{"paper_id":"7269ae15-5bf2-4d7d-b893-aa0c0990ebd3","body_text":"Society of Medical Education & Research \n \n2022, Volume 2, Issue 1, Page No: 58-84 \nISSN: 3108-4826 \n \n \nRegulation of GLUT1 Deubiquitination by UBE2S–USP10 Reprograms Metabolism and \nImmunity in Endometriosis \nLaura Beatrice Conti1*, Marco Antonio De Santis1, Chiara Lucia Bianchi1 \n1Department of Management, Catholic University of the Sacred Heart, Milan, Italy. \n \n*E-mail  l.conti.ucsc@outlook.com \n \n \nEndometriosis (EM) represents a persistent inflammatory condition marked by the presence of endometrium-like tissue outside \nits normal location and associated fibrotic changes. Alterations in metabolism, notably heightened glycolytic processes, alon g \nwith disruptions in the immune milieu, constitute prominent aspects of EM advancement. Nonetheless, the precise molecular \npathways involved are still inadequately clarified. The present investigation employed a combination of transcriptome profiling, \nimmunoprecipitation-mass spectrometry (IP -MS), co -immunoprecipitation, and ubiquitination experiments to thoroughly \nexplore the function of Ubiquitin-Conjugating Enzyme E2S (UBE2S) in controlling glucose metabolism and immune regulation \nwithin EM. Cellular assays in  vitro, along with murine models, were utilized to confirm its influence on glycolytic pathways, \nmacrophage polarization states, and fibrotic development. Expression of UBE2S was markedly elevated in stromal cells from \nectopic endometriotic sites. Through IP-MS, GLUT1 and USP10 emerged as principal partners interacting with UBE2S. \nDetailed mechanistic examinations demonstrated that UBE2S facilitates K48 -linked deubiquitination of GLUT1 via USP10, \nthereby maintaining GLUT1 protein stability and augmenting gl ycolytic flux. Such metabolic shifts result in increased lactate \nbuildup, which in turn triggers M2-type macrophage polarization and release of TGF-β1, consequently driving the conversion \nof fibroblasts to myofibroblasts and hastening lesion fibrosis. Admi nistration of the UBE2S inhibitor cephalomannine notably \nreduced GLUT1 levels, curtailed glycolysis, impeded M2 polarization, and mitigated fibrosis in ectopic sites.  The research \nelucidates the pathway whereby the UBE2S –USP10–GLUT1 axis modulates the imm une surroundings and advances fibrosis \nin EM via alterations in metabolism. These observations yield fresh perspectives on EM pathophysiology and establish a \nrationale for pursuing UBE2S as a target in treatment approaches.   \n \nKeywords: Endometriosis, UBE2S, GLUT1, USP10, Glycolysis, M2 macrophage polarization \nIntroduction \nEndometriosis (EM) constitutes a longstanding \ngynecological condition defined by the development of \nendometrium-resembling tissue beyond the uterus, \nresulting in ongoing inflammation, fibrotic alterations, \nand clinical issues like pelvic discomfort and subfertility \n[1, 2]. Despite extensive prior research into its origins, \nthe core molecular processes continue to be elusive. \nMounting data point to critical involvement of metabolic \nshifts and immune imbalances in EM evolution, chiefly \nthrough modified glucos e handling and impaire d \nimmune cell activity [3, 4]. \nLesions in endometriosis (EM) show a metabolic pattern \ndominated by glycolysis, where lactate arising from \nirregular glucose processing acts as an essential link \nconnecting ectopic tissues to immune components. \nElevated lactate fosters M2 macrophage polari zation, \nmodifying the immune context and facilitating immune \nescape [5]. These M2 macrophages additionally drive \nfibrosis via stimulation of fibroblast transformation into \nmyofibroblasts [4, 5]. Accordingly, immune regulation \nmediated by lactate potentiall y bridges glucose \n \nReceived: 12 November 2021; Accepted: 08 February 2022 \nCopyright CC BY-NC-SA 4.0 \nHow to cite this article:  Conti LB, Santis MAD, Bianchi  CL. Regulation of \nGLUT1 Deubiquitination by UBE2S –USP10 Reprograms Metabolism and \nImmunity in Endometriosis . J Med Sci Interdiscip Res . 2022;2(1):58-84. \nhttps://doi.org/10.51847/DMofvX2jn9 \nJournal of Medical Sciences and Interdisciplinary Research \n \nAbstract \n \nAccess this article online                              https://smerpub.com/ \n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n59 \nmetabolism to fibrotic advancement in EM. Inhibiting \nkey metabolic components may yield innovative options \nfor diagnosis and therapy, although the exact control \nprocesses require further definition.   \nUbiquitination serves as an important post -translational \nalteration that governs protein stability and activity, \nexerting significant influence on cell metabolism and \nimmune equilibrium in diverse disorders [6, 7]. UBE2S \nstands out as a distinctive E2 ubiq uitin-conjugating \nenzyme featuring E3 -dependent as well as independent \nligase capabilities [8], and it has been linked to protein \nbreakdown and cancer advancement [9, 10]. Yet, its \ninvolvement in endometriosis remains unclarified. Via \nIP/MS analysis, an as sociation was uncovered between \nUBE2S and the glucose transporter GLUT1. This vital \nglycolysis controller supports glucose entry and boosts \nlactate production [11]. Beyond metabolism, lactate \nfunctions as a pivotal immunomodulatory agent capable \nof promoting M2 macrophage polarization [12]. While \nUBE2S and GLUT1 are each connected separately to \nubiquitination processes and metabolic control, their \ninteractive role in endometriosis is undetermined. The \ncurrent work seeks to determine if UBE2S influences \nGLUT1 ubiquitination and protein stability, thus \naffecting the metabolic and immunological shifts \nobserved in endometriosis. \nIn the present investigation, we examined the function of \nUBE2S in endometriosis (EM) and put forward the \nconcept that UBE2S increases GLUT1 protein stability \nby facilitating its deubiquitination via Ubiquitin-Specific \nPeptidase 10 (USP10). This mechanism supports \nalterations in cellular metabolism, shifts in macrophage \nphenotypes, and fibrotic development in lesions. \nThrough detailed exploration of this axis both in cellular \nsystems and animal models, our objective was to reveal \na novel understanding of th e metabolism -immune \ninteraction in EM and highlight possible intervention \npoints for treatment. \nMaterials and Methods  \nPatients and samples \nSamples of endometrial tissue were obtained from 20 \npatients diagnosed with ovarian endometriotic cysts and \nfrom individuals with other non -endometriotic \nconditions (such as leiomyomas of the uterus and cervical \nintraepithelial lesions) who received total hysterectomy \nat the First Affiliated Hospital of Harbin Medical \nUniversity. The collection comprised 20 specimens of \nectopic endometrium (EC), 20 of eutopic endometrium \n(Eu), and 20 of normal endometrium (NM) from patients \nwithout endometriosis. From the 2 0 clinical samples in \neach category, 5 were designated for \nimmunohistochemistry, while the other 15 were \nprocessed for primary stromal cell isolation. After strict \ndouble-marker phenotyping (Vimentin⁺/Cytokeratin⁻), \nonly cultures achieving >95% purity acro ss passages 2–\n3 were employed in further procedures, encompassing \nRNA sequencing, nucleic acid/protein isolation, assays \nfor cell growth and movement, and metabolic \nevaluations. Inclusion requirements included: age \nbetween ≥18 and ≤50 years, premenopausal status with \nregular cycles (28 ± 7 days), absence of oral \ncontraceptives, injectable or implantable contraception, \nintrauterine devices, or hormone therapy for at least three \nmonths before collection. Exclusion factors were: age \noutside 18–50 years, postme nopausal condition, lack of \npostoperative pathological confirmation of EM in the \nstudy group, or any endometrial abnormalities in \ncontrols. All procedures received approval from the \nEthics Committee of the First Affiliated Hospital of \nHarbin Medical Univer sity, with written informed \nconsent provided by every participant. \n \nIsolation and culture of endometrial stromal cells   \nStromal cells from ectopic endometrium (EESCs), \neutopic endometrium (EuSCs), and normal endometrium \n(NESCs) were separated via enzymatic treatment