Sp140 is a multi-SUMO-1 target and its PHD finger promotes SUMOylation of the adjacent Bromodomain.

Abstract Background Human Sp140 protein is a leukocyte-specific member of the speckled protein (Sp) family (Sp100, Sp110, Sp140, Sp140L), a class of multi-domain nuclear proteins involved in intrinsic immunity and transcriptional regulation. Sp140 regulates macrophage transcriptional program and is implicated in several haematologic malignancies. Little is known about Sp140 structural domains and its post-translational modifications. Methods We used mass spectrometry and biochemical experiments to investigate endogenous Sp140 SUMOylation in Burkitt's Lymphoma cells and Sp140 SUMOylation sites in HEK293T cells, FLAG-Sp140 transfected and His6-SUMO-1T95K infected. NMR spectroscopy and in vitro SUMOylation reactions were applied to investigate the role of Sp140 PHD finger in the SUMOylation of the adjacent BRD. Results Endogenous Sp140 is a SUMO-1 target, whereby FLAG-Sp140 harbors at least 13 SUMOylation sites distributed along the protein sequence, including the BRD. NMR experiments prove direct binding of the SUMO E2 ligase Ubc9 and SUMO-1 to PHD-BRDSp140. In vitro SUMOylation reactions show that the PHDSp140 behaves as SUMO E3 ligase, assisting intramolecular SUMOylation of the adjacent BRD. Conclusions Sp140 is multi-SUMOylated and its PHD finger works as versatile protein-protein interaction platform promoting intramolecular SUMOylation of the adjacent BRD. Thus, combinatorial association of Sp140 chromatin binding domains generates a multifaceted interaction scaffold, whose function goes beyond the canonical histone recognition. General significance The addition of Sp140 to the increasing lists of multi-SUMOylated proteins opens new perspectives for molecular studies on Sp140 transcriptional activity, where SUMOylation could represent a regulatory route and a docking surface for the recruitment and assembly of leukocyte-specific transcription regulators

A persulfidation-based mechanism controls aquaporin-8 conductance

Upon engagement of tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases release H2O2 in the extracellular space. We reported previously that aquaporin-8 (AQP8) transports H2O2 across the plasma membrane and is reversibly gated during cell stress, modulating signal strength and duration. We show that AQP8 gating is mediated by persulfidation of cysteine 53 (C53). Treatment with H2S is sufficient to block H2O2 entry in unstressed cells. Silencing cystathionine beta-synthase (CBS) prevents closure, suggesting that this enzyme is the main source of H2S. Molecular modeling indicates that C53 persulfidation displaces a nearby histidine located in the narrowest part of the channel. We propose that H2O2 molecules transported through AQP8 sulfenylate C53, making it susceptible to H2S produced by CBS. This mechanism tunes H2O2 transport and may control signaling and limit oxidative stress.This work was supported in part through grants from the Associazione Italiana Ricerca sul Cancro (IG 2016-18824 to R.S. and IG 2016-15434 to A.R.), the Fondazione Cariplo (2015-0591 to R.S.), the Ministero della Salute (PE-2011-02352286 to R.S. and RF-2013-02354880 to G.M.), the Telethon (GGP15059 to R.S.), and the "Cinque per mille"; (to A.R.). G.P.B. was supported by an Emmy Noether grant 1668/1-1 from the Deutsche Forschungsgemeinschaft

SMARCA5 interacts with NUP98-NSD1 oncofusion protein and sustains hematopoietic cells transformation

BACKGROUND: Acute myeloid leukemia (AML) is characterized by accumulation of aberrantly differentiated hematopoietic myeloid progenitor cells. The karyotyping-silent NUP98-NSD1 fusion is a molecular hallmark of pediatric AML and is associated with the activating FLT3-ITD mutation in &gt; 70% of the cases. NUP98-NSD1 fusion protein promotes myeloid progenitor self-renewal in mice via unknown molecular mechanism requiring both the NUP98 and the NSD1 moieties.METHODS: We used affinity purification coupled to label-free mass spectrometry (AP-MS) to examine the effect of NUP98-NSD1 structural domain deletions on nuclear interactome binding. We determined their functional relevance in NUP98-NSD1 immortalized primary murine hematopoietic stem and progenitor cells (HSPC) by inducible knockdown, pharmacological targeting, methylcellulose assay, RT-qPCR analysis and/or proximity ligation assays (PLA). Fluorescence recovery after photobleaching and b-isoxazole assay were performed to examine the phase transition capacity of NUP98-NSD1 in vitro and in vivo.RESULTS: We show that NUP98-NSD1 core interactome binding is largely dependent on the NUP98 phenylalanine-glycine (FG) repeat domains which mediate formation of liquid-like phase-separated NUP98-NSD1 nuclear condensates. We identified condensate constituents including imitation switch (ISWI) family member SMARCA5 and BPTF (bromodomain PHD finger transcription factor), both members of the nucleosome remodeling factor complex (NURF). We validated the interaction with SMARCA5 in NUP98-NSD1+ patient cells and demonstrated its functional role in NUP98-NSD1/FLT3-ITD immortalized primary murine hematopoietic cells by genetic and pharmacological targeting. Notably, SMARCA5 inhibition did not affect NUP98-NSD1 condensates suggesting that functional activity rather than condensate formation per se is crucial to maintain the transformed phenotype.CONCLUSIONS: NUP98-NSD1 interacts and colocalizes on the genome with SMARCA5 which is an essential mediator of the NUP98-NSD1 transformation in hematopoietic cells. Formation of NUP98-NSD1 phase-separated nuclear condensates is not sufficient for the maintenance of transformed phenotype, which suggests that selective targeting of condensate constituents might represent a new therapeutic strategy for NUP98-NSD1 driven AML.</p

Metabolic determinants of the immune modulatory function of neural stem cells.

