (8), G6658 (9) were tested at 50 µM and 100 µM, compounds A3875-H6 (11) and A3875-I6 (12) were studied at 20 and 50 µM, while A3875-G6 (10) was evaluated at 10 and 25 µM. All investigated heparin derivatives were able to inhibit adenosine formation from ATP in U87 glioblastoma cells Figure 7The non-commercial heparin derivatives showed a potent, but lower inhibitory activity on the formation of adenosine in U87 tumor cells compared to the commercial derivatives )’ or those preincubated with 20 µM of the ectonucleotidase substrate ATP ‘(U87 (+))’. The latter solution induced a stronger differentiation of CD4+ T cells in Treg cells than the controls, indicating the formation of extracellular adenosine from ATP by the U87 cells. This value was considered as maximum effect, defined as 100%  of 1-1.5 h  reach the following Cmax values: Tinzaparin (2) dosed 175 IU/kg body weight s.c. once daily reaches ca. 0.5-1.0 IU/ml . Daltepa50 value determined for NPP1 inhibition. However, the negatively charged sulfopolyglycosides may accumulate at the extracellular cell membrane, where NPP1 is located, and local concentrations might actually be higher.The therapeutic dosage for fondaparinux (6) is typically 2.5 mg daily, and plasma concentrations have been reported to range from 0.1 to 0.5 mg/L for adult patients on thromboprophylaxis treatment, and from 0.6 to 1.5 mg/L for adults on therapeutic doses, the compound showing a half-life of 17 h . These cin vivo studies in suitable animal models.NPP1 inhibition may be beneficial for personalized medicine to treat cancers that show high NPP1 expression. Besides some brain tumors , certaini, and its inhibition will not only reduce adenosine, but also PPi concentrations. Since PPi is a key inhibitor of mineralization  SUB12934589 ENPP1_U87_K173Q (OQ569912).CM designed and supervised the study; VL performed experiments on NPP1, NPP4 and U87 membrane preparations. RI, CR, LS, SM, S-YL and VL performed selectivity experiments on various ectonucleotidases. HS and MS performed cell-based experiments under the supervision of GB. HS established and performed the immunological studies and performed with VV cDNA sequencing experiments of U87. AN synthesized and provided non-commercially available synthetic heparins. JP and JS produced and provided NTPDase preparations. BS collected and provided patient-derived cell samples. KS performed qRT-PCR analysis experiments. VL and CM wrote the manuscript with contribution from all coauthors. All authors have given approval to the final version of the manuscript.