Metabolic profiling of tryptophan pathways: Implications for obesity and metabolic dysfunction-associated steatotic liver disease.

BACKGROUND AND AIMS: The rise in obesity highlights the need for improved therapeutic strategies, particularly in addressing metabolic dysfunction-associated steatotic liver disease (MASLD). We aim to assess the role of tryptophan metabolic pathways in the pathogenesis of obesity and in the different histological stages of MASLD. MATERIALS AND METHODS: We used ultra-high performance liquid chromatography to quantify circulating levels of 15 tryptophan-related metabolites from the kynurenine, indole and serotonin pathways. A cohort of 76 subjects was analysed, comprising 18 subjects with normal weight and 58 with morbid obesity, these last being subclassified into normal liver (NL), simple steatosis (SS) and metabolic dysfunction-associated steatohepatitis (MASH). Then, we conducted gene expression analysis of hepatic IDO-1 and kynyrenine-3-monooxygenase (KMO). RESULTS: Key findings in obesity revealed a distinct metabolic signature characterized by a higher concentration of different kynurenine-related metabolites, a decrease in indole-3-acetic acid and indole-3-propionic acid, and an alteration in the serotonin pathway. Elevated tryptophan levels were associated with MASLD presence (37.659 (32.577-39.823) µM of tryptophan in NL subjects; 41.522 (38.803-45.276) µM in patients with MASLD). Overall, pathway fluxes demonstrated an induction of tryptophan catabolism via the serotonin pathway in SS subjects and into the kynurenine pathway in MASH. We found decreased IDO-1 and KMO hepatic expression in NL compared to SS. CONCLUSIONS: We identified a distinctive metabolic signature in obesity marked by changes in tryptophan catabolic pathways, discernible through altered metabolite profiles. We observed stage-specific alterations in tryptophan catabolism fluxes in MASLD, highlighting the potential utility of targeting these pathways in therapeutic interventions.

Platelet-associated biomarkers in nonalcoholic steatohepatitis: Insights from a female cohort with obesity.

BACKGROUND: There is a lack of noninvasive diagnostic methods for nonalcoholic steatohepatitis (NASH), the severe condition of metabolic dysfunction-associated steatotic liver disease (MASLD). Platelet activation, evaluated through certain related parameters, is associated with liver disease and inflammation, but previous results are inconclusive. AIM: To investigate the potential utility of platelet-related indices as noninvasive diagnostic markers for the detection and prediction of MASLD, focusing on NASH. RESULTS: We found that mean platelet volume (MPV), plateletcrit (PCT) and platelet distribution width (PDW) were increased in the severe and morbidly obese (SMO) group compared to the normal weight (NW) group. We found decreased levels of MPV in steatosis and NASH patients. MPV and PCT values were decreased in the presence of mild liver inflammation. Platelet count (PLA) and PCT values were lower in the presence of ballooning. We obtained an area under the ROC curve of 0.84 using MPV and three other variables to predict MASLD. CONCLUSIONS: Some platelet-related indices vary depending on liver condition. Here, we reported decreased MPV in MASLD presence. Moreover, we presented for the first time a predictive model using MPV, ALT levels and the presence of diabetes mellitus and metabolic syndrome to predict MASLD in obese women. Also, MPV is closely related to early liver inflammation in NASH, and PLA and PCT are related to hepatic ballooning. These indices could be widely used for the early detection of NASH since they are usually determined in routine laboratory tests.