using \ntype IV collagenase (1 mg/mL, Biosharp) at 37 °C. \nFollowing filtration and centrifugation steps, cells were \nplaced in DMEM/F12 medium (GIBCO, NY, USA) \nsupplemented with 10% FBS and maintained at 37 °C in \nan atmosphere containing 5% CO₂. \n \nRNA sequencing (RNA-seq)   \nTotal RNA isolation was carried out with Trizol reagent \n(Invitrogen, CA, USA). Assessment of RNA quality and \ncompleteness involved NanoDrop 2000 (Thermo Fisher, \nUSA) and Bioanalyzer 2100 (Agilent, USA), alongside \nverification of integrity on 1.5% agarose g els. mRNA \nenrichment utilized Poly -T magnetic beads, and library \nconstruction employed the VAHTS Universal V6 RNA -\nseq Library Kit for MGI (Vazyme, China). Library \nvalidation occurred via Qubit 3.0 (Thermo Fisher, USA) \nand Bioanalyzer 2100, with subsequent sequencing \nperformed on the MGI -SEQ 2000 system (Frasergen, \nWuhan, China). Initial reads underwent filtering using \nSOAPnuke (v2.1.0) to eliminate adapters, poor -quality \n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n60 \nsequences, and those with excessive unknown bases. \nProcessed reads were mapped to the reference genome \nthrough HISAT2 (v2.1.0) and Bowtie2 (v2.3.5). \nExpression levels were calculated with RSEM (v1.3.1) \nand expressed as FPKM. Identification of differentiall y \nexpressed genes relied on DESeq2 (v1.22.2), applying \ncriteria of |log2FC| >1 and false discovery rate (FDR) < \n0.05 for significance. \n \nGene ontology (GO) and gene set enrichment analysis \n(GSEA)   \nEnrichment analysis of GO terms for differentially \nexpressed genes was conducted via the enrichGO tool in \nthe R package clusterProfiler (v3.6.3). Terms achieving \np value ≤ 0.05 were deemed significantly enriched. \nGraphical representation of GO findings was  generated \nwith ggplot2 (v3.5.1) from R. For GSEA, a ranked list of \nall detected genes served as input, evaluated against \nHallmark gene sets from the Molecular Signatures \nDatabase (MSigDB). Analysis used the GSEA tool \nwithin clusterProfiler (v3.6.3), with settings: minimum \ngene set size = 0, maximum = 20,000, no p value cutoff, \nBenjamini–Hochberg adjustment for multiple testing, \nand 1,000 permutations. Gene sets showing normalized \nenrichment score (NES) > 1 (or < −1), p value < 0.05, \nand FDR < 0.25 were classified as significantly enriched. \nPlots of GSEA outcomes were created using the gseaNb \nfunction in the R package GseaVis (v0.0.5). \n \nSTRING protein –protein interaction (PPI) network \nanalysis   \nInteractions among proteins encoded by differentially \nexpressed genes were explored through the STRING \ndatabase (https://string -db.org/). The constructed \nnetwork was displayed and examined further in \nCytoscape (v3.9.1) to detect central hub genes and \nimportant regulatory clusters. \n \nMacrophage polarization   \nTHP-1 cells (ScienCell ) were induced to become M0 \nmacrophages by exposure to 100 ng/ml phorbol 12 -\nmyristate 13 -acetate (PMA; Sigma -Aldrich, St. Louis, \nMO, USA) for 24 h. Upon microscopic confirmation of \nadhesion and extended morphology, the PMA-containing \nmedium was replaced with standard complete medium to \nhalt induction. A transwell system featuring 0.4 μm pores \n(Corning, NY, USA) was applied. Differentially treated \nEESCs were placed in the upper insert, whereas M0 \nmacrophages occupied the lower compartment. \nFollowing 48 h of co-incubation, lower -chamber cells \nwere collected for evaluation of M2 markers (CD163, \nCD206, ARG-1). \n \nMass spectrometry analysis   \nProtein bands were removed from Coomassie Brilliant \nBlue–stained gels, reduced using DTT, alkylated with \niodoacetamide, and subjected to overnight tryptic \ndigestion. Peptides were recovered in acetonitrile, \npurified on ZipTip C18 columns, and examined by L C–\nMS/MS using a nanoLC-Q Exactive instrument (Thermo \nScientific, USA). Raw spectra were queried against the \nUniProt human proteome database (2022 release) via the \nSEQUEST HT search engine in Proteome Discoverer \n(v1.4). Parameters included: full trypsin spe cificity \nallowing up to two missed cleavages, 10 ppm precursor \ntolerance, 0.02 Da fragment tolerance, fixed \ncarbamidomethylation on cysteine, and variable \nmethionine oxidation. Identifications were refined with \nPercolator to achieve a 1% FDR for high -confidence \npeptides. \nCreation of cell lines deficient in USP10 or UBE2S  \nLines devoid of USP10 and UBE2S were developed via \nthe CRISPR/Cas9 methodology. To produce cells \nwithout USP10, guide RNAs aimed at USP10 were \nincorporated into the PX459 plasmid and delivered into \nHEK293T cells employing Lipofectamine 3000. \nFollowing a 24  h period, selection involved puromycin \n(2 µg/mL) over 48 h. Isolated single clones underwent \nexpansion and verification via PCR combined with \nWestern blot procedures. An analogous strategy was \napplied for knocking out UBE2S in HEK293T as well as \nEESCs. Guide RNAs designed for UBE2S were placed \ninto PX459, with subsequent delivery and puromycin -\nbased screening. Confirmation of effective gene \ndisruption in chosen clones relied on P CR and Western \nblot techniques. \nIntroduction of plasmids and development of stable lines  \nPlasmids utilized recombinantly in this work comprised \npCAGGS-UBE2S (bearing Flag, HA, and Myc tags), \npCMV-Flag-UBE2S ∆N, pCMV -Flag-UBE2S ∆C, and \npCMV-Flag-UBE2S ∆Core, supplied courtesy of \nProfessor Changjiang Weng from the Harbin Veterinary \nResearch Institute. Custom short hairpin RNAs directed \nagainst UBE2S and GLUT1 were produced synthetically, \naccompanied by nonspecific control sequences. \nAnnealing of these synthetic pieces was followed by \n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n61 \nrestriction using BamH I and EcoR I, then insertion into \npLVX-shRNA1 (Clontech, Palo Alto, USA). Delivery \ninto cells occurred through Lipofectamine 3000. \nProduction of lentiviruses required joint introduction into \nHEK293T of desired constructs together wit h auxiliary \nvectors psPAX2 and pMD2.G. \nCCK8 assay for assessing cell growth  \nSeeding occurred at 2000 cells per well across 96 -well \nformats, with incubation spanning 24, 48, and 72 h. \nAddition of 10 µL CCK -8 reagent (Meilunbio, Dalian, \nChina) preceded measurement of absorbance values at \n450 nm. \nEdU-based evaluation of proliferative activity  \nPlacement involved 1 × 10⁵ cells per well in 12 -well \nsetups, followed by 4 h exposure to EdU (Beyotime \nBiotechnology, Shanghai, China), fixation, and dye \napplication. Resulting fluorescent patterns were \ndocumented via microscopy. \nScratch wound assay  \nCells after treatment were transferred to six-well formats. \nAt near 90% density, a linear injury was inflicted using a \ntip. Removal of detached material via PBS wash \npreceded initial imaging at 0 h. Further cultivation for 24 \nh allowed repeat photography. \nTranswell system for assessing cell movement  \nSuspensions at 2.5 × 10⁵ cells/mL occupied upper \ncompartments of 24 -well transwell units (Corning, NY, \nUSA) featuring 8 μm membranes, under conditions \nlacking serum. Lower sections held DMEM enriched \nwith 20% FBS. Incubation at 37 °C lasted 24 h, after \nwhich fixation was employed 4% paraformaldehyde (15 \nmin), PBS rinses, crystal violet application (20 min), \nadditional washes, and imaging for quantitation. \nAssays for glucose consumption and levels of \nlactate/pyruvate  \nCells following plasmid introduction were moved to six-\nwell dishes and allowed 12 –16 h growth. Collection \nenabled determination of glucose, lactate, and pyruvate \namounts via specialized kits (mlbio, Shanghai, China). \nImmunohistochemical procedures (IHC)  \nH&E involved xylene -based deparaffinization of 5 μm \nsections, hematoxylin -eosin sequence, and bright -field \nviewing. IHC on 4 μm slices included deparaffinization, \nhydration steps, and 15 min exposure to 3% H2O2 for \nperoxidase blocking. Retrieval used citrate (pH 9.0), with \n30 min goat serum block. Overnight 4 °C exposure to \nprimaries—UBE2S (1:200, Proteintech) or GLUT1 \n(1:200, Proteintech) —preceded 1 h secondary (HR P-\nlinked), DAB revelation, and viewing. ImageJ (NIH, \nBethesda, MD, USA) quantified intensity and positive \nregions. Per