BACKGROUND: Neural stem cells (NSCs) display tissue trophic and immune modulatory therapeutic activities after transplantation in central nervous system disorders. The intercellular interplay between stem cells and target immune cells is increased in NSCs exposed to inflammatory cues. Here, we hypothesize that inflammatory cytokine signalling leads to metabolic reprogramming of NSCs regulating some of their immune modulatory effects. METHODS: NSC lines were prepared from the subventricular zone (SVZ) of 7-12-week-old mice. Whole secretome-based screening and analysis of intracellular small metabolites was performed in NSCs exposed to cocktails of either Th1-like (IFN-γ, 500 U/ml; TNF-α, 200 U/ml; IL-1β, 100 U/ml) or Th2-like (IL-4, IL-5 and IL-13; 10 ng/ml) inflammatory cytokines for 16 h in vitro. Isotopologues distribution of arginine and downstream metabolites was assessed by liquid chromatography/mass spectrometry in NSCs incubated with U-(13)C6 L-arginine in the presence or absence of Th1 or Th2 cocktails (Th1 NSCs or Th2 NSCs). The expression of arginase I and II was investigated in vitro in Th1 NSCs and Th2 NSCs and in vivo in the SVZ of mice with experimental autoimmune encephalomyelitis, as prototypical model of Th1 cell-driven brain inflammatory disease. The effects of the inflammatory cytokine signalling were studied in NSC-lymph node cells (LNC) co-cultures by flow cytometry-based analysis of cell proliferation following pan-arginase inhibition with N(ω)-hydroxy-nor-arginine (nor-NOHA). RESULTS: Cytokine-primed NSCs showed significantly higher anti-proliferative effect in co-cultures vs. control NSCs. Metabolomic analysis of intracellular metabolites revealed alteration of arginine metabolism and increased extracellular arginase I activity in cytokine-primed NSCs. Arginase inhibition by nor-NOHA partly rescued the anti-proliferative effects of cytokine-primed NSCs. CONCLUSIONS: Our work underlines the use of metabolic profiling as hypothesis-generating tools that helps unravelling how stem cell-mediated mechanisms of tissue restoration become affected by local inflammatory responses. Among different therapeutic candidates, we identify arginase signalling as novel metabolic determinant of the NSC-to-immune system communication.This work has received support from the National Multiple Sclerosis Society (NMSS, partial grants RG-4001-A1), the Italian Multiple Sclerosis Association (AISM, grant 2010/R/31 and grant 2014/PMS/4), the Italian Ministry of Health (GR08-7), the European Research Council (ERC) under the ERC-2010-StG Grant agreement n° 260511-SEM_SEM and the UK Regenerative Medicine Platform Acellular hub (Partnership award RG69889) and core support grant from the Wellcome Trust and MRC to the Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute. LPJ was supported by a Wellcome Trust Research Training Fellowship (RG79423).This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s12974-016-0667-

The autoimmune regulator PHD finger binds to non-methylated histone H3K4 to activate gene expression

Mutations in the gene autoimmune regulator (AIRE) cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of tissue-restricted antigens. By the combined use of biochemical and biophysical methods, we show that AIRE selectively interacts with histone H3 through its first plant homeodomain (PHD) finger (AIRE–PHD1) and preferentially binds to non-methylated H3K4 (H3K4me0). Accordingly, in vivo AIRE binds to and activates promoters containing low levels of H3K4me3 in human embryonic kidney 293 cells. We conclude that AIRE–PHD1 is an important member of a newly identified class of PHD fingers that specifically recognize H3K4me0, thus providing a new link between the status of histone modifications and the regulation of tissue-restricted antigen expression in thymus

Laboratori verso la resilienza

Partendo dalle agende urbane per la sostenibilità, il Rapporto sulle Città cerca di tratteggiare un percorso con il quale i centri urbani possano proporsi quali luoghi privilegiati per l’avvio di una non più procrastinabile transizione verso nuovi modelli di sviluppo economico e rinnovate forme di convivenza e cittadinanza

A stapled chromogranin A-derived peptide is a potent dual ligand for integrins αvβ6 and αvβ8

Combining 2D STD-NMR, computation, biochemical assays and click-chemistry, we have identified a chromogranin-A derived compound (5) that has high affinity and bi-selectivity for αvβ6 and αvβ8 integrins and is stable in microsomal preparations

The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus

Milk From Cow Fed With High Forage/Concentrate Ratio Diet: Beneficial Effect on Rat Skeletal Muscle Inflammatory State and Oxidative Stress Through Modulation of Mitochondrial Functions and AMPK Activity

Milk and dairy products are relevant components of daily diet and are part of dietary recommendation in many countries due to their content of key nutrients. However, the relatively high content of saturated fat of the milk and its extensive usage for every age group raises concerns about its potential negative health effects. Therefore, in the last years, several researchers dedicated their attention to milk production and quality. Milk fatty acids profile depend on cow feeding and in particular on the type of forage and concentrate and forage/concentrate ratio. It was demonstrated that feeding dairy cows with a 70/30 forage/concentrate ratio yields milk with a low ω6:ω3 ratio and high CLA levels. In this work, we demonstrated that the supplementation of rats diet with this high forage milk (HFM) results, in the skeletal muscle of these animals, in a reduced lipid content and inflammation levels, and an improved mitochondrial lipid oxidation, and redox status through modulation of AMPK activity