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Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

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Glioblastoma (GBM) is a common and deadly form of brain tumor in adults. Dysregulated metabolism in GBM offers an opportunity to deploy metabolic interventions as precise therapeutic strategies. To identify the molecular drivers and the modalities by which different molecular subgroups of GBM exploit metabolic rewiring to sustain tumor progression, we interrogated the transcriptome, the metabolome, and the glycoproteome of human subgroup-specific GBM sphere-forming cells (GSC). L-fucose abundance and core fucosylation activation were elevated in mesenchymal (MES) compared with proneural GSCs; this pattern was retained in subgroup-specific xenografts and in subgroup-affiliated human patient samples. Genetic and pharmacological inhibition of core fucosylation significantly reduced tumor growth in MES GBM preclinical models. Liquid chromatography-mass spectrometry (LC-MS)–based glycoproteomic screening indicated that most MES-restricted core-fucosylated proteins are involved in therapeutically relevant GBM pathological processes, such as extracellular matrix interaction, cell adhesion, and integrin-mediated signaling. Selective L-fucose accumulation in MES GBMs was observed using preclinical minimally invasive PET, implicating this metabolite as a potential subgroup-restricted biomarker.Overall, these findings indicate that L-fucose pathway activation in MES GBM is a subgroup-specific dependency that could provide diagnostic markers and actionable therapeutic targets.Significance:Metabolic characterization of subgroup-specific glioblastoma (GBM) sphere-forming cells identifies the L-fucose pathway as a vulnerability restricted to mesenchymal GBM, disclosing a potential precision medicine strategy for targeting cancer metabolism.

Glioblastoma (GBM) is a common and deadly form of brain tumor in adults. Dysregulated metabolism in GBM offers an opportunity to deploy metabolic interventions as precise therapeutic strategies. To identify the molecular drivers and the modalities by which different molecular subgroups of GBM exploit metabolic rewiring to sustain tumor progression, we interrogated the transcriptome, the metabolome, and the glycoproteome of human subgroup-specific GBM sphere-forming cells (GSC). L-fucose abundance and core fucosylation activation were elevated in mesenchymal (MES) compared with proneural GSCs; this pattern was retained in subgroup-specific xenografts and in subgroup-affiliated human patient samples. Genetic and pharmacological inhibition of core fucosylation significantly reduced tumor growth in MES GBM preclinical models. Liquid chromatography-mass spectrometry (LC-MS)–based glycoproteomic screening indicated that most MES-restricted core-fucosylated proteins are involved in therapeutically relevant GBM pathological processes, such as extracellular matrix interaction, cell adhesion, and integrin-mediated signaling. Selective L-fucose accumulation in MES GBMs was observed using preclinical minimally invasive PET, implicating this metabolite as a potential subgroup-restricted biomarker. Overall, these findings indicate that L-fucose pathway activation in MES GBM is a subgroup-specific dependency that could provide diagnostic markers and actionable therapeutic targets. Significance: Metabolic characterization of subgroup-specific glioblastoma (GBM) sphere-forming cells identifies the L-fucose pathway as a vulnerability restricted to mesenchymal GBM, disclosing a potential precision medicine strategy for targeting cancer metabolism.

Objective: Significant efforts are currently being made to identify novel biomarkers for the diagnosis and risk stratification of prostate cancer (PCa). Metabolomics can be a very useful approach in biomarker discovery because metabolites are an important read-out of the disease when characterized in biological samples. We aimed to determine a metabolomic signature which can accurately distinguish men with clinically significant PCa from those affected by benign prostatic hyperplasia (BPH). Methods: We first performed untargeted metabolomics using ultrahigh-performance liquid chromatography tandem mass spectrometry on expressed prostatic secretion urine (EPS-urine) from 25 patients affected by BPH and 25 men with clinically significant PCa (defined as Gleason score ≥ 3 + 4). Diagnosis was histologically confirmed after surgical treatment. The EPS-urine metabolomic approach was then applied to a larger, prospective cohort of 92 consecutive patients undergoing multiparametric magnetic resonance imaging for clinical suspicion of PCa prior to biopsy. Results: We established a novel metabolomic signature capable of accurately distinguishing PCa from benign tissue. A metabolomic signature was associated with clinically significant PCa in all subgroups of the Prostate Imaging Reporting and Data System (PI-RADS) classification (100% and 89.13% of accuracy when the PI-RADS was in range of 1–2 and 4–5, respectively, and 87.50% in the more critical cases when the PI-RADS was 3). Conclusions: A combination of metabolites and clinical variables can effectively help in identifying PCa patients that might be overlooked by current imaging technologies. Metabolites from EPS-urine should help in defining the diagnostic pathway of PCa, thus improving PCa detection and decreasing the number of unnecessary prostate biopsies.