specimen, five unrelated fields at 20× \nyielded averages. Analyses maintained blinding. \nCo-Immunoprecipitation Protocol (Co-IP)  \nHarvested cells underwent disruption in buffer (50 mM \nTris, pH 7.6, 0.5 mM EDTA, 0.1% NP40, 0.5 mM \nPMSF). Centrifugation allowed BCA-based quantitation \n(Beyotime, Shanghai, China). A fifth portion acted as \ninput; the rest received target antibody and agarose for an \novernight 4 °C rotation. Buffer washes are preceded by \n10 min boil, then Western blot or spectrometric \nexamination. \nGST-based affinity isolation  \nExpression of pGEX -4T-1-UBE2S occurred in E. coli \nBL21 (DE3) via 1 mM IPTG induction. Harvested \nmaterial received inhibitor -supplemented buffer, \nsonication, and spin (12,000 × g, 20 min, 4 °C). Cleared \nfraction with GST -UBE2S bound glutathione beads \novernight at 4 °C. His-USP10 (from pET-24a(+)-USP10) \njoined immobilized fusion for 2 h at 4 °C. Multiple \nwashes (4 –5) allowed SDS buffer elution/boil, \nelectrophoretic separation, and His-directed immunoblot. \nIsolation of RNA, cDNA preparation, and quantitative \nPCR  \nCell-derived total RNA came via Trizol (Invitrogen, CA, \nUSA), with NanoDrop  quantitation (Thermo Fisher \nScientific, MA, USA). Conversion to cDNA used a \ndedicated kit (Seven, Beijing, China). Amplification \nrelied on S6 Universal SYBR qPCR Mix (Enzy Artisan, \nShanghai, China).  \nMolecular docking evaluation  \nTo investigate the interaction preference of \ncephalomannine (CPM) for UBE2S and detect possible \nnonspecific bindings, computational docking was carried \nout employing AutoDock Vina (v1.2.5). Protein \ncoordinates were retrieved from the UniProt repository \n(www.uniprot.org) and prepared via ADFRsuite -1.0. \nTarget files in PDBQT format retained native ionization \nand charge distributions. The small-molecule structure of \nCPM was similarly handled with ADFRsuite -1.0 to \n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n62 \nproduce its PDBQT representation. Simulations utilized \n32 parallel processing cores, a grid interval of 0.375, with \nremaining settings unchanged. Interaction strengths were \nestimated from Vina scores (ΔG), wherein more negative \nfigures signify greater anticipated affinity. \nIn vivo studies   \nFor assessing UBE2S contribution to endometriosis \nwithin a living system, a homologous graft model was \nadopted. Mice lacking UBE2S (UBE2S –/–) on the \nC57BL/6J strain were produced through targeted \nrecombination (Biocytogen, Beijing, China. Female \nanimals, either wild-type C57BL/6J or UBE2S–/–, aged \n6–8 weeks, were given intraperitoneal estrogen (1 \nµg/mL) on days 1, 4, and 7. On day eight, donors were \nhumanely sacrificed under sedation, the uterine horn \nendometrium was harvested, and it was resuspended in \nPBS. Aliquots of 0.6 mL suspension were administered \nintraperitoneally into pairs of immunocompetent \nC57BL/6J recipients. Weekly estrogen continued \nthereafter. Ectopic growths were recovered after four \nweeks. In therapeutic trials, animals were allocated \nrandomly to weekly intraperitoneal CPM (10 mg/kg) or \nvehicle starting seven days post -implantation (n = 6 per \ncohort). Lesions were excised following four weeks of \nadministration for examination. Every procedure \ninvolving animals gained approval from the Anima l \nEthics Committee of the First Affiliated Hospital of \nHarbin Medical University and adhered to relevant \nregulatory standards. \n \nData interpretation  \nAnalyses employed SPSS (version 20.0) alongside \nGraphPad Prism (version 8.0). Group comparisons \nutilized one -way ANOVA with subsequent Tukey \nadjustment. Results achieving p < 0.05 were deemed \nsignificant. \nResults and Discussion \nUBE2S emerges as the primary ubiquitin -proteasome \ncomponent linked to EESCs  \nIn pursuit of candidate markers for endometriosis, RNA \nsequencing was performed on three normal and three \nectopic stromal cell samples. P er the filtration pipeline \n(Figure 1a), duplicate entries were eliminated, retaining \ndominant isoforms for multi -transcript genes. Applying \ncutoffs of |log2FC| >1 and FDR < 0.001 yielded 4,404 \ngenes showing altered expression. Enrichment \nassessments via GO and GSEA highlighted critical \nactivities and routes in the condition. GO terms for these \ngenes prominently featured processes lik e “positive \ncontrol of proteolysis”, “control of protein \nubiquitination”, “enhanced ubiquitin -reliant protein \nbreakdown”, and “proteolysis of membrane proteins” . \nConcurrent GSEA on ranked gene lists disclosed notable \nactivation of sets including “Positive Control of Protein \nMetabolism”, “Activator Role in Ubiquitin Transfer”, \nand “Enhanced Proteolysis” . Such patterns indicate \ntranscriptional perturbation of ubiquitin -driven \ndegradation routes in endometriosis, potentially driving \npathogenesis. A compiled set of 181 ubiquitin -\nproteasome pathway genes [13] was cross -referenced \nwith the 4,404 altered genes, uncovering 28 differentially \nregulated members (8 elevated, 20 reduced). These \ninformed a volcanic display (Figure 1b) marking the 28 \ncandidates, plus a clu stered heat map i llustrating their \nprofiles (Figure 1c). Immune infiltration estimates here \nderive from xCell computations via the IOBR R package \n[14]. Abundance scores for immune populations were \ncorrelated (Pearson) with levels of the 28 pathway genes. \nStringent criteria (p-value < 0.0001, absolute correlation \n>0.9) isolated three top -associated genes, presented in a \nVenn layout (Figure 1d). Protein interaction mapping of \nthe 28 candidates through STRING positioned UBE2S as \nthe most connected node (Figure 1e). Network centrality \nalone, however, does not prove disease relevance. \nAccordingly, UBE2S levels were confirmed in ectopic \ncells, followed by targeted assays to define its \ninvolvement in endometriosis biology. \n \n\n \n \n \na) \n  \nb) c) \n  \nd) e) \n  \nf) g) \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n64 \n \nh) \nFigure 1. Elevated levels of UBE2S in endometriosis relative to healthy endometrial tissue. (a) Schematic \noutlining the approach for pinpointing UBE2S as a preferentially involved gene within endometriosis \ntranscriptome datasets. (b) Volcanic representation highlighting altered expression of ubiquitin-proteasome \npathway components across compared cohorts. (c) Clustered heat map portraying expression variances of \nubiquitin-proteasome pathway elements between cohorts. (d) Venn illustration showing intersections among \nendometriosis transcriptome findings, ubiquitin-proteasome pathway components, and immune-related profiles. \n(e) Protein interaction network derived from STRING for the 28 selected genes. (f) Immunoblot examination of \nUBE2S protein in normal (NM), eutopic (Eu), and ectopic (EC) endometrial samples. (g) Quantitative RT-PCR \nmeasurement of UBE2S transcript abundance in normal stromal cells (NESCs), eutopic stromal cells (EuSCs), \nand ectopic stromal cells (EESCs). (h) Immunoblot detection of UBE2S protein abundance in NESCs, EuSCs, \nand EESCs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 \nFor verification of UBE2S abundance in endometriosis, \nimmunoblotting was conducted across 12 matched sets \nof ectopic (EC), eutopic (Eu), and normal (NM) \nendometrial specimens. Outcomes demonstrated \nmarkedly higher UBE2S protein content in ectopic \nsamples versus normal counterparts (Figure 1f). Parallel \nassessments of transcript and protein in isolated stromal \ncells disclosed substantial increases of UBE2S both \ntranscriptionally (Figure 1g) and translationally (Figure \n1h) within ectopic stromal populations. Collectively, \nthese observations point to overexpression of UBE2S in \nendometriotic lesions and derived cells.  \n \nUBE2S influences key cellular activities in EESCs   \nTo delineate the functional contribution of UBE2S in \nectopic stromal cells, lines with enforced expression or \nsilenced UBE2S were established, and impacts on growth \nand motility were examined. Growth effects were \ninitially probed via CCK -8 and EdU methodologies. \nEnforced UBE2S led to notable enhancement of cellular \nexpansion ( Figures 2a and 2c; p < 0.05), while \nsuppression markedly reduced it (Figures 2b and d; p < \n0.01). Motility was further interrogated through \nTranswell and wound closure tests. Elevated UBE2S \nsubstantially augmented migratory potential (Figures 2e \nand 2g; p < 0.05), whereas depletion curtailed it (Figures \n2f and 2h; p < 0.001). Such cellular studies underscore \nthat UBE2S drives both expansion and movement of \nectopic stromal cells, implicating it centrally in \nendometriosis development.   \n \n \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n65 \n  \na) b) \n  \nc) d) \n  \ne) f) \n  \ng) h) \nFigure 2. UBE2S enhances the growth and motility of ectopic stromal cells under culture conditions. (a-b) CCK-\n8 evaluation of proliferative rates in EESCs following UBE2S gain or loss. (c-d) EdU incorporation assay \nreflecting proliferative changes after UBE2S modulation. (e-f) Transwell quantification of migratory behavior \npost-UBE2S alteration. (g-h) Wound healing assessment of motility under UBE2S overexpression or reduction. * \np < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 \nUBE2S associates with GLUT1 in a complex and \ncontrols lactate production pathways  \nIn pursuit of deeper mechanistic insights into UBE2S \nactions in endometriosis, Flag -tagged UBE2S was \nintroduced into ectopic stromal cells, with an empty Flag \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n66 \nvector serving as a control. Immunoprecipitation \nuncovered a distinct band exc lusive to the tagged group \n(Figure 3a). Targeted proteomic sequencing of this band \nvia LC –MS/MS pinpointed GLUT1 as a candidate \ninteractor. Endogenous confirmation employed \nmonoclonal antibodies against each protein in cell \nlysates, verifying GLUT1 capture by UBE2S pull-down \n(Figure 3b) and reciprocal detection of UBE2S in \nGLUT1 isolates (Figure 3c). Additional validation \ninvolved dual delivery of Flag -GLUT1 and HA-UBE2S \ninto 293T  cells, with bead -based isolation reve aling \nmutual co -enrichment. Domain mapping via truncated \nHA-UBE2S variants (∆C, ∆N, ∆Core) in co-precipitation \nshowed binding of GLUT1 to wild-type, ∆C, and ∆Core \nforms, but absence with ∆N. These data establish cellular \ncomplex formation between UBE2S and GLUT1, \ndependent critically on the N-terminal region of UBE2S. \n \n \n \n \na) b) \n  \nc) d) \n \ne) \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n67 \n \nf) \n \ng) \n   \nh) i) j) \n   \nk) l) m) \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n68 \n   \nn) o) p) \nFigure 3. UBE2S associates with GLUT1 to drive glycolytic activity in ectopic stromal cells. (a) Proteomic \nidentification of GLUT1 as an interactor of UBE2S. (b-c) Endogenous co-immunoprecipitation in EESC lysates \nemploying antibodies specific to UBE2S or GLUT1, with subsequent immunoblot detection of reciprocal \npartners. (d) Assessments of glucose consumption, lactate output, and pyruvate generation in endometriosis-\nderived cells following enforced UBE2S expression. (e) Corresponding metabolic parameters in cells after \nUBE2S depletion. (f) Reintroduction of Flag-UBE2S into knockout EESCs and immunoblot evaluation of \nGLUT1 abundance. (g) Introduction of UBE2S-directed shRNA into EESCs and immunoblot quantification of \nGLUT1. (h-j) Metabolic readouts (glucose consumption, pyruvate, lactate) in EESCs after specified plasmid \ndeliveries. (k-m) Parallel metabolic evaluations under different transfection conditions. (n-p) Further metabolic \nassays across indicated genetic manipulations. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 \nStaining via immunohistochemistry disclosed greater \nabundance of both UBE2S and GLUT1 within ectopic \nendometrial specimens relat ive to normal counterparts . \nImmunoblotting across 12 ectopic (EC), 12 eutopic (Eu), \nand 12 normal (NM) samples verified markedly  \nheightened GLUT1 in EC tissues and derived EESCs  \nversus Eu or NM controls (p < 0.001). As the primary \nfacilitator of cellular glucose entry, GLUT1 plays a \npivotal role in enhanced aerobic glycolysis. To determine \nif UBE2S governs this pathway in ectopic cells, impacts \non glucose intake and production of lactate/pyruvate \nwere examined. Enforced UBE2S substantiall y boosted \nthese parameters (Figure 3d), while its reduction \ndiminished them (Figure 3e). \nIn UBE2S -deficient EESCs restored with varying \nUBE2S levels, GLUT1 protein showed notable elevation \n(Figure 3f). Parallel experiments with control or UBE2S \nshRNA in EESCs demonstrated a clear decline in \nGLUT1 upon UBE2S silencing ( Figure 3g; p < 0.05). \nDedicated GLUT1 suppression constructs were \ngenerated to probe dependency . Combined depletion of \nGLUT1 and UBE2S markedly lowered glucose intake \nand lactate/pyruvate output (Figures 3h–j). GLUT1 loss \nabrogated the metabolic elevation  triggered by excess \nUBE2S (Figures 3k–m), whereas GLUT1 enrichment \nrestored parameters d iminished by UBE2S absence \n(Figures 3n–p). Collectively, these observations \nestablish that UBE2S augments glycolytic activity and \nlactate generation in EESCs via control over GLUT1. \nTo evaluate UBE2S effects on metabolism amid \ncontrolled substrate availability, cultures were shifted to \nglucose-deprived media supplemented at 0, 5, or 25 mM \nglucose. Lactate yield was tracked in contexts of UBE2S \ngain or loss. Baseline cells exhibited gl ucose dose -\nresponsive lactate rises. Added UBE2S amplified \naccumulation at every level, indicating enhanced flux. In \ncontrast, UBE2S removal subs tantially curtailed output. \nSuch patterns highlight UBE2S as a substrate -sensitive \nenhancer of glycolysis and lactate formation.   \n \nUBE2S diminishes K48 -linked ubiquitin chains on \nGLUT1 \nAs an E2 ubiquitin -conjugating component, UBE2S \nextends polyubiquitin modifications on targets, \ninfluencing their proteasomal clearance [15, 16]. To test \ninvolvement in GLUT1 turnover, proteasome blockade \nwas applied via MG132 (10 µM, 8 h). In UBE2S -null \nEESCs, GLUT1 levels rose significantly post -inhibitor \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n69 \nexposure versus vehicle ( Figure 4a; p < 0.05). No \ncomparable shift occurred in wild -type UBE2S EESCs \nirrespective of treatment (Figure 4a; p > 0.05). \n \n \n  \na) c) \n \n \nb) d) \n \n \ne) f) \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n70 \n  \ng) h) \n \ni) \nFigure 4. UBE2S decreases K48-linked polyubiquitination of GLUT1. (a) EESCs UBE2S knockout (KO) and \nwild-type (WT) cells were treated with the proteasome inhibitor MG132 (10 µM, 8 h), and GLUT1 protein levels \nwere evaluated by western blotting. (b) EESCs UBE2S-KO and WT cells were exposed to cycloheximide (CHX, \n50 µg/mL) for varying durations, followed by western blot analysis of GLUT1 protein expression. (c) GLUT1 \nubiquitination was examined in EESCs after transfection with shUBE2S. (d) GLUT1 ubiquitination was assessed \nin EESCs following Flag-UBE2S overexpression. (e) 293T cells were co-transfected with HA-Ub-WT, Myc-\nUBE2S, and Flag-GLUT1 plasmids, and GLUT1 ubiquitination was detected. (f) 293T cells were transfected \nwith HA-Ub mutants (K11R, K48R, K63R), Flag-GLUT1, and Myc-UBE2S, and GLUT1 ubiquitination was \nevaluated by Co-IP. (g) 293T cells received Myc-UBE2S, Flag-GLUT1, and HA-Ub variants (K11-only, K48-\nonly, K63-only), with GLUT1 ubiquitination analyzed via Co-IP. (h) UBE2S-KO 293T cells were transfected \nwith Myc-UBE2S WT or its mutants (C95S, C118A, DM), and GLUT1 expression was determined by western \nblotting. (i) UBE2S-KO 293T cells were transfected with Myc-UBE2S WT or mutants (C95S, C118A, DM), \ntogether with Flag-GLUT1 and HA-Ub-K48, and GLUT1 ubiquitination was measured by Co-IP. * p < 0.05, ** p \n< 0.01, *** p < 0.001, **** p < 0.0001 \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n71 \nWe subsequently treated EESCs with the protein \nsynthesis inhibitor cycloheximide (CHX, 50 µg/mL) and \nmonitored GLUT1 protein levels at multiple time points. \nThe data showed that, relative to UBE2S-WT EESCs, the \nhalf-life of GLUT1 was markedly shortened in UBE2S-\nKO cells after CHX exposure ( Figure 4b; p < 0.01). \nThese observations suggest that UBE2S enhances \nGLUT1 stability by suppressing its proteasome-mediated \ndegradation, leading to higher GLUT1 levels in \nendometrial stromal cells during endometriosis.   \nTo investigate if UBE2S modulates GLUT1 \nubiquitination, we examined ubiquitination status in \nEESCs with either reduced or elevated UBE2S \nexpression. Knockdown of UBE2S in EESCs resulted in \nelevated GLU T1 ubiquitination (Figure 4c), whereas \nUBE2S overexpression markedly lowered it (Figure 4d). \nIn 293T cells co-transfected with Myc-UBE2S, HA-Ub-\nWT, and Flag -GLUT1, Co -IP analysis using anti -HA \nrevealed that UBE2S overexpression substantially \ndiminished GLUT1 ubiquitination (Figure 4e).   \nGiven that K11-, K48-, and K63-linked ubiquitin chains \ncan drive protein degradation or signaling [17], we \nexplored linkage specificity in 293T cells using ubiquitin \nmutants (K11R, K48R, K63R). Results indicated that \nUBE2S influences GLUT1 ubiquitination p rimarily via \nthe K48 pathway (Figure 4f). Further experiments in \n293T cells overexpressing Myc -UBE2S, Flag -GLUT1, \nand single -lysine ubiquitin constructs (K11, K48, or \nK63) confirmed that UBE2S selectively suppresses K48-\nlinked polyubiquitination of GLUT1 (Figure 4g). Thus, \nUBE2S specifically targets K48-linked chains rather than \nbroadly affecting other linkage types.   \nUBE2S possesses E2 ubiquitin -conjugating activity and \nalso functions as an E3 ligase [18, 19]. Its dual enzymatic \nroles rely on two cysteine residues (Cys95 and Cys118) \nwithin the UBC domain. To determine whether GLUT1 \nregulation by UBE2S requires E2 or E3 activity, we \ngenerated three mutants: C95S (loss of E2 activity), \nC118A (loss of E3 activity), and a double mutant (DM, \nloss of both). These, along with WT Myc -UBE2S, were \nintroduced into UBE2S-KO 293T cells. Both WT and all \nmutants increased GLUT1 protein levels (Figure 4h) and \neffectively re duced GLUT1 ubiquitination (Figure 4i). \nThese results imply that UBE2S -mediated K48 \nmodification of GLUT1 is independent of its E2 or E3 \ncatalytic functions.   \n \nUBE2S enlists the deubiquitinase USP10 to eliminate \nK48-linked polyubiquitination from GLUT1   \nAlthough UBE2S primarily acts as an E2 enzyme, our \ndata indicate it may facilitate GLUT1 deubiquitination \nvia an unidentified deubiquitinase, prompting deeper \nmechanistic studies. Mass spectrometry revealed USP10 \nas an interacting partner of UBE2S in EESCs . We \ntherefore proposed that UBE2S recruits USP10 to cleave \nK48-linked ubiquitin chains from GLUT1 in EESCs. To \ntest for direct binding, we conducted GST pull -down \nassays with recombinant proteins produced in E. coli: \nGST-UBE2S from pGEX-4T-1-UBE2S and His-USP10 \nfrom pET-24a(+)-USP10. Incubation showed that GST -\nUBE2S efficiently pulled down His -USP10, verified by \nanti-His immunoblotting, confirming direct interaction in \nvitro (Figure 5a). In EESC lysates, immunoprecipitation \nwith anti -USP10 antibody and Protein A+G beads \ndemonstrated that USP10 associates with both UBE2S \nand GLUT1 intrace llularly (Figure 5b). Reciprocal \nimmunoprecipitation using anti-UBE2S antibody further \nvalidated the UBE2S –USP10 interaction in EESCs \n(Figure 5c). \n \n \n \n \na) b) \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n72 \n \n \nc) d) \n \n \ne) f) \n  \ng) h) \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n73 \n \ni) \nFigure 5. UBE2S enlists USP10 to diminish K48-linked polyubiquitination of GLUT1. (a) Recombinant GST or \nGST-UBE2S was incubated with recombinant His-USP10. Pull-down was performed using glutathione agarose \nbeads, and eluted samples were analyzed by SDS-PAGE and immunoblotting with anti-His antibody. Input and \npull-down samples are displayed, with GST as a negative control. (b-c) Whole-cell lysates from EESCs were \nimmunoprecipitated using monoclonal antibodies against USP10 or UBE2S, followed by western blotting to \nexamine USP10 and UBE2S protein levels. (d-e) Co-IP performed on lysates from 293T cells transfected with the \nspecified plasmids. (f) Co-IP on lysates from 293T cells transfected with the indicated plasmids. (g) Diagram of \nfull-length UBE2S and its truncation mutants (top), along with Co-IP results showing binding of FLAG-tagged \nUSP10 to HA-tagged wild-type or truncated UBE2S variants (∆N, ∆C, and ∆Core) in HEK293T cells (bottom). \n(h) UBE2S-KO 293T cells transfected with Myc-UBE2S, Flag-GLUT1, Myc-USP10, and HA-Ub-K48 plasmids, \nfollowed by Co-IP on cell lysates. (i) USP10-KO 293T cells transfected with Myc-UBE2S, Flag-GLUT1, Myc-\nUSP10, and HA-Ub-K48 plasmids, followed by Co-IP on cell lysates. \nIn 293T cells, co -expression of Flag -GLUT1 and HA -\nUSP10 followed by Co -IP verified direct bindi ng \nbetween GLUT1 and USP10 (Figure 5d). Likewise, co-\nexpression of Flag -UBE2S and HA -USP10 confirmed \nbinding between UBE2S and USP10 (Figure 5e).  \nAdditionally, triple transfection with Flag-GLUT1, Myc-\nUBE2S, and HA-USP10 in 293T cells showed that both \nMyc-UBE2S and HA-USP10 co-precipitated with Flag-\nGLUT1, reinforcing the association amo ng GLUT1, \nUSP10, and UBE2S (Figure 5f).   \nTo determine which regions of UBE2S mediate its \nbinding to USP10, we introduced three HA -tagged \ntruncation mutants (∆C, ∆N, and ∆Core) along with Flag-\nUSP10 into 293T cells and conducted Co -IP. Data \nshowed that USP10 bound to wild-type UBE2S, ∆C, and \n∆Core mutants, but not to the ∆N mutant (Figure 5g). \nThis indicates that the N -terminal region of UBE2S is \ncritical for USP10 interaction.   \nTo examine the cooperative effects of UBE2S and \nUSP10 on K48-linked ubiquitination of GLUT1, we first \ntested USP10–GLUT1 association. In UBE2S-KO 293T \ncells co-transfected with Flag-GLUT1, Myc-UBE2S, and \nMyc-USP10, USP10 failed to bind GLUT1 without \nUBE2S, but associated with GL UT1 when UBE2S was \npresent (Figure 5h). In USP10 -KO 293T cells \ntransfected with Flag -GLUT1, Myc-UBE2S, and Myc -\nUSP10, UBE2S alone did not lower K48 -linked \nubiquitination of GLUT1, whereas combined expression \nof UBE2S and USP10 markedly reduced it (Figure 5i). \nThese results indicate that neither protein suffices \nindependently for K48 deubiquitination of GLUT1; both \nare required concurrently. UBE2S acts as a scaffold via \nits N -terminal domain to assemble a ternary complex \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n74 \nwith GLUT1 and USP10, thereby removing K48 -linked \nchains from GLUT1 and preserving its protein stability.   \n \nUBE2S promotes glycolysis and drives M2 macrophage \npolarization   \nUBE2S shows elevated immune scores in breast cancer, \nglioma, bladder cancer, and liver cancer, reflecting close \nties to the tumor immune microenvironment [20, 21]. \nExcessive M2 macrophage infiltration characterizes the \nchronic inflammation in endometriosis  (EM). To assess \nUBE2S impact on M2 polarization in EM, THP -1 cells \nwere differentiated into M0 macrophages with PMA and \nthen co -cultured with differentially treated EESCs. \nMacrophages were harves ted to evaluate M2 markers \n(Figure 6a).  Results demonstrated  that, relative to co -\nculture with control EESCs, macrophages exposed to \nUBE2S-overexpressing EESCs displayed markedly \nhigher mRNA levels of CD163 (Figure 6b),  Arg-1 \n(Figure 6c),  and CD206 (Figure 6d).  