Establishing the interactome of the cancer associated stress protein NUPR1 (NUclear PRotein 1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1 thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further reported that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system indicating NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation dependent functions of key proteins involved in the DDR.
Wenjun Lan, Patricia Santofimia-Castaño, Mirna Swayden, Yi Xia, Zhengwei Zhou, Stephane Audebert, Luc Camoin, Can Huang, Ling Peng, Ana Jiménez-Alesanco, Adrián Velázquez-Campoy, Olga Abian, Gwen Lomberk, Raul Urrutia, Bruno Rizzuti, Vincent Geli, Philippe Soubeyran, Jose Luis Neira, Juan L. Iovanna
Dysregulated sensing of self nucleic acid is a leading cause of autoimmunity in multifactorial and monogenic diseases. Mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells, cause autoimmune manifestations and increased production of type-I interferons by innate cells. Here we show that complexes of self-DNA and autoantibodies (DNA-IC) contribute to elevated interferon levels via activation of the cGAS-STING pathway of cytosolic sensing. Mechanistically, lack of endosomal F-actin nucleation by WASp causes a delay in endolysosomal maturation and prolongs the transit time of ingested DNA-IC. Stalling in maturation-defective organelles facilitates leakage of DNA-IC into the cytosol, promoting activation of the TBK1-STING pathway. Genetic deletion of STING, STING and cGAS chemical inhibitors abolish interferon production and rescue systemic activation of interferon stimulated genes in vivo. These data unveil the contribution of cytosolic self-nucleic acid sensing in WAS and underscore the importance of WASp-mediated endosomal actin remodelling to prevent innate activation.
Giulia Maria Piperno, Asma Naseem, Giulia Silvestrelli, Roberto Amadio, Nicoletta Caronni, Karla Evelia Cervantes Luevano, Nalan Liv, Judith Klumperman, Andrea Colliva, Hashim Ali, Francesca Graziano, Philippe Benaroch, Hans Haecker, Richard N. Hanna, Federica Benvenuti
Histone deacetylase (HDAC) enzymes regulate transcription through epigenetic modification of chromatin structure, but their specific functions in the kidney remain elusive. We discovered that the human kidney expresses class I HDACs. Kidney medullary-specific inhibition of class I HDACs in the rat during high salt feeding results in hypertension, polyuria, hypokalemia, and nitric oxide (NO) deficiency. Three new, inducible murine models were used to determine that HDAC1 and HDAC2 in the kidney epithelium are necessary for maintaining epithelial integrity and maintaining fluid-electrolyte balance during increased dietary sodium intake. Moreover, single nucleus RNA sequencing determined that epithelial HDAC1 and HDAC2 are necessary for expression of many sodium or water transporters and channels. In performing a systematic review and meta-analysis of serious adverse events associated with clinical HDAC inhibitor use, we found that HDAC inhibitors increased the odds ratio of experiencing fluid-electrolyte disorders such as hypokalemia. This study provides insight on the mechanisms of potential serious adverse events with HDAC inhibitors, which may be fatal to critically ill patients. In conclusion, kidney tubular HDACs provide a link between the environment, such as consumption of high salt diets, with regulation of homeostatic mechanisms to remain in fluid-electrolyte balance.
Kelly A. Hyndman, Joshua S. Speed, Luciano D. Mendoza, John Allan, Jackson Colson, Randee Sedaka, Chunhua Jin, Hyun Jun Jung, Samir El-Dahr, David Pollock, Jennifer Pollock.
Over the last years, a number of drugs have been approved for the treatment of cystic fibrosis (CF). Among them, the new Trikafta, a combination of three drugs, holds great promises to radically improve the quality of life for a large part of CF patients carrying one copy of the most frequent CFTR mutation: F508del. Currently available, disease-modifying, CF drugs work by rescuing the function of mutated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) anion channel. Recent research work shows that membrane lipids, and the cell lipidome in general, play a significant role in the mechanism of CFTR defective trafficking and, on the other hand, in its rescue. In this paper, by using untargeted lipidomics on CFBE41o– cells, we identified distinctive changes in bronchial epithelial cell lipidome associated with treatment with the triple combination VX-661/VX-445/VX-770 (drug name: Trikafta) and other CF drugs. Particularly interesting is the reduction of ceramide levels, known molecular players in the induction of apoptosis, that appears to be associated with a decrease in cell susceptibility to undergo apoptosis. This evidence could account for additional beneficial role of the triple combination on CF phenotypes.
Nara Liessi, Emanuela Pesce, Clarissa Braccia, Sine Mandrup Bertozzi, Alessandro Giraudo, Tiziano Bandiera, Nicoletta Pedemonte, Andrea Armirotti
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a relative paucity of cancer cells that are surrounded by an abundance of non-tumor cells and extracellular matrix, known as stroma. The interaction between stroma and cancer cells contributes to poor outcome, but how proteins from these individual compartments drive aggressive tumor behavior is not known. Here, we report the proteomic analysis of laser-capture microdissected (LCM) PDAC samples. We isolated stroma, tumor, and bulk samples from a cohort with long- and short-term survivors. Compartment-specific proteins were measured by mass spectrometry, yielding the largest PDAC proteome landscape to date. These analyses revealed that in bulk analysis, tumor-derived proteins were typically masked and that LCM was required to reveal biology and novel prognostic markers. We validated tumor CALB2 and stromal COL11A1 expression as compartment-specific prognostic markers. We identified and functionally addressed the contributions of the tumor cell receptor EPHA2 to tumor cell viability and motility, underscoring the value of compartment-specific protein analysis in PDAC.