Aims Biliary diseases represent around 10% of all chronic liver diseases and affect both adults and children. Currently available biochemical tests detect cholestasis but not early liver fibrosis. Circulating extracellular vesicles (EVs) provide a non-invasive, real-time molecular snapshot of the injured organ. We thus aimed at searching for a panel of EV-based biomarkers for cholestasis-induced early liver fibrosis using mouse models. Results: Progressive and detectable histological evidence of collagen deposition and liver fibrosis was observed as from Day 8 after bile duct ligation (BDL) in mice. Whole transcriptome and small RNA-seq analyses of circulating EVs revealed differentially enriched RNA species after BDL versus sham controls. Unsupervised hierarchical clustering identified a signature that allowed for discrimination between BDL and controls. In particular, 151 microRNAs enriched in BDL-derived EVs were identified, of which 66 were conserved in humans. The liver was an important source of circulating EVs in BDL animals as evidenced by the enrichment of several hepatic mRNAs, such as Albumin and Haptoglobin. Interestingly, among experimentally validated miRNAs, miR192-5p, miR194-5p, miR22-3p and miR29a-3p showed similar enrichment patterns also in EVs derived from DDC-treated (drug-induced severe cholestasis) but not in mice with mild phenotype or non-cholestatic liver fibrosis. Innovation A panel of mRNAs and miRNAs contained in circulating EVs, when combined, indicate hepatic damage and fibrosis in mice, and represent promising biomarkers for human severe cholestasis-induced liver fibrosis. Conclusion Analysis of EV-based miRNAs, in combination with hepatic injury RNA markers, can detect early cholestatic liver injury and fibrosis in mice.

Lymph nodes (LNs) are secondary lymphoid tissues that play a critical role in filtering the lymph and promoting adaptive immune responses. Surgical resection of LNs, radiation therapy, or infections may damage lymphatic vasculature and compromise immune functions. Here, we describe the generation of functional synthetic lympho-organoids (LOs) using LN stromal progenitors and decellularized extracellular matrix-based scaffolds, two basic constituents of secondary lymphoid tissues. We show that upon transplantation at the site of resected LNs, LOs become integrated into the endogenous lymphatic vasculature and efficiently restore lymphatic drainage and perfusion. Upon immunization, LOs support the activation of antigen-specific immune responses, thus acquiring properties of native lymphoid tissues. These findings provide a proof-of-concept strategy for the development of functional lympho-organoids suitable for restoring lymphatic and immune cell functions., Stem Cell Reports, 12 (6)

