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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)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer. The characteristic excessive stromatogenesis accompanying the growth of this tumor is believed to contribute to chemoresistance which, together with drug toxicity, results in poor clinical outcome. An increasing number of studies are showing that gut microbiota and their metabolites are implicated in cancer pathogenesis, progression and response to therapies. In this study we tested butyrate, a product of dietary fibers' bacterial fermentation, whose anticancer and anti-inflammatory functions are known. We provided in vitro evidence that, beside slowing proliferation, butyrate enhanced gemcitabine effectiveness against two human pancreatic cancer cell lines, mainly inducing apoptosis. In addition, we observed that, when administered to a PDAC mouse model, alone or combined with gemcitabine treatment, butyrate markedly reduced the cancer-associated stromatogenesis, preserved intestinal mucosa integrity and affected fecal microbiota composition by increasing short chain fatty acids producing bacteria and decreasing some pro-inflammatory microorganisms. Furthermore, a biochemical serum analysis showed butyrate to ameliorate some markers of kidney and liver damage, whereas a metabolomics approach revealed a deep modification of lipid metabolism, which may affect tumor progression or response to therapy. Such results support that butyrate supplementation, in addition to conventional therapies, can interfere with pancreatic cancer biology and response to treatment and can alleviate some damages associated to cancer itself or to chemotherapy.

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.

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

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

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 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.

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.

The complete primary structure of human erythrocyte acylpeptide hydrolase has been determined by using a combination of different mass spectrometric procedures and sequencing techniques. These data allowed us to correct the incomplete nucleotide sequence of the DNF15S2 locus on the short arm of human chromosome 3 at region 21, coding for the enzyme. The protein consists of 732 amino acid residues and is acetylated at the N-terminus. Alkylation experiments on the native enzyme demonstrated that all 17 cysteine residues present in the polypeptide chain are in reduced form. Multiple sequence alignment did not reveal striking similarity with proteases of known tertiary structure with the exception of members of the serine oligopeptidase family. Limited proteolysis experiments generated a C-terminal portion, containing all the catalytic triad elements responsible for proteolytic activity, and an N-terminal domain of unknown function, both still strongly associated in a completely active nicked form. The site of tryptic hydrolysis was identified as Arg193. The secondary structural organization of the protease domain of the enzyme is consistent with the alpha/beta hydrolase fold.

SummaryProteolytic cleavage of the urokinase plasminogen activator receptor (uPA(R)) prevents the binding of uPA and vitronectin while generating biologically active uPAR fragments. We have recently shown that matrix metalloproteinase-12 (MMP-12) releases cellular uPAR-antigen from stimulated human micro-vascular endothelial cells providing a novel feedback mechanism between the plasminogen activation and MMP systems. We now show that MMP-12 and other MMPs directly and efficiently cleave uPAR at the Thr86||Tyr87 peptide bond located in the linker region connecting uPAR domains 1 and 2, releasing the major ligand binding domain 1 from the rest of the receptor. The possible biological importance of uPAR cleavage by MMPs is supported by the observation that also murine uPAR is cleaved by MMP-12 (at the Pro89||Gln90 peptide bond), despite the limited sequence homology between the linker regions. Using an antibody raised against the human uPAR linker region we show that this region of uPAR, which contains the chemotactic SRSRY epitope, is exposed upon MMP cleavage.