Identification of the Potential Molecular Mechanisms Linking RUNX1 Activity with Nonalcoholic Fatty Liver Disease, by Means of Systems Biology.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic hepatic disease; nevertheless, no definitive diagnostic method exists yet, apart from invasive liver biopsy, and nor is there a specific approved treatment. Runt-related transcription factor 1 (RUNX1) plays a major role in angiogenesis and inflammation; however, its link with NAFLD is unclear as controversial results have been reported. Thus, the objective of this work was to determine the proteins involved in the molecular mechanisms between RUNX1 and NAFLD, by means of systems biology. First, a mathematical model that simulates NAFLD pathophysiology was generated by analyzing Anaxomics databases and reviewing available scientific literature. Artificial neural networks established NAFLD pathophysiological processes functionally related to RUNX1: hepatic insulin resistance, lipotoxicity, and hepatic injury-liver fibrosis. Our study indicated that RUNX1 might have a high relationship with hepatic injury-liver fibrosis, and a medium relationship with lipotoxicity and insulin resistance motives. Additionally, we found five RUNX1-regulated proteins with a direct involvement in NAFLD motives, which were NFκB1, NFκB2, TNF, ADIPOQ, and IL-6. In conclusion, we suggested a relationship between RUNX1 and NAFLD since RUNX1 seems to regulate NAFLD molecular pathways, posing it as a potential therapeutic target of NAFLD, although more studies in this field are needed.

Ditopic Aza-Scorpiand Ligands Interact Selectively with ds-RNA and Modulate the Interaction upon Formation of Zn2+ Complexes.

Nucleic acids are essential biomolecules in living systems and represent one of the main targets of chemists, biophysics, biologists, and nanotechnologists. New small molecules are continuously developed to target the duplex (ds) structure of DNA and, most recently, RNA to be used as therapeutics and/or biological tools. Stimuli-triggered systems can promote and hamper the interaction to biomolecules through external stimuli such as light and metal coordination. In this work, we report on the interaction with ds-DNA and ds-RNA of two aza-macrocycles able to coordinate Zn2+ metal ions and form binuclear complexes. The interaction of the aza-macrocycles and the Zn2+ metal complexes with duplex DNA and RNA was studied using UV thermal and fluorescence indicator displacement assays in combination with theoretical studies. Both ligands show a high affinity for ds-DNA/RNA and selectivity for ds-RNA. The ability to interact with these duplexes is blocked upon Zn2+ coordination, which was confirmed by the low variation in the melting temperature and poor displacement of the fluorescent dye from the ds-DNA/RNA. Cell viability assays show a decrease in the cytotoxicity of the metal complexes in comparison with the free ligands, which can be associated with the observed binding to the nucleic acids.

Cell Death Triggered by the Autophagy Inhibitory Drug 3-Methyladenine in Growing Conditions Proceeds With DNA Damage.

Macroautophagy (hereafter autophagy) is a multistep intracellular catabolic process with pleiotropic implications in cell fate. Attending to its activation, autophagy can be classified into inducible or constitutive. Constitutive, or basal autophagy, unfolds under nutrient-replete conditions to maintain the cellular homeostasis. Autophagy inhibitory drugs are powerful tools to interrogate the role of autophagy and its consequences on cell fate. However, 3-methyladenine and various of these compounds present an intrinsic capacity to trigger cell death, for instance the broadly-employed 3-methyladenine. To elucidate whether the inhibition of basal autophagy is causative of cell demise, we have employed several representative compounds acting at different phases of the autophagic process: initiation (SBI0206965 and MHY1485), nucleation (3-methyladenine, SAR405, Spautin-1 and Cpd18), and completion (Bafilomycin A1 and Chloroquine). These compounds inhibited the basal autophagy of MEF cultures in growing conditions. Among them, 3-methyladenine, SBI-0206965, Chloroquine, and Bafilomycin A1 triggered BAX- and/or BAK-dependent cytotoxicity and caspase activation. 3-methyladenine was the only compound to induce a consistent and abrupt decrease in cell viability across a series of ontologically unrelated human cell lines. 3-methyladenine-induced cytotoxicity was not driven by the inhibition of the AKT/mTOR axis. Autophagy-deficient Fip200-/- MEFs displayed an increased sensitivity to activate caspases and to undergo cell death in response to 3-methyladenine. The cytotoxicity induced by 3-methyladenine correlated with a massive DNA damage, as shown by γ-H2A.X. This genotoxicity was observed at 10 mM 3-methyladenine, the usual concentration to inhibit autophagy and was maximized in Fip200-/- MEFs. In sum, our results suggest that, in growing conditions, autophagy acts as a protective mechanism to diminish the intrinsic cytotoxicity of 3-methyladenine. However, when the cellular stress exerted by 3-methyladenine surpasses the protective effect of basal autophagy, caspase activation and DNA damage compromise the cell viability.