Furthermore, \nGLUT1 knockdown in EESCs abrogated the UBE2S -\ninduced i ncrease in M2 polarization (Figures 6b–d). \nProtein-level confirmation was obt ained via western \nblotting (Figure 6e). \n \n \n  \na) b) \n  \nc) d) \n \ne) \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n75 \n  \n \nf) g) h) \n \ni) \nFigure 6. Elevated UBE2S levels in EESCs stimulate glycolytic activity and favor M2-type macrophage \ndifferentiation. (a) Illustration of the co-culture setup. (b-d) Quantitative RT-PCR quantification of CD163, \nCD206, and Arg-1 transcript abundance in macrophages following 48 h co-culture with EESCs harboring various \ntransfections. (e) Immunoblot detection of CD163, CD206, and Arg-1 proteins in macrophages from co-cultures \nwith differently transfected EESCs. (f-h) UBE2S-overexpressing EESCs were exposed to 2-DG or CPM for 24 h; \nthe resulting conditioned media (CM) were harvested and applied to M0 macrophages. Quantitative RT-PCR \nevaluation of CD163, CD206, and Arg-1 transcripts in treated macrophages. (i) Parallel setup as above, with \nimmunoblot assessment of CD163, CD206, and Arg-1 proteins in macrophages. * p < 0.05, ** p < 0.01, *** p < \n0.001, **** p < 0.0001 \nCephalomannine (CPM), a small -molecule agent, \ndownregulates UBE2S protein abundance [22]. EESCs \ntransfected with Flag -UBE2S (labeled OE -UBE2S-\nEESCs) were shifted to serum -free conditions for 24 h, \nthen exposed to vehicle (DMSO), 1 mM 2-deoxyglucose \n(2-DG), or 100 µM CPM for 24 h. Media were renewed, \nculture extended another 24 h, and supernatants \ncollected, diluted 1:1 with fresh media, then used to \nstimulate M0 macrophages for 48 h prior to M2 marker \nanalysis. Inclusion of 2-DG or CPM during OE-UBE2S-\nEESC conditioning strongly suppressed subsequent \nmacrophage M2 differentiation, evident at both transcript \nand protein levels (Figures 6f–i), achieving statistical \nsignificance. \nTo establish lactate as a critical downstream component \nin UBE2S/GLUT1 -orchestrated M2 differentiation, \nexogenous sodium lactate rescue studies were performed. \nM0 macrophages co -cultured with EESCs bearing \nUBE2S or GLUT1 shRNA received vehicle or 10 mM \nsodium lactate. Immunoblotting showed a marked \ndecline in M2 -associated proteins (CD206, Arg1, \nCD163) upon either knockdown, but partial recovery of \nthese proteins occurred with lactate addition . This \nsupports lactate’s role as a key executor in the \nUBE2S/GLUT1 pathway driving M2 polarization.   \nOverall, these data establish that UBE2S augments \nGLUT1 abundance, accelerates glycolysis within \nEESCs, and thereby sustains M2 macrophage presence in \nendometriotic tissues. Disruption of UBE2S function or \nglycolytic flux in EESCs curbs M2 differentiation at \nlesion sites.   \n \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n76 \nM2 macrophage shifts in endometriotic tissues influence \nEESC functional properties   \nEndometriosis progresses as a fibrosing disorder \nfeaturing tissue rigidity, primarily from abundant \nextracellular matrix buildup during fibrotic remodeling \n[23]. Evidence links endogenous macrophage \nphenotypes to remodeling dynamics in endometriosis, \nwhere M2 cells correlate positively with fibrotic extent. \nThese M2 cells produce TGF -β1, fostering fibrogenesis \nin endometrial lineages [24]. Heightened stromal cell \nmotility and invasiveness hallmark fibrotic progression. \nWe quantified TGF -β1 and observed subs tantial \nupregulation in macrophages after exposure to UBE2S -\noverexpressing EESCs (OE -UBE2S-EESCs), while 2 -\nDG or CPM exposure notably suppressed it. \nHaving examined stromal impact on macrophages, we \nreversed the direction to probe macrophage influence on \nendometriotic stromal behavior. Macrophages co -\ncultured for 48 h with either UBE2S -overexpressing \nEESCs (M0-UBE2S) or vector-control EESCs (M0-NC), \nfollowed by stromal cell removal and 24 h further \nincubation in fresh serum -free media. Harvested \nsupernatants, diluted 1:1 with serum -free media to yield \nconditioned medium (CM), were applied to naive EESCs \n(Figure 7a). M0-UBE2S-derived CM potently \naugmented EESC proliferative capacity (Figures 7b–c) \nand motility (Figure 7d). Such gains were largely \nabolished by prior 2-DG exposure in the M0-UBE2S arm \n(Figures 7b–d), yielding significant reductions. \nConversely, CM from macrophages co -cultured with \nUBE2S-depleted EESCs (M0 -sh-UBE2S) impaired \nEESC proliferation and migration, effects partially \noverridden by 10 mmol/L lactate supplementation to the \nCM (Figures 7e–g; p < 0.05). \n \n \n \na) \n  \nb) e) \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n77 \n  \nc) f) \n  \nd) g) \n \nh) \n \ni) \nFigure 7. M2-type macrophages drive cell proliferation, motility, and fibrotic changes in endometriotic stromal \ncells. (a) Schematic overview of conditioned medium (CM) treatment applied to EESCs. (b) EdU incorporation \nassay measuring EESC proliferative activity. (c) CCK-8 assay quantifying EESC proliferative rates. (d) Wound-\nhealing scratch test examining EESC migratory capacity following CM exposure. (e) EdU incorporation assay \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n78 \nevaluating EESC proliferative response. (f) CCK-8 assay determining EESC growth rates. (g) Wound-healing \nscratch test assessing EESC motility after CM application. (h) Immunoblot detection of fibrotic tissue remodeling \n(FMT) indicators (α-SMA, FN, Col-1) in EESCs exposed to CM for 48 h. (i) Immunoblot examination of FMT \nindicators in EESCs after 48 h CM incubation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 \nSubsequent analysis focused on fibrotic remodeling \nmarkers across EESC treatment conditions. Data showed \nthat CM from M0-UBE2S co-cultures markedly elevated \nα-SMA, Col -1, and FN protein ab undance in recipient \nEESCs (Figure 7h) . Exposure to 2 -DG notably \nattenuated this fibrotic induction (Figure 7h). In contrast, \nCM from M0-sh-UBE2S systems lowered expression of \nthese markers, while lactate supplementation to the CM \nlargely counteracted the decline (Figure 7i), yielding \nsignificant statistical outcomes. Coll ectively, these \nobservations reveal that M2 macrophages, induced by \nUBE2S-high EESCs, accelerate stromal proliferation, \nmotility, and fibrotic remodeling in endometriosis.   \nTo confirm CPM selectivity toward UBE2S, \ncomputational docking was conducted against a set of \nproteins involved in ubiquitination, glycolytic pathways, \nand fibrogenesis [25, 26]. Results highlighted strong \nbinding preference for UBE2S , whereas substantially \nweaker affinities (higher ΔG values) were calculated for \nalternative targets including ubiquitin enzymes (UBE2C, \nUBE2D1, USP10, USP7, OTUB1), glycolytic \ncomponents (HK2, PFKFB3, PKM2, LDHA), and \nfibrosis-linked kinases (TGF -βR1, SMA D3, MAPK1, \nJNK1, mTOR), implying minimal off-target risks.   \nRescue studies in UBE2S -deficient EESCs clarified the \nCPM mechanism. Genetic ablation of UBE2S alone \ndecreased lactate output, and CPM failed to impose \nadditional suppression in this knockout context, \nconfirming UBE2S dependency for CPM -mediated \nglycolytic blockade . Antifibrotic specificity was tested \nvia macrophage-EESC co -culture (Figure 7a). CM \nharvested from macrophage interactions with UBE2S -\nKO (M0 -KO) or wild -type (M0 -WT) EESCs was \ntransferred to fresh EESCs. Immunoblotting \ndemonstrated that M0 -KO-derived CM substantially \ndiminished fibrotic markers (α-SMA, Col-1, FN) relative \nto M0 -WT CM . CPM exposure in UBE2S -absent co -\ncultures produced no further marker reduction, whereas \nCPM application to wild -type systems potently \nsuppressed fibrosis indicators compared to vehicle \ncontrols, with clea r statistical significance . Thus, CPM \nantifibrotic action relies explicitly on UBE2S presence, \nreinforcing targeted modulation of glycolysis -fibrosis \naxes.   \n \nUBE2S facilitates endometriosis progression in vivo   \nA global UBE2S knockout (UBE2S−/−) mouse line was \ngenerated to probe UBE2S contributions to endometrial \nfunction in living organisms. Homozygous knockout \nfemales exhibited sharply reduced fertility, prompting \nthe use of UBE2S−/− males crossed with heterozy gous \nfemales for propagation. Endometriosis lesions were \ninduced using endometrial fragments from UBE2S−/− \ndonors to assess in vivo consequences (Figure 8a). \nLesion dimensions were notably smal ler in knockout \nrecipients (Figure 8b). Quantitative assessment of total \nlesion volume and mass across cohorts confirmed \nsubstantial growth restraint in  the UBE2S -deficient \ngroup (Figures 8c-d). Hence, systemic UBE2S depletion \neffectively impedes endometriosis establishment and \nexpansion in murine models.