Despite research progresses, the chance to accurately predict the risk for diabetic nephropathy (DN) is still poor. So far, the first evidence of DN is micro-albuminuria, which is detected only 10–20 years after the onset of diabetes. Our goal is to develop new predictive tools of nephropathy starting from urine, which can be easily obtained using noninvasive procedures and it is directly related to kidney. Since it is reasonable to suppose that, in predisposed patients, the mechanisms leading to nephropathy start acting since the diabetes onset, urine from children with recent diagnosis of type 1 diabetes was subjected to proteomic analysis in comparison to age-matched controls. Targeted confirmation was performed on children with a longer history of diabetes using Western Blotting and applying a urinary lipidomic approach. To definitively understand whether the observed alterations could be related to diabetic nephropathy, urine from diabetic adults with or without albuminuria was also examined. For the first time, lipid metabolisms of prostaglandin and ceramide, which are significantly and specifically modified in association with DN, are shown to be already altered in children with a recent diabetes diagnosis. Future studies on larger cohorts are needed to improve the validity and generalizability of these findings. Data are available via ProteomeXchange with identifier PXD011183 Submission details: Project Name: Urinary proteomics by 2DE and LC-MS/MS. Project accession: PXD011183 Project DOI: https://doi.org/10.6019/PXD011183 Significance Nephropathy is a very common diabetic complication. Once established, its progression can only be slowed down but full control or remission is achieved in very few cases, thus posing a large burden on worldwide health. The first evidence of diabetic nephropathy (DN) is micro-albuminuria, but only 30% of patients with micro-albuminuria progress to proteinuria, while in some patients it spontaneously reverts to normo-albuminuria. Thus, there is clear need for biomarkers that can accurately predict the risk to develop DN. Herein, by applying proteomic and lipidomic approaches on urine samples, we show that alteration of prostaglandin and ceramide metabolisms specifically occurs in association with DN. Interestingly, we demonstrate that the modification of these metabolic pathways is an early event in diabetic patients, suggesting the identified changed proteins as possible predictive biomarkers of diabetes-induced renal function decline.

ERBB2 is a ligand-less tyrosine kinase receptor expressed at very low levels in normal tissues; when overexpressed, it is involved in malignant transformation and tumorigenesis in several carcinomas. In cancer cells, ERBB2 represents the preferred partner of other members of the ERBB receptor family, leading to stronger oncogenic signals, by promoting both ERK and AKT activation. The identification of the specific signaling downstream of ERBB2 has been impaired by the lack of a ligand and of an efficient way to selectively activate the receptor. In this paper, we found that antibodies (Abs) targeting different epitopes on the ERBB2 extracellular domain foster the activation of ERBB2 homodimers, and surprisingly induce a unique cytostatic signaling cascade promoting an ERK-dependent ERBB2 Thr701 phosphorylation, leading to AKT de-phosphorylation, via PP2A Ser/Thr phosphatases. Furthermore, the immunophilin Cyclophilin A plays a crucial role in this pathway, acting as a negative modulator of AKT de-phosphorylation, possibly by competing with Ser/Thr phosphatases for binding to AKT. Altogether, our data show that Ab recognizing ERBB2 extracellular domain function as receptor agonists, promoting ERBB2 homodimer activation, leading to an anti-proliferative signaling. Thus, the ultimate outcome of ERBB2 activity might depend on the dimerization status: pro-oncogenic in the hetero-, and anti-oncogenic in the homo-dimeric form.

ERBB2 is a ligand-less tyrosine kinase receptor expressed at very low levels in normal tissues; when overexpressed, it is involved in malignant transformation and tumorigenesis in several carcinomas. In cancer cells, ERBB2 represents the preferred partner of other members of the ERBB receptor family, leading to stronger oncogenic signals, by promoting both ERK and AKT activation. The identification of the specific signaling downstream of ERBB2 has been impaired by the lack of a ligand and of an efficient way to selectively activate the receptor. In this paper, we found that antibodies (Abs) targeting different epitopes on the ERBB2 extracellular domain foster the activation of ERBB2 homodimers, and surprisingly induce a unique cytostatic signaling cascade promoting an ERK-dependent ERBB2 Thr

INTRODUCTION AND OBJECTIVES:The lack of non-invasive diagnostic tests capable to distinguish between indolent and aggressive prostate cancer (PCa) may lead to over-diagnosis and overtreatment. Alth...