Circular DNA intermediates in the generation of large human segmental duplications.

BACKGROUND: Duplications of large genomic segments provide genetic diversity in genome evolution. Despite their importance, how these duplications are generated remains uncertain, particularly for distant duplicated genomic segments. RESULTS: Here we provide evidence of the participation of circular DNA intermediates in the single generation of some large human segmental duplications. A specific reversion of sequence order from A-B/C-D to B-A/D-C between duplicated segments and the presence of only microhomologies and short indels at the evolutionary breakpoints suggest a circularization of the donor ancestral locus and an accidental replicative interaction with the acceptor locus. CONCLUSIONS: This novel mechanism of random genomic mutation could explain several distant genomic duplications including some of the ones that took place during recent human evolution.

Mitochondrial lipid droplet formation as a detoxification mechanism to sequester and degrade excessive urothelial membranes.

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due to its coverage by urothelial plaques consisting of 2D crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Because mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism.

Uroplakins play conserved roles in egg fertilization and acquired additional urothelial functions during mammalian divergence.

Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique two-dimensional crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mammalian urothelium and are often referred to as being urothelium specific, they are also expressed in several mouse nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms, and ovary/oocytes. In oocytes, uroplakins colocalize with CD9 on cell-surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs' fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require two-dimensional-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form two-dimensional-crystalline plaques during mammalian divergence, enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, to protect/modify the apical surface of the modern-day mammalian urothelium.

Homo- and Heterobinuclear Cu2+ and Zn2+ Complexes of Ditopic Aza Scorpiand Ligands as Superoxide Dismutase Mimics.

Two polytopic aza-scorpiand-like ligands, 6-[7-(diaminoethyl)-3,7-diazaheptyl]-3,6,9-triaza-1-(2,6-pyridina)cyclodecaphane (L1) and 6-[6'-[3,6,9-triaza-1-(2,6-pyridina)cyclodecaphan-6-yl]-3-azahexyl]-3,6,9-triaza-1-(2,6-pyridina)cyclodecaphane (L2), have been synthesized. The acid-base behavior and Cu2+, Zn2+, and Cu2+/Zn2+ mixed coordination have been analyzed by potentiometry, cyclic voltammetry, and UV-vis spectroscopy. The resolution of the crystal structures of [Cu2L2Cl2](ClO4)2·1.67H2O (1), [Cu2HL2Br2](ClO4)3·1.5H2O (2), and [CuZnL2Cl2](ClO4)2·1.64H2O (3) shows, in agreement with the solution data, the formation of homobinuclear Cu2+/Cu2+ and heterobinuclear Cu2+/Zn2+ complexes. The metal ions are coordinated within the two macrocyclic cavities of the ligand with the involvement of a secondary amino group of the bridge in the case of 1 and 3. Energy-dispersive X-ray spectroscopy confirms the 1:1 Cu2+/Zn2+ stoichiometry of 3. The superoxide dismutase (SOD) activities of the Cu2+/Cu2+ and Cu2+/Zn2+ complexes of L1 and L2 have been evaluated using nitro blue tetrazolium assays at pH 7.4. The IC50 and kcat values obtained for the [Cu2L1]4+ complex rank among the best values reported in the literature for Cu-SOD mimics. Interestingly, the binuclear Cu2+ complexes of L1 and L2 have low toxicity in cultures of mammalian cell lines and show significant antioxidant activity in a copper-dependent SOD (SOD1)-defective yeast model. The results are rationalized by taking into account the binding modes of the Cu2+ ions in the different complexes.

Phosphotyrosine phosphatase R3 receptors: Origin, evolution and structural diversification.