\n \n \na) \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n79 \n \n \nb) c) \n \n \nd) f) \n \n \ng) \n \nh) \n\n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n80 \n \n \ne) i) \n \nj) \nFigure 8. UBE2S drives endometriosis progression in animal models. (a) Generation of the UBE2S knockout \nmouse model for endometriosis induction. (b-d) Body weight measurements and endometriotic lesion volumes in \ntreatment (n = 6) and control (n = 6) cohorts post-lesion excision. (e) Hematoxylin-eosin (H&E, 10x) and \nimmunohistochemistry (IHC, 20x) staining for UBE2S, GLUT1, CD163, and α-SMA (scale bar = 50 μm). \n\n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n81 \nStaining intensity quantified via ImageJ analysis. (f) Administration of CPM in the C57BL/6J endometriosis \nmodel. (g) Representative photographs of the most prominent ectopic implants per animal. (h-i) Quantification of \nectopic lesion volume (h) and mass (i). (j) Overview diagram illustrating UBE2S-orchestrated metabolic shifts \nand immune regulation in endometriosis. * p < 0.05, ** p < 0.01, *** p < 0.001 \nIHC staining was subsequently conducted to measure \nUBE2S and GLUT1 abundance. Lesions from the \nknockout group displayed markedly lower UBE2S and \nGLUT1 expression compared to controls (Figure 8e; p < \n0.001). Markers for fibrosis (α -SMA) and M2 \nmacrophages (CD163) were also diminished, supporting \nUBE2S’s role in fostering M2 polarization and fibrotic \nprocesses during in vivo  endometriosis advancement \n(Figure 8e; p < 0.01). Immunofluorescence on lesion \nsections from wild -type and UBE2S⁻/⁻ mice further \nrevealed that in wild -type tissues, GLUT1 -positive \nstromal cells were closely associated with CD163 -\npositive M2 macrophages. This association was greatly \nattenuated in knockout lesions, which showed reduced \nGLUT1 and fewer CD163⁺ i nfiltrates. Quantification \nconfirmed decreased CD163⁺ cell d ensity in knockout \nsamples. Likewise, wild -type lesions featured enriched \nα-SMA⁺ fibroblasts near GLUT1⁺ areas, indicative of \nongoing fibrosis, whereas knockout lesions exhibited \nlower GLUT1 and α -SMA signals . Quantitative data \ncorroborated these observations. Together, these in vivo \nfindings substantiate UBE2S’s promotion of M2 \nmacrophage recruitment and fibrotic changes via \nGLUT1-mediated pathways.   \nTo test the therapeutic potential of the UBE2S inhibitor \nCPM in vivo, an endometriosis model wa s induced in \nC57BL/6J mice (Figure 8f). The treatment arm received \nweekly intraperitoneal CPM (10 mg/kg). After four \nweeks, ectopic tissues were harves ted and evaluated \n(Figure 8g). Treatment resulted in significantly smaller \nlesion volumes and weights re lative to vehicle controls \n(Figures 8h-i). These data highlight CPM’s potent in \nvivo efficacy against endometriosis through UBE2S \ninhibition.   \nGlycolytic dysregulation in endometriotic tissues is well-\ndocumented [27, 28], yet the underlying molecular \ndrivers remain poorly defined. Transcriptomic profiling \nrevealed substantial UBE2S upregulation in \nendometriotic stromal cells (EESCs). Prior resear ch \nlinked UBE2S to metabolic reprogramming in tumors, \nsuch as stabilizing VHL in hepatocellular carcinoma to \nindirectly bolster HIF -1α and upregulate glycolytic \ngenes, thereby increasing glucose uptake and lactate \noutput under hypoxia [10]. In contrast, ou r work \nuncovered a distinct mechanism: UBE2S recruits USP10 \nto catalyze K48 -linked deubiquitination of GLUT1, \npreventing its degradation and enhancing stability. This \npathway likely operates independently of severe hypoxia, \ngiven the partial oxygenation in endome triotic lesions. \nFunctional assays confirmed that UBE2S -stabilized \nGLUT1 markedly boosts glucose uptake and glycolytic \nrates, pushing EESCs toward a hyper -glycolytic state. \nThis represents novel evidence for UBE2S’s direct \ninvolvement in glycolytic control, broadening insights \ninto metabolic aberrations in endometriosis.   \nUBE2S typically exerts pathogenic effects via its E2/E3 \nenzymatic activities, targeting substrates for \nubiquitination. Examples include K11 -linked \nmodification of APC/C to hasten cell cycle progression \n[29] and stabilization of β -catenin to enhance Wnt \nsignaling and metastasis in colorectal cancer [30]. \nEmerging evidence indicates UBE2S can also recruit \ndeubiquitinases like USP15 for opposing functions [19]. \nOur findings extend this dual role, demonstrating that \nUBE2S engages USP10 via its N -terminal region  to \nfacilitate K48-linked deubiquitination of GLUT1. USP10 \nrequires UBE2S to form a functional complex for this \nactivity. This discovery unveils a previously \nunrecognized deubiquitination -dependent role for \nUBE2S in GLUT1 regulation, enriching its multifaceted \nbiological profile.   \nEndometriosis is a persistent inflammatory condition \nwith profound immune microenvironment alterations \n[31]. M2 macrophages predominate in lesions, fueling \nchronic inflammation and fibrosis. We observed that \nUBE2S-enhanced GLUT1 stability elevates glycolyt ic \noutput in EESCs, leading to lactate accumulation that \npromotes M2 polarization. Lactate influences \nmacrophage phenotype via HIF-1α and STAT3 pathways \n[5, 32]. Additional experiments showed that UBE2S -\ndriven GLUT1 overexpression raised lactate and induced \nM2 markers (CD206, Arg -1, IL -10). Suppression of \nUBE2S or glycolytic inhibition reversed these changes, \nunderscoring the pathway’s critical role in macrophage \nreprogramming. \nPersistent inflammation and scarring in endometriosis \n(EM) lesions play a central role in driving disease \nadvancement and relapse [33-35]. M2-type macrophages \n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n82 \nrelease TGF-β1 and PDGF, which stimulate fibroblasts \nand drive their transformation into myofibroblasts via the \nTGF-β1/Smad pathway, thereby hastening tissue fibrosis \n[4, 36, 37]. Experiments conducted in vitro and in animal \nmodels showed that UBE2S -driven polarization of M2 \nmacrophages facilitated the shift of EM stromal \nfibroblasts toward a myofibroblast phenotype, with \nelevated levels of α -SMA and Col1. Administration of \nthe UBE2S inhibitor CPM markedly suppressed this \nprocess, indicating that UBE2S coul d serve as a \npromising target for treating fibrosis associated with EM. \nIt is worth noting that, although UBE2S knockout \nimpairs fertility in female mice, this effect stems mainly \nfrom early embryonic death due to APC/C inactivation \nand meiotic blockage, not from hormonal disruption or \novarian defects [38]. In our study, UBE2S –/– females \nexhibited no alterations in sexual development, estrous \ncycle patterns, or ovarian follicle reserve. Additionally, \nto minimize hormonal fluctuations during model \nestablishment, both recipient and donor mice underwent \nuniform estrogen priming and cycle synchronization. \nSuch standardization ensures that the observed fibrotic \nand metabolic changes are not influenced by hormonal or \ndevelopmental variations, but instead highlight the direct \ninvolvement of UBE2S in mature endometrial function. \nNevertheless, certain limitations exist in this work. The \ninvestigation relied predominantly on cellular and murine \nmodels, and validation using human patient specimens is \nrequired to strengthen its clinical relevance. The \nmetabolic shift driven by GLUT1 m ight engage \nadditional downstream cascades, such as the \nPI3K/Akt/mTOR pathway [39, 40]. Subsequent research \nshould explore these signaling networks in greater detail. \nWhile in silico docking and rescue experiments in \nUBE2S-knockout cells support CPM’s sele ctivity, \ncomprehensive profiling of deubiquitinases or kinases \nwas not performed. Predictive computational models \ncannot completely replicate intracellular binding \nbehavior. Future chemical proteomics approaches could \nverify CPM’s precise targets in physiological settings. \nConclusion \nIn summary, the present study reveals a previously \nunrecognized pathway in which UBE2S -dependent \ndeubiquitination of GLUT1 modulates glucose uptake, \nimmune milieu, and fibrogenesis in EM, positioning \nUBE2S as a ca ndidate therapeutic target (Figure 8j). \nThese results provide fresh perspectives on EM \npathogenesis and establish a conceptual basis for \nupcoming therapeutic strategies in patients. \nAcknowledgments: None \nConflict of Interest: None \nFinancial Support: None \nEthics Statement: None \nReferences \n1. Lu C, Qiao P, Fu R, Wang Y, Lu J, Ling X, et al. \nPhosphorylation of PFKFB4 by PIM2 promotes \nanaerobic Glycolysis and cell proliferation in \nendometriosis. Cell Death Dis. 2022;13(9):790–801. \n2. Esfandiari F, Chitsazian F, Jahromi MG, Favaedi R, \nBazrgar M, Aflatoonian R, et al. HOX cluster and \ntheir cofactors showed an altered expression pattern \nin eutopic and ectopic endometriosis tissues. Reprod \nBiol Endocrinol. 2021;19(1):132–42. \n3. Ivashkiv LB. The hypoxia -lactate axis tempers \ninflammation. Nat Rev Immunol. 2020;20(2):85–6. \n4. Nishimoto-Kakiuchi A, Sato I, Nakano K, Ohmori \nH, Kayukawa Y, Tanimura H, et al. A long -acting \nanti-IL-8 antibody improves inflammation and \nfibrosis in endometriosis. Sci Transl Med. \n2023;15(684):eabq5858. \n5. Gou Y, Wang H, Wang T, Wang H, Wang B, Jiao N, \net al. Ectopic endometriotic stromal cells -derived \nlactate induces M2 macrophage polarization via \nMettl3/Trib1/ERK/STAT3 signalling pathway in \nendometriosis. Immunology. 2023;168(3):389–402. \n6. Ling X, Lu J, Wang X, Liu L, Liu L, Wang Y, et al. \nOvarian tumorB1-mediated heat shock transcription \nfactor 1 deubiquitination is critical for Glycolysis \nand development of endometriosis. iScience. \n2022;25(11):105363. \n7. Popovic D, Vucic D, Dikic I. Ubiquitination in \ndisease pathogenesis and treatment. Nat Med. \n2014;20(11):1242–53. \n8. Jung CR, Hwang KS, Yoo J, Cho WK, Kim JM, Kim \nWH, et al. E2 -EPF UCP targets pVHL for \ndegradation and associates with tumor growth and \nmetastasis. Nat Med. 2006;12(7):809–16. \n9. Han Z, Xu L, Wang A, Wang B, Liu Q, Liu H, et al. \nUBE2S facilitates glioblastoma progression through \nactivation of the NF-kappaB pathway via attenuating \n\nJ Med Sci Interdiscip Res, 2022, 2(1):58-84                                                                                           Conti et al. \n \n \n \n83 \nK11-linked ubiquitination of AKIP1. Int J Biol \nMacromol. 2024;278(Pt 1):134426. \n10. Zhang R, Li C, Zhang S, Kong L, Liu Z, Guo Y, et \nal. UBE2S promotes Glycolysis in hepatocellular \ncarcinoma by enhancing E3 enzyme -independent \npolyubiquitination of VHL. Clin Mol Hepatol. \n2024;30(4):771–92. \n11. De Leo A, Ugolini A, Yu X, Scirocchi F, Scocozza \nD, Peixoto B, et al. Glucose-driven histone \nlactylation promotes the immunosuppressive \nactivity of monocyte -derived macrophages in \nglioblastoma. Immunity. 2024;57(5):1105–23. \n12. Colegio OR, Chu NQ, Szabo AL, Chu T, Rhebergen \nAM, Jairam V, et al. Functional polarization of \ntumour-associated macrophages by tumour -derived \nlactic acid. Nature. 2014;513(7519):559–63. \n13. Ren L, Xu B, Xu J, Li J, Jiang J, Ren Y, et al. A \nmachine learning model to predict survival and \ntherapeutic responses in multiple myeloma. Int J \nMol Sci. 2023;24(7):6683–700. \n14. Aran D, Hu Z, Butte AJ. xCell: digitally portraying \nthe tissue cellular heterogeneity landscape. Genome \nBiol. 2017;18(1):220–34. \n15. Pickart CM. Back to the future with ubiquitin. Cell. \n2004;116(2):181–90. \n16. Trulsson F, Akimov V, Robu M, van Overbeek N, \nBerrocal DAP, Shah RG, et al. Deubiquitinating \nenzymes and the proteasome regulate Preferential \nsets of ubiquitin substrates. Nat Commun. \n2022;13(1):2736–53. \n17. Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, \nRobert J, et al. Quantitative proteomics reveals the \nfunction of unconventional ubiquitin chains in \nproteasomal degradation. Cell. 2009;137(1):133–45. \n18. Lim JH, Shin HW, Chung KS, Kim NS, Kim JH, \nJung HR, et al. E2-EPF UCP possesses E3 ubiquitin \nligase activity via its cysteine 118 residue. PLoS \nONE. 2016;11(9):e0163710. \n19. Huang L, Liu H, Zhang K, Meng Q, Hu L, Zhang Y, \net al. Ubiquitin -Conjugating enzyme 2S enhances \nviral replication by inhibiting type I IFN production \nthrough recruiting USP15 to deubiquitinate TBK1. \nCell Rep. 2020;32(7):108044. \n20. Yue H, Wang J, Hou S, Zhang M. As a potential \npredictor of pan-cancer, UBE2S is related to tumor-\nassociated macrophage infiltration. Future Oncol. \n2023;19(29):1973–90. \n21. Qiu L, Wang Y, Li Z, Tu Z, Liu H. The landscape of \nUBE2S in hepatocellular carcinoma: prognostic \nsignificance, immuno -oncology feature and drug \nresponse. Genes Dis. 2023;10(2):363–5. \n22. Peng S, Chen X, Huang C, Yang C, Situ M, Zhou Q, \net al. UBE2S as a novel ubiquitinated regulator of \np16 and beta-catenin to promote bone metastasis of \nprostate cancer. Int J Biol Sci. 2022;18(8):3528–43. \n23. Muraoka A, Suzuki M, Hamaguchi T, Watanabe S, \nIijima K, Murofushi Y, et al. Fusobacterium \ninfection facilitates the development of \nendometriosis through the phenotypic transition of \nendometrial fibroblasts. Sci Transl Med. \n2023;15(700):eadd1531. \n24. Bokhari AA, Lee LR, Raboteau D, Hamilton CA, \nMaxwell GL, Rodriguez GC, et al. Progesterone \ninhibits endometrial cancer invasiveness by \ninhibiting the TGFbeta pathway. Cancer Prev Res \n(Phila). 2014;7(10):1045–55. \n25. McKinnon BD, Kocbek V, Nirgianakis K, Bersinger \nNA, Mueller MD. Kinase signalling pathways in \nendometriosis: potential targets for non -hormonal \ntherapeutics. Hum Reprod Update. 2016;22(3):382–\n403. \n26. Zhang M, Xu T, Tong D, Li S, Yu X, Liu B, et al. \nResearch advances in endometriosis -related \nsignaling pathways: A review. Biomed \nPharmacother. 2023;164:114909. \n27. Garcia-Gomez E, Vazquez -Martinez ER, Reyes -\nMayoral C, Cruz -Orozco OP, Camacho -Arroyo I, \nCerbon M. Regulation of inflammation pathways \nand inflammasome by sex steroid hormones in \nendometriosis. Front Endocrinol (Lausanne). \n2019;10:935–52. \n28. Chen Q, Jiao Y, Yin Z, Fu X, Guo S, Zhou Y, et al. \nEstablishment of a novel glycolysis-immune-related \ndiagnosis gene signature for endometriosis by \nmachine learning. J Assist Reprod Genet. \n2023;40(5):1147–61. \n29. Williamson A, Wickliffe KE, Mellone BG, Song L, \nKarpen GH, Rape M. Identification of a \nphysiological E2 module for the human anaphase -\npromoting complex. Proc Natl Acad Sci U S A. \n2009;106(43):18213–8. \n30. Li Z, Wang Y, Li Y, Yin W, Mo L, Qian X, et al. \nUbe2s stabilizes beta -Catenin through K11 -linked \npolyubiquitination to promote mesendoderm \nspecification and colorectal cancer development. \nCell Death Dis. 2018;9(5):456–69. \n31. Symons LK, Miller JE, Kay VR, Marks RM, Liblik \nK, Koti M, et al. The immunopathophysiology of \n\nConti et al.                                                                                           J Med Sci Interdiscip Res, 2022, 2(1):58-84  \n \n \n \n84 \nendometriosis. Trends Mol Med. 2018;24(9):748 –\n62. \n32. Chen J, Huang Z, Chen Y, Tian H, Chai P, Shen Y, \net al. Lactate and lactylation in cancer. Signal \nTransduct Target Ther. 2025;10(1):38–64. \n33. Chapron C, Marcellin L, Borghese B, Santulli P. \nRethinking mechanisms, diagnosis and management \nof endometriosis. Nat Rev Endocrinol. \n2019;15(11):666–82. \n34. Maulitz L, Stickeler E, Stickel S, Habel U, \nTchaikovski SN, Chechko N. Endometriosis, \npsychiatric comorbidities and neuroimaging: \nestimating the odds of an endometriosis brain. Front \nNeuroendocrinol. 2022;65:100988. \n35. Salliss ME, Farland LV, Mahnert ND, Herbst -\nKralovetz MM. The role of gut and genital \nmicrobiota and the estrobolome in endometriosis, \ninfertility and chronic pelvic pain. Hum Reprod \nUpdate. 2021;28(1):92–131. \n36. Duan J, Liu X, Wang H, Guo SW. The M2a \nmacrophage subset May be critically involved in the \nfibrogenesis of endometriosis in mice. Reprod \nBiomed Online. 2018;37(3):254–68. \n37. Wu S, Han L, Zhou M, Li X, Luo L, Wang Z, et al. \nLncRNA AOC4P recruits TRAF6 to regulate EZH2 \nubiquitination and participates in trophoblast \nGlycolysis and M2 macrophage polarization which \nis associated with recurrent spontaneous abortion. \nInt Immunopharmacol. 2023;125(Pt B):111201–15. \n38. Sun SM, Zhao BW, Li YY, Liu HY, Xu YH, Yang \nXM, et al. Loss of UBE2S causes meiosis I arrest \nwith normal spindle assembly checkpoint dynamics \nin mouse oocytes. Development. 2024;151(6). \n39. Bao YY, Zhong JT, Shen LF, Dai LB, Zhou SH, Fan \nJ, et al. Effect of Glut -1 and HIF -1alpha double \nknockout by CRISPR/CAS9 on radiosensitivity in \nlaryngeal carcinoma via the PI3K/Akt/mTOR \npathway. J Cell Mol Med. 2022;26(10):2881–94. \n40. Deng Y, Ma J, Zhao S, Yang M, Sun Y, Zhang Q. \nExpression of glucose transporter -1 in follicular \nlymphoma affected tumor -infiltrating immunocytes \nand was related to progression of disease within 24 \nmonths. Transl Oncol. 2023;28:101614–24.","source_license":"CC0","license_restricted":false}