Objective To study pathogenic features of the somatic testicular microenvironment associated with idiopathic germ cell aplasia. Design Cross-sectional study. Setting Tertiary referral center for reproductive medicine. Patient(s) Testicular specimens from men with idiopathic nonobstructive azoospermia (iNOA) prospectively submitted to microdissection testicular sperm extraction. Of 20 specimens used for histology, 10 were also available for proteomic analysis. Primary Sertoli cells with normal karyotype and phenotype were also used. Intervention(s) Patients with iNOA were dichotomized according to a positive versus negative sperm retrieval at microdissection testicular sperm extraction, and on the isolated extracellular matrix (ECM) the proteomic analysis was performed. Main Outcome Measure(s) Proteomic analysis of the ECM from testicular specimens with positive versus negative sperm retrieval. Gene ontology enrichment was used to identify upstream regulators based on the 11 deregulated ECM proteins, which were validated by immunohistochemistry and quantitative polymerase chain reaction. Continuous variables were expressed as medians and interquartile range. Result(s) Germ cell aplasia was characterized by an increased signaling of the retinoic acid in Sertoli cells and associated with decreased expression of the basal membrane markers nidogen-2 and heparan sulfate proteoglycan-2. Decreased levels of the interstitial matrisome-associated factor IX and its regulator VKORC1 were, instead, coupled with decreased signaling of vitamin K in Leydig cells. An altered expression of a further eight ECM proteins was also found, including laminin-4 and laminin-5. Peripheral levels of the two vitamins were within the reference range in the two cohorts of iNOA men. Conclusion(s) We identified the pathogenetic signature of the somatic human testicular microenvironment, providing two vitamin-related mechanistic insights related to the molecular determinants of the idiopathic germ cell aplasia.

Conditioned medium (CM) and extracellular vesicles (EV) from Adipose-derived Stem/stromal cells (ASC) and Dermal fibroblasts (DF) represent promising tools for therapeutic applications. Which one should be preferred is still under debate and no direct comparison of their proteome has been reported yet. Here, we apply quantitative proteomics to explore the protein composition of CM and EV from the two cell types. Data are available via ProteomeXchange (identifier PXD020219). We identified 1977 proteins by LC-MS/MS proteomic analysis. Unsupervised clustering analysis and PCA recognized CM and EV as separate groups. We identified 68 and 201 CM and EV specific factors. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation factors. The analysis of ASC and DF secretomes revealed the presence of cell type-specific proteins. ASC-CM and -EV carried factors involved in ECM organization and immunological regulation, respectively. Conversely, DF-CM and -EV were enriched in epithelium development associated factors and -EV in Wnt signaling factors. In conclusion, this analysis provides evidence of a different protein composition between CM and EV and of the presence of cell type-specific bioactive mediators suggesting their specific future use as advanced therapy medicinal products. SIGNIFICANCE: The use of cell secretome presents several advantages over cell therapy such as the lower risks associated to the administration step and the avoidance of any potential risk of malignant transformation. The main secretome preparations consist in concentrated conditioned medium (CM) and extracellular vesicles (EV). Both of them showed well-documented therapeutic potentials. However, it is still not clear in which case it should be better to use one preparation over the other and an exhaustive comparison between their proteome has not been performed yet. The choice of the cell source is another relevant aspect that still needs to be addressed. In order to shed light on these questions we explored the protein composition of CM and EV obtained from Adipose-derived Stem/stromal Cells (ASC) and Dermal Fibroblasts (DF), by a comprehensive quantitative proteomics approach. The analysis showed a clear distinction between CM and EV proteome. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation-related factors. Furthermore, the analysis of ASC and DF secretomes revealed specific biological processes for the different cell products. ASC secretome presented factors involved in ECM organization (hyaluronan and glycosaminoglycan metabolism) and immunological regulation (e.g. macrophage and IkB/NFkB signaling regulation), respectively. On the other hand, DF-CM and -EV were both enriched in epithelium development associated factors, whilst DF-CM in proteins involved in cellular processes regulation and -EV in Wnt signaling factors. In conclusion, our study shed a light on the different protein composition of CM and EV of two promising cell types, spanning from basic processes involved in secretion to specific pathways supporting their therapeutic potential and their possible future use as advanced therapy medicinal products.

White adipose tissue (WAT) can undergo a phenotypic switch, known as browning, in response to environmental stimuli such as cold. Post-translational modifications of histones have been shown to regulate cellular energy metabolism, but their role in white adipose tissue physiology remains incompletely understood. Here we show that histone deacetylase 3 (HDAC3) regulates WAT metabolism and function. Selective ablation of Hdac3 in fat switches the metabolic signature of WAT by activating a futile cycle of de novo fatty acid synthesis and β-oxidation that potentiates WAT oxidative capacity and ultimately supports browning. Specific ablation of Hdac3 in adipose tissue increases acetylation of enhancers in Pparg and Ucp1 genes, and of putative regulatory regions of the Ppara gene. Our results unveil HDAC3 as a regulator of WAT physiology, which acts as a molecular brake that inhibits fatty acid metabolism and WAT browning., Histone deacetylases, such as HDAC3, have been shown to alter cellular metabolism in various tissues. Here the authors show that HDAC3 regulates WAT metabolism by activating a futile cycle of fatty acid synthesis and oxidation, which supports WAT browning.

Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface ((568)NGR) and the other buried in the tertiary structure ((962)NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed (568)NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden (962)NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion.

Broncho-pulmonary dysplasia (BPD) is a chronic pulmonary disorder that follows premature birth. It is preceded by respiratory distress syndrome (RDS), characterized by acute respiratory failure due to deficiency of surfactant at birth. Clinical characteristics of infants affected by BPD have widely changed in the last decades: they are extraordinarly immature, with impaired alveolar and vascular lung development. To build up new therapeutic strategies for BPD babies, it is necessary to understand the pathogenic mechanisms, which are complicated by environmental risk factors and genetic predisposition. Therefore, the aim of this study was to highlight protein changes in the broncho-alveolar lavage fluid (BALF), thus providing an appropriate picture on what is happening in the locus of injury. We analyzed BALF samples from preterm babies, born at different stages of lung development. We confirmed that gestational age is relevant for BPD progression, but we also detected few de-regulated proteins in the younger babies; we discovered less abundant calcium signaling-related proteins, consistent with BPD severity, comparing severe to mild BPD babies with matched gestational age. In conclusion, this study suggests a subset of proteins to be investigated to better treat BPD babies and facilitate the definition of potential drug targets for novel therapies. Biological significance Pulmonary biomarkers are needed to predict the clinical course of lung disease, status, progression and response to treatment. A key aspect in biomarker discovery is uncovering molecules that appear early during disease initiation, when the natural history of the disease can be modified. Using a proteomic-based approach we compared broncho-alveolar lavage fluid (BALF) protein profile from preterm neonates at different postmenstrual ages, to have a molecular description of broncho-pulmonary dysplasia (BPD) progression. BALF provided a snapshot of local molecular changes, which are relevant for early diagnosis, assessment and characterization of lung disorders. We showed that even if the studied patients had similar clinical phenotype (they all developed severe BPD and they were all cured in the same way in terms of mechanical ventilation, surfactant administration, antenatal steroid treatment and ibuprofen treatment for patent ductus arteriosus), however their BALF protein profiling displayed significant differences in a subset of proteins, which could be exploited to facilitate the development of novel effective therapies, distinct for age and severity of the disease.

The extracellular matrix (ECM) from perilesional and colorectal carcinoma (CRC), but not healthy colon, sustains proliferation and invasion of tumor cells. We investigated the biochemical and physical diversity of ECM in pair-wised comparisons of healthy, perilesional and CRC specimens. Progressive linearization and degree of organization of fibrils was observed from healthy to perilesional and CRC ECM and was associated with a steady increase of stiffness and collagen crosslinking. In the perilesional ECM these modifications coincided with increased vascularization, whereas in the neoplastic ECM they were associated with altered modulation of matrisome proteins, increased content of hydroxylated lysine and lysyl oxidase. This study identifies the increased stiffness and crosslinking of the perilesional ECM predisposing an environment suitable for CRC invasion as a phenomenon associated with vascularization. The increased stiffness of colon areas may represent a new predictive marker of desmoplastic region predisposing to invasion, thus offering new potential application for monitoring adenoma with invasive potential.

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery–Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer-oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein-uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA-plasminogen activator inhibitor type 1-mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. © The Rockefeller University Press.

Expression of the membrane receptor uPAR induces profound changes in cell morphology and migration, and its expression correlates with the malignant phenotype of cancers. To identify the molecular interactions essential for uPAR function in these processes, we carried out a complete functional alanine scan of uPAR in HEK293 cells. Of the 255 mutant receptors characterized, 34 failed to induce changes in cell morphology. Remarkably, the molecular defect of all of these mutants was a specific reduction in integrin-independent cell binding to vitronectin. A membrane-tethered plasminogen activator inhibitor-1, which has the same binding site in vitronectin as uPAR, replicated uPAR-induced changes. A direct uPAR–vitronectin interaction is thus both required and sufficient to initiate downstream changes in cell morphology, migration, and signal transduction. Collectively these data demonstrate a novel mechanism by which a cell adhesion molecule lacking inherent signaling capability evokes complex cellular responses by modulating the contact between the cell and the matrix without the requirement for direct lateral protein–protein interactions.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins. DOI: http://dx.doi.org/10.7554/eLife.08887.001, eLife digest Several proteins in humans and other animals contain a region called a 'zona pellucida domain'. This domain enables these proteins to associate with each other and form long filaments. Uromodulin is one such protein that was first identified more than fifty years ago. This protein is known to play a role in human diseases such as hypertension and kidney failure, but uromodulin’s biological purpose still remains elusive. Uromodulin is only made in the kidney and it is the most abundant protein in the urine of healthy individuals. Uromodulin also contains a so-called 'external hydrophobic patch' that must be removed before the zona pellucida domain can start to form filaments. This hydrophobic patch is removed when uromodulin is cut by an unknown enzyme; this cutting releases the rest of the uromodulin protein from the surface of the cells that line the kidney into the urine. Brunati et al. have now tested a panel of candidate enzymes and identified that one called hepsin is able to cut uromodulin. Hepsin is embedded in the cell membrane of the cells that line the kidney. When the level of hepsin was artificially reduced in cells grown in the laboratory, uromodulin remained anchored to the cell surface, its processing was altered and it did not form filaments. Brunati et al. next analysed mice in which the gene encoding hepsin had been deleted. While these animals did not have any major defects in their internal organs, they had much lower levels of uromodulin in their urine. Furthermore, this residual urinary protein was not cut properly and it did not assemble into filaments. Thus, these findings reveal that hepsin is the enzyme that is responsible for releasing uromodulin in the urine. This discovery could be exploited to alter the levels of uromodulin release, and further studies using mice lacking hepsin may also help to understand uromodulin’s biological role. Finally, it will be important to understand if hepsin, or a similar enzyme, is also responsible for the release of other proteins containing the zona pellucida domain. DOI: http://dx.doi.org/10.7554/eLife.08887.002

Figure S1. Western blot analysis for contaminant hemoglobin detection. Figure S2. Immunoprecipitation of both resting- and oxidized-ceruloplasmin.Table S1. Spectrophotometric analysis for hemoglobin contaminant detection in CSF samples. (PDF 1445 kb)

SummaryThe role of circulating factors in regulating colonic stem cells (CoSCs) and colonic epithelial homeostasis is unclear. Individuals with long-standing type 1 diabetes (T1D) frequently have intestinal symptoms, termed diabetic enteropathy (DE), though its etiology is unknown. Here, we report that T1D patients with DE exhibit abnormalities in their intestinal mucosa and CoSCs, which fail to generate in vitro mini-guts. Proteomic profiling of T1D+DE patient serum revealed altered levels of insulin-like growth factor 1 (IGF-I) and its binding protein 3 (IGFBP3). IGFBP3 prevented in vitro growth of patient-derived organoids via binding its receptor TMEM219, in an IGF-I-independent manner, and disrupted in vivo CoSC function in a preclinical DE model. Restoration of normoglycemia in patients with long-standing T1D via kidney-pancreas transplantation or in diabetic mice by treatment with an ecto-TMEM219 recombinant protein normalized circulating IGF-I/IGFBP3 levels and reestablished CoSC homeostasis. These findings demonstrate that peripheral IGF-I/IGFBP3 controls CoSCs and their dysfunction in DE.Video Abstract

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of β-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native β-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of β-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of β-glycosidase from S. solfataricus.

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA.suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.

Three related nucleotide sequences, encoding mature proteins of 108-113 amino acids, have been obtained from antennal cDNA of the Phasmid Eurycantha calcarata. Among these, one is also expressed in the tarsi as demonstrated by N-terminal sequence and mass spectrometric analyses of protein samples isolated from both organs. PCR experiments performed with specific primers, showed that this species is also expressed in the mouth organs and in the cuticle, while the other two are antennal specific. All three isoforms are similar to Drosophila OS-D and other proteins reported in several insect orders, but one of them is significantly different from the other two. The best conserved elements are the N-terminal region and the four cysteine residues. Accurate ESMS measurements indicated that all cysteines are involved in two disulphide bonds and ruled out the occurrence of additional post-translational modifications. Polyclonal antibodies, raised against the purified protein, did not react with proteins of the same class expressed in another Phasmid species, Carausius morosus, and in the orthopteran Schistocerca gregaria, nor did antibodies against these proteins recognise those of E. calcarata.

The structure of ecto-5 0 -nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for < 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the highmannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosylphosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S‐S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51‐Cys57, Cys353‐Cys358, Cys365‐Cys387 and Cys476‐Cys479. This work resolves details of the structure of ecto-5 0 -nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.

Human alpha1-microglobulin (alpha1-m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily, was isolated by sequential anion-exchange chromatography and gel filtration from the urine of hemodialized patients and from amniotic fluid collected in the week 16-18 of pregnancy. The carbohydrate chains of the protein purified from the two sources, which are organized in two Asn-linked and one Thr-linked oligosaccharides, were structurally characterized using matrix-assisted laser desorption ionization and electrospray mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and O-glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary structures attached in the case of urinary alpha1-m and only diantennary glycans in the amniotic fluid protein. The main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary glycans. The position of the oligosaccharide chains in a three-dimensional model of the protein, produced using the automated Swiss-Model service, is also discussed.

The carbohydrate chains of nine isoforms of chicken egg-white riboflavin-binding protein (RfBP) and six isoforms each of quail egg-white and yolk RfBP have been structurally characterized. The two N-glycosylation sites, Asn36 and Asn147, of the most abundant isoform of each of the three proteins were analyzed in further detail leading to the identification of different glycosylation patterns. In both chicken and quail egg-white RfBP the carbohydrates attached to position 36 had a lower degree of branching and, in the case of the quail protein, this site was only partially glycosylated. A very heterogeneous mixture of complex structures was characteristic of the other glycosylation site. Analysis of the two sites in quail yolk RfBP confirmed this result which agrees with what has been established for hen yolk RfBP. The presence in the three proteins of a highly heterogeneous mixture of differently branched glycans suggests that the differences in isoelectric points, which is a peculiarity of the different isoforms, are probably indeed due to differences in carbohydrate structure.

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor,called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has beenreported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yetbeen clearly established. In the present study we examined structural and membrane-binding properties ofsaposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionicphospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization ofthe lipid surface and, conversely, the association with the membrane markedly affected the fluorescenceproperties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsictyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues.The structural properties of saposin D were investigated in detail using advanced MS analysis. It was foundthat the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked inthe following order: Cys5–Cys78, Cys8–Cys72 and Cys36–Cys47. The disulfide pattern of saposin D isidentical with that previously established for two other saposins, B and C, which also exhibit a strong affinity forlipids. The common disulfide structure probably has an important role in the interaction of these proteins withmembranes.The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in themolecule is mainly modified by high-mannose-type structures varying from two to six hexose residues.Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that theglycosylation site is not related to the lipid-binding site.The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neitherceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding,while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured theinteraction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles wheresaposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely,saposin D may affect the lipid organization of anionic phospholipid-containing membranes.Keywords: disulfide bridges; mass spectrometry, membrane interaction; phospholipids; saposin D.Saposin D is released together with three other similar proteins,saposins A, B and C, from a common precursor calledprosaposin [1–5]. This precursor is proteolytically processedinto mature saposins in late endosomes and lysosomes [6,7].Genetic defects of saposins have provided insights into theirrole in the degradation of sphingolipids. When all saposins aremissing, as seen in a patient carrying a mutation in theprosaposin initiation codon, accumulation of several sphingo-lipids such as ceramide, glucosylceramide, lactosylceramideand ganglioside GM3 has been observed [8,9]. A mutant mouseline in which the prosaposin gene was inactivated exhibited aclinical, pathological and biochemical phenotype closelyresembling that of the human disease [10]. Saposins B and Cappear to be involved in the catabolism of specific sphingo-lipids, as indicated by the storage of sulfatide caused by aselective deficiency of saposin B in an atypical form ofmetachromatic leukodystrophy and the storage of glucosylcer-amide caused by a selective deficiency of saposin C in a variantform of Gaucher’s disease [11–13]. An isolated deficiency insaposin A or D has not been reported so far and thus their actualphysiological functions are still not known. In vitro, saposin Ais able to activate the degradation of glucosylceramide [14,15]and galactosylceramide [14], and saposin D that of sphingo-myelin and ceramide [16,17]. The addition of saposin D to theculture medium of fibroblasts from patients with prosaposindeficiency leads to a decrease in the accumulated ceramide[18], and thus the function of this saposin seems to be related toceramide degradation rather than sphingomyelin degradation.As saposins are involved in the catabolism of membranecomponents such as sphingolipids, a knowledge of theirinteractions with lipid bilayers is of critical importance to ourunderstanding of their physiological behaviour. We haverecently found that at least two saposins, C and D, are able tobind to phospholipid large unilamellar vesicles (LUVs) at lowEur. J. Biochem. 263, 486–494 (1999) q FEBS 1999

Acetylation of α-tubulin at lysine 40 (K40) is a well-conserved posttranslational modification that marks long-lived microtubules but has poorly understood functional significance. Recently, αTAT1, a member of the Gcn5-related N-acetyltransferase superfamily, has been identified as an α-tubulin acetyltransferase in ciliated organisms. Here, we explored the function of αTAT1 with the aim of understanding the consequences of αTAT1-mediated microtubule acetylation. We demonstrate that α-tubulin is the major target of αTAT1 but that αTAT1 also acetylates itself in a regulatory mechanism that is required for effective modification of tubulin. We further show that in mammalian cells, αTAT1 promotes microtubule destabilization and accelerates microtubule dynamics. Intriguingly, this effect persists in an αTAT1 mutant with no acetyltransferase activity, suggesting that interaction of αTAT1 with microtubules, rather than acetylation per se, is the critical factor regulating microtubule stability. Our data demonstrate that αTAT1 has cellular functions that extend beyond its classical enzymatic activity as an α-tubulin acetyltransferase.

The mechanisms of BM hematopoietic stem/progenitor cell (HSPC) adhesion, engraftment, and mobilization remain incompletely identified. Here, using WT and transgenic mice, we have shown that membrane-anchored plasminogen activator, urokinase receptor (MuPAR) marks a subset of HSPCs and promotes the preservation of the size of this pool of cells in the BM. Loss or inhibition of MuPAR increased HSPC proliferation and impaired their homing, engraftment, and adhesion to the BM microenvironment. During mobilization, MuPAR was inactivated by plasmin via proteolytic cleavage. Cell-autonomous loss of the gene encoding MuPAR also impaired long-term engraftment and multilineage repopulation in primary and secondary recipient mice. These findings identify MuPAR and plasmin as regulators of the proliferation, marrow pool size, homing, engraftment, and mobilization of HSPCs and possibly also of HSCs.

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of beta-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native beta-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of beta-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of beta-glycosidase from S. solfataricus.