Subtype R3 phosphotyrosine phosphatase receptors (R3 RPTPs) are single-spanning membrane proteins characterized by a unique modular composition of extracellular fibronectin repeats and a single cytoplasmatic protein tyrosine phosphatase (PTP) domain. Vertebrate R3 RPTPs consist of five members: PTPRB, PTPRJ, PTPRH and PTPRO, which dephosphorylate tyrosine residues, and PTPRQ, which dephosphorylates phophoinositides. R3 RPTPs are considered novel therapeutic targets in several pathologies such as ear diseases, nephrotic syndromes and cancer. R3 RPTP vertebrate receptors, as well as their known invertebrate counterparts from animal models: PTP52F, PTP10D and PTP4e from the fruitfly Drosophila melanogaster and F44G4.8/DEP-1 from the nematode Caenorhabditis elegans, participate in the regulation of cellular activities including cell growth and differentiation. Despite sharing structural and functional properties, the evolutionary relationships between vertebrate and invertebrate R3 RPTPs are not fully understood. Here we gathered R3 RPTPs from organisms covering a broad evolutionary distance, annotated their structure and analyzed their phylogenetic relationships. We show that R3 RPTPs (i) have probably originated in the common ancestor of animals (metazoans), (ii) are variants of a single ancestral gene in protostomes (arthropods, annelids and nematodes); (iii) a likely duplication of this ancestral gene in invertebrate deuterostomes (echinodermes, hemichordates and tunicates) generated the precursors of PTPRQ and PTPRB genes, and (iv) R3 RPTP groups are monophyletic in vertebrates and have specific conserved structural characteristics. These findings could have implications for the interpretation of past studies and provide a framework for future studies and functional analysis of this important family of proteins.

Correction: The Tetraspanin-Associated Uroplakins Family (UPK2/3) Is Evolutionarily Related to PTPRQ, a Phosphotyrosine Phosphatase Receptor.

[This corrects the article DOI: 10.1371/journal.pone.0170196.].

The Tetraspanin-Associated Uroplakins Family (UPK2/3) Is Evolutionarily Related to PTPRQ, a Phosphotyrosine Phosphatase Receptor.

Uroplakins are a widespread group of vertebrate integral membrane proteins that belong to two different families: UPK1a and UPK1b belong to the large tetraspanin (TSPAN) gene family, and UPK3a, UPK3b, UPK3c, UPK3d, UPK2a and UPK2b form a family of their own, the UPK2/3 tetraspanin-associated family. In a previous study, we reported that uroplakins first appeared in vertebrates, and that uroplakin tetraspanins (UPK1a and UPK1b) should have originated by duplication of an ancestor tetraspanin gene. However, the evolutionary origin of the UPK2/3 family remains unclear. In this study, we provide evidence that the UPK2/3 family originated by gene duplication and domain loss from a protoPTPRQ-like basal deuterostome gene. PTPRQs are members of the subtype R3 tyrosine phosphatase receptor (R3 PTPR) family, which are characterized by having a unique modular composition of extracellular fibronectin (FN3) repeats, a transmembrane helix, and a single intra-cytoplasmic phosphotyrosine phophatase (PTP) domain. Our assumption of a deuterostome protoPTPRQ-like gene as an ancestor of the UPK2/3 family by gene duplication and loss of its PTP and fibronectin (FN3) domains, excluding the one closest to the transmembrane helix, is based on the following: (i) phylogenetic analyses, (ii) the existence of an identical intron/exon gene pattern between UPK2/3 and the corresponding genetic region in R3 PTPRs, (iii) the conservation of cysteine patterns and protein motifs between UPK2/3 and PTPRQ proteins and, (iv) the existence in tunicates, the closest organisms to vertebrates, of two sequences related to PTPRQ; one with the full subtype R3 modular characteristic and another without the PTP domain but with a short cytoplasmic tail with some sequence similarity to that of UPK3a. This finding will facilitate further studies on the structure and function of these important proteins with implications in human diseases.

Significant in vivo anti-inflammatory activity of Pytren4Q-Mn a superoxide dismutase 2 (SOD2) mimetic scorpiand-like Mn (II) complex.