Tessa Y.S. Le Large, G. Mantini, Laura L. Meijer, T.V. Pham, N. Funel, Nicole C.T. van Grieken, B. Kok, Jaco C. Knol, H.W.M. van Laarhoven, S.R. Piersma, C.R. Jimenez, G. Kazemier, E. Giovannetti, M.F. Bijlsma
Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient’s risk of severe COPD. We determined 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in IGSF3 (immunoglobulin superfamily, member 3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs of which, rs1414272 and rs12066192 were directly- and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide- and sphingosine-1 phosphate-, but low glycosphingolipids- and gangliosides levels; were less apoptotic and more adherent; with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and a weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.
Kelly S. Schweitzer, Natini Jinawath, Raluca Yonescu, Kevin Ni, Natalia Rush, Varodom Charoensawan, Irina Bronova, Evgeny Berdyshev, Sonia M. Leach, Lucas A. Gillenwater, Russell P. Bowler, David B. Pearse, Constance A. Griffin, Irina Petrache
Free light chains (FLCs) induce inflammatory pathways in proximal tubule cells (PTCs). The role of toll-like receptors (TLR) in these responses is unknown. Here we present findings on the role of TLRs in FLC-induced PTC injury. We exposed human kidney PTC cultures to κ and λ FLCs, and used cell supernatants and pellets for ELISA and gene expression studies. We also analyzed tissues from Stat1–/– and littermate control mice treated with daily intraperitoneal injections of a κ-FLC for 10 days. FLCs increased the expression of TLRs 2, 4, 6 via HMGB1, a damage-associated molecular pattern. Countering TLRs 2, 4, and 6 through GIT-27 or specific TLR-siRNAs reduced downstream cytokine responses. Blocking HMGB1 through siRNA or pharmacologic inhibition, or via STAT1 inhibition reduced FLC-induced TLRs 2, 4, and 6 expression. Blocking endocytosis of FLCs through silencing of megalin/cubilin, with bafilomycin-A1, or hypertonic sucrose attenuated FLC-induced cytokine responses in PTCs. Immunohistochemistry showed decreased TLR 4 and 6 expression in kidney sections from Stat1–/– mice compared to their littermate controls. PTCs exposed to FLCs released HMGB1, which induced TLRs 2, 4, 6 expression and downstream inflammation. Blocking FLCs’ endocytosis, Stat1 knock-down, HMGB1 inhibition, and TLR knock-down each rescued PTCs from FLC-induced injury.
Rohit Upadhyay, Wei-Zhong Ying, Zannatul Nasrin, Hana Safah, Edgar A. Jaimes, Wenguang Feng, Paul W. Sanders, Vecihi Batuman
Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus non-active tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secrete factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of pro-angiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGFBR2, thereby sensitizing tumor cells to TGF-β ligands. Together these findings suggest that Wnt/β-catenin active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.
Allegra G. Hawkins, Elisabeth A. Pedersen, Sydney Treichel, Kelsey Temprine, Colin Sperring, Jay A. Read, Brian Magnuson, Rashmi Chugh, Elizabeth R. Lawlor
Tissue regeneration capacity declines with aging in association with heightened oxidative stress. Expression of the oxidant-generating enzyme, NADPH oxidase 4 (Nox4) is elevated in aged mice with diminished capacity for fibrosis resolution. Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal (BET) family of proteins that function as epigenetic “readers” of acetylated lysine groups on histones. In this study, we explored the role of Brd4 and its interaction with the p300 acetyltransferase in the regulation of Nox4, and the in-vivo efficacy of a BET inhibitor to reverse established age-associated lung fibrosis. BET inhibition interferes with the association of Brd4, p300, and acetylated histone H4K16 with the Nox4 promoter in lung fibroblasts stimulated with the pro-fibrotic cytokine, transforming growth factor-β1 (TGF-β1). This Brd4-Nox4 epigenetic axis is constitutively upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. A number of BET inhibitors, including I-BET-762, JQ1, and OTX015, downregulate Nox4 gene expression and activity. Aged mice with established and persistent lung fibrosis recovered capacity for fibrosis resolution with OTX015 treatment. This study implicates epigenetic regulation of Nox4 by Brd4 and p300, and supports BET/Brd4 inhibition as an effective strategy for the treatment of age-related fibrotic lung disease.
Yan Y. Sanders, Xing Lyu, Q. Jennifer Zhou, Zheyi Xiang, Denise Stanford, Sandeep Bodduluri, Steven M. Rowe, Victor J. Thannickal
Increased microvascular leakage is a cardinal feature of many critical diseases. Regular exercise is associated with improved endothelial function and reduced risk of cardiovascular disease. Irisin, secreted during exercise, contributes to many health benefits of exercise. However, the effects of irisin on endothelial function and microvascular leakage remain unknown. In this study, we found that irisin remarkably strengthened endothelial junctions and barrier function via binding to integrin αVβ5 receptor in LPS-treated endothelial cells. The beneficial effect of irisin was associated with suppression of the Src-MLCK-β-catenin pathway, activation of the AMPK-Cdc42/Rac1 pathway and improvement of mitochondrial function. In preclinical models of microvascular leakage, exogenous irisin improved pulmonary function, decreased lung edema and injury, suppressed inflammation, and increased survival. In ARDS patients, serum irisin levels were decreased and inversely correlated with disease severity and mortality. In conclusion, irisin enhances endothelial barrier function and mitigates microvascular leakage related diseases.
Jianbin Bi, Jia Zhang, Yifan Ren, Zhaoqing Du, Yuanyuan Zhang, Chang Liu, Yawen Wang, Lin Zhang, Zhihong Shi, Zheng Wu, Yi Lv, Rongqian Wu
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