BACKGROUND: The clinical use of purified SOD enzymes has strong limitations due to their large molecular size, high production cost and immunogenicity. These limitations could be compensated by using instead synthetic SOD mimetic compounds of low molecular weight. BACKGROUND/METHODOLOGY: We have recently reported that two SOD mimetic compounds, the Mn(II) complexes of the polyamines Pytren2Q and Pytren4Q, displayed high antioxidant activity in bacteria and yeast. Since frequently molecules with antioxidant properties or free-radical scavengers also have anti-inflammatory properties we have assessed the anti-inflammatory potential of Pytren2Q and Pytren4Q Mn(II) complexes, in cultured macrophages and in a murine model of inflammation, by measuring the degree of protection they could provide against the cellular injury produced by lipopolisacharide, a bacterial endotoxin. PRINCIPAL FINDINGS: In this report we show that the Mn(II) complex of Pytren4Q but not that of Pytren2Q effectively protected human cultured THP-1 macrophages and whole mice from the inflammatory effects produced by LPS. These results obtained with two molecules that are isomers highlight the importance of gathering experimental data from animal models of disease in assessing the potential of candidate molecules. CONCLUSION/SIGNIFICANCE: The effective anti-inflammatory activity of the Mn(II) complex of Pytren4Q in addition to its low toxicity, water solubility and ease of production would suggest it is worth taking into consideration for future pharmacological studies.

Generation of divergent uroplakin tetraspanins and their partners during vertebrate evolution: identification of novel uroplakins.

BACKGROUND: The recent availability of sequenced genomes from a broad array of chordates (cephalochordates, urochordates and vertebrates) has allowed us to systematically analyze the evolution of uroplakins: tetraspanins (UPK1a and UPK1b families) and their respective partner proteins (UPK2 and UPK3 families). RESULTS: We report here: (1) the origin of uroplakins in the common ancestor of vertebrates, (2) the appearance of several residues that have statistically significantly positive dN/dS ratios in the duplicated paralogs of uroplakin genes, and (3) the existence of strong coevolutionary relationships between UPK1a/1b tetraspanins and their respective UPK2/UPK3-related partner proteins. Moreover, we report the existence of three new UPK2/3 family members we named UPK2b, 3c and 3d, which will help clarify the evolutionary relationships between fish, amphibian and mammalian uroplakins that may perform divergent functions specific to these different and physiologically distinct groups of vertebrates. CONCLUSIONS: Since our analyses cover species of all major chordate groups this work provides an extremely clear overall picture of how the uroplakin families and their partner proteins have evolved in parallel. We also highlight several novel features of uroplakin evolution including the appearance of UPK2b and 3d in fish and UPK3c in the common ancestor of reptiles and mammals. Additional studies of these novel uroplakins should lead to new insights into uroplakin structure and function.

Analysis of the Ush2a gene in medaka fish (Oryzias latipes).

Patients suffering from Usher syndrome (USH) exhibit sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. USH is the most common genetic disorder affecting hearing and vision and is included in a group of hereditary pathologies associated with defects in ciliary function known as ciliopathies. This syndrome is clinically classified into three types: USH1, USH2 and USH3. USH2 accounts for well over one-half of all Usher cases and mutations in the USH2A gene are responsible for the majority of USH2 cases, but also for atypical Usher syndrome and recessive non-syndromic RP. Because medaka fish (Oryzias latypes) is an attractive model organism for genetic-based studies in biomedical research, we investigated the expression and function of the USH2A ortholog in this teleost species. Ol-Ush2a encodes a protein of 5.445 aa codons, containing the same motif arrangement as the human USH2A. Ol-Ush2a is expressed during early stages of medaka fish development and persists into adulthood. Temporal Ol-Ush2a expression analysis using whole mount in situ hybridization (WMISH) on embryos at different embryonic stages showed restricted expression to otoliths and retina, suggesting that Ol-Ush2a might play a conserved role in the development and/or maintenance of retinal photoreceptors and cochlear hair cells. Knockdown of Ol-Ush2a in medaka fish caused embryonic developmental defects (small eyes and heads, otolith malformations and shortened bodies with curved tails) resulting in late embryo lethality. These embryonic defects, observed in our study and in other ciliary disorders, are associated with defective cell movement specifically implicated in left-right (LR) axis determination and planar cell polarity (PCP).

DOR/Tp53inp2 and Tp53inp1 constitute a metazoan gene family encoding dual regulators of autophagy and transcription.

Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28-42; region 2, 66-112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription.