Research Article
Abstract
Hepatic insulin resistance is central to type 2 diabetes (T2D) and metabolic syndrome, but defining the molecular basis of this defect in humans is challenging because of limited tissue access. Utilizing inducible pluripotent stem cells differentiated into hepatocytes from control individuals and patients with T2D and liquid chromatography with tandem mass spectrometry–based (LC-MS/MS–based) phosphoproteomics analysis, we identified a large network of cell-intrinsic alterations in signaling in T2D. Over 300 phosphosites showed impaired or reduced insulin signaling, including losses in the classical insulin-stimulated PI3K/AKT cascade and their downstream targets. In addition, we identified over 500 phosphosites of emergent, i.e., new or enhanced, signaling. These occurred on proteins involved in the Rho-GTPase pathway, RNA metabolism, vesicle trafficking, and chromatin modification. Kinome analysis indicated that the impaired phosphorylation sites represented reduced actions of AKT2/3, PKCθ, CHK2, PHKG2, and/or STK32C kinases. By contrast, the emergent phosphorylation sites were predicted to be mediated by increased action of the Rho-associated kinases 1 and 2 (ROCK1/2), mammalian STE20-like protein kinase 4 (MST4), and/or branched-chain α-ketoacid dehydrogenase kinase (BCKDK). Inhibiting ROCK1/2 activity in T2D induced pluripotent stem cell–derived hepatocytes restored some of the alterations in insulin action. Thus, insulin resistance in the liver in T2D did not simply involve a loss of canonical insulin signaling but the also appearance of new phosphorylations representing a change in the balance of multiple kinases. Together, these led to altered insulin action in the liver and identified important targets for the therapy of hepatic insulin resistance.
Authors
Arijeet K. Gattu, Maria Tanzer, Tomer M. Yaron-Barir, Jared L. Johnson, Ashok Kumar Jayavelu, Hui Pan, Jonathan M. Dreyfuss, Lewis C. Cantley, Matthias Mann, C. Ronald Kahn
Abstract
Chronic liver injury triggers the activation and recruitment of immune cells, causing antigen-independent tissue damage and liver disease progression. Tissue inflammation can reshape macrophage composition through monocyte replacement. Replacement of tissue macrophages with monocytes differentiating in an inflammatory environment can potentially imprint a phenotype that switches the liver from an immune-tolerant organ to one predisposed to tissue damage. We longitudinally sampled the liver of patients with chronic hepatitis B who had active liver inflammation and were starting antiviral therapy. Antiviral therapy suppressed viral replication and liver inflammation, which coincided with decreased myeloid activation markers. Single-cell RNA-Seq mapped peripheral inflammatory markers to a monocyte-derived macrophage population, distinct from Kupffer cells, with an inflammatory transcriptional profile. The inflammatory macrophages (iMacs) differentiated from blood monocytes and were unique from macrophage found in healthy or cirrhotic liver. iMacs retained their core transcriptional signature after inflammation resolved, indicating inflammation-mediated remodeling of the macrophage population in the human liver that may affect progressive liver disease and immunotherapy.
Authors
Juan Diego Sanchez Vasquez, Shirin Nkongolo, Daniel Traum, Valentin Sotov, Samuel C. Kim, Deeqa Mahamed, Aman Mehrotra, Anjali Patel, Diana Y. Chen, Scott Fung, Anuj Gaggar, Jordan J. Feld, Kyong-Mi Chang, Jeffrey J. Wallin, Ben X. Wang, Harry L.A. Janssen, Adam J. Gehring
Abstract
Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the activated B cell–like subtype (ABC-DLBCL) is associated with particularly poor outcome. Many ABC-DLBCLs harbor gain-of-function mutations that cause inappropriate assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, a cytoplasmic complex that drives downstream NF-κB signaling. MALT1 is the effector protein of the CBM signalosome such that its recruitment to the signalosome via interaction with BCL10 allows it to exert both protease and scaffolding activities that together synergize in driving NF-κB. Here, we demonstrate that a molecular groove located between two adjacent immunoglobulin-like domains within MALT1 represents a binding pocket for BCL10. Leveraging this discovery, we performed an in silico screen to identify small molecules that dock within this MALT1 groove and act as BCL10-MALT1 protein-protein interaction (PPI) inhibitors. We report the identification of M1i-124 as a first-in-class compound that blocks BCL10-MALT1 interaction, abrogates MALT1 scaffolding and protease activities, promotes degradation of BCL10 and MALT1 proteins, and specifically targets ABC-DLBCLs characterized by dysregulated MALT1. Our findings demonstrate that small-molecule inhibitors of BCL10-MALT1 interaction can function as potent agents to block MALT1 signaling in selected lymphomas, and provide a road map for clinical development of a new class of precision-medicine therapeutics.
Authors
Heejae Kang, Lisa M. Maurer, Jing Cheng, Mei Smyers, Linda R. Klei, Dong Hu, Juliana Hofstatter Azambuja, Marcelo J. Murai, Ahmed Mady, Ejaz Ahmad, Matthew Trotta, Hanna B. Klei, Minda Liu, Prasanna Ekambaram, Zaneta Nikolovska-Coleska, Bill B. Chen, Linda M. McAllister-Lucas, Peter C. Lucas
Abstract
Glioblastoma (GBM) is a highly aggressive form of brain tumor characterized by dysregulated metabolism. Increased fatty acid oxidation (FAO) protects tumor cells from lipid peroxidation–induced cell death, although the precise mechanisms involved remain unclear. Here, we report that loss of TNF receptor–associated factor 3 (TRAF3) in GBM critically regulated lipid peroxidation and tumorigenesis by controlling the oxidation of polyunsaturated fatty acids (PUFAs). TRAF3 was frequently repressed in GBM due to promoter hypermethylation. TRAF3 interacted with enoyl-CoA hydratase 1 (ECH1), an enzyme that catalyzes the isomerization of unsaturated FAs (UFAs) and mediates K63-linked ubiquitination of ECH1 at Lys214. ECH1 ubiquitination impeded TOMM20-dependent mitochondrial translocation of ECH1, which otherwise promoted the oxidation of UFAs, preferentially the PUFAs, and limited lipid peroxidation. Overexpression of TRAF3 enhanced the sensitivity of GBM to ferroptosis and anti–programmed death–ligand 1 (anti–PD-L1) immunotherapy in mice. Thus, the TRAF3/ECH1 axis played a key role in the metabolism of PUFAs and was crucial for lipid peroxidation damage and immune elimination in GBM.
Authors
Yu Zeng, Liqian Zhao, Kunlin Zeng, Ziling Zhan, Zhengming Zhan, Shangbiao Li, Hongchao Zhan, Peng Chai, Cheng Xie, Shengfeng Ding, Yuxin Xie, Li Wang, Cuiying Li, Xiaoxia Chen, Daogang Guan, Enguang Bi, Jianyou Liao, Fan Deng, Xiaochun Bai, Ye Song, Aidong Zhou
Abstract
Steatotic liver enhances liver metastasis of colorectal cancer (CRC), but this process is not fully understood. Steatotic liver induced by a high-fat diet increases cancer-associated fibroblast (CAF) infiltration and collagen and hyaluronic acid (HA) production. We investigated the role of HA synthase 2 (HAS2) in the fibrotic tumor microenvironment in steatotic liver using Has2ΔHSC mice, in which Has2 is deleted from hepatic stellate cells. Has2ΔHSC mice had reduced steatotic liver–associated metastatic tumor growth of MC38 CRC cells, collagen and HA deposition, and CAF and M2 macrophage infiltration. We found that low–molecular weight HA activates Yes-associated protein (YAP) in cancer cells, which then releases connective tissue growth factor to further activate CAFs for HAS2 expression. Single-cell analyses revealed a link between CAF-derived HAS2 and M2 macrophages and CRC cells through CD44; these cells were associated with exhausted CD8+ T cells via programmed death–ligand 1 and programmed cell death protein 1 (PD-1). HA synthesis inhibitors reduced steatotic liver–associated metastasis of CRC, YAP expression, and CAF and M2 macrophage infiltration, and improved response to anti–PD-1 antibody. In conclusion, steatotic liver modulates a fibrotic tumor microenvironment to enhance metastatic cancer activity through a bidirectional regulation between CAFs and metastatic tumors, enhancing the metastatic potential of CRC in the liver.
Authors
Yoon Mee Yang, Jieun Kim, Zhijun Wang, Jina Kim, So Yeon Kim, Gyu Jeong Cho, Jee Hyung Lee, Sun Myoung Kim, Takashi Tsuchiya, Michitaka Matsuda, Vijay Pandyarajan, Stephen J. Pandol, Michael S. Lewis, Alexandra Gangi, Paul W. Noble, Dianhua Jiang, Akil Merchant, Edwin M. Posadas, Neil A. Bhowmick, Shelly C. Lu, Sungyong You, Alexander M. Xu, Ekihiro Seki
Abstract
Sleep disturbance is bidirectionally associated with an increased risk of Alzheimer’s disease and other tauopathies. While the sleep-wake cycle regulates interstitial and cerebrospinal fluid (CSF) tau levels, the underlying mechanisms remain unknown. Understanding these mechanisms is crucial, given the evidence that tau pathology spreads through neuron-to-neuron transfer, involving the secretion and internalization of pathological tau forms. Here, we combined in vitro, in vivo, and clinical methods to reveal a pathway by which changes in body temperature (BT) over the sleep-wake cycle modulate extracellular tau levels. In mice, a higher BT during wakefulness and sleep deprivation increased CSF and plasma tau levels, while also upregulating unconventional protein secretion pathway I (UPS-I) events including (a) intracellular tau dephosphorylation, (b) caspase 3–mediated cleavage of tau (TauC3), and (c) membrane translocation of tau through binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and syndecan 3. In humans, the increase in CSF and plasma tau levels observed after wakefulness correlated with BT increases during wakefulness. By demonstrating that sleep-wake variation in BT regulates extracellular tau levels, our findings highlight the importance of thermoregulation in linking sleep disturbances to tau-mediated neurodegeneration and the preventative potential of thermal interventions.
Authors
Geoffrey Canet, Felipe Da Gama Monteiro, Emma Rocaboy, Sofia Diego-Diaz, Boutheyna Khelaifia, Kelly Godbout, Aymane Lachhab, Jessica Kim, Daphne I. Valencia, Audrey Yin, Hau-Tieng Wu, Jordan Howell, Emily Blank, Francis Laliberté, Nadia Fortin, Emmanuelle Boscher, Parissa Fereydouni-Forouzandeh, Stéphanie Champagne, Isabelle Guisle, Sébastien S. Hébert, Vincent Pernet, Haiyan Liu, William Lu, Ludovic Debure, David M. Rapoport, Indu Ayappa, Andrew W. Varga, Ankit Parekh, Ricardo S. Osorio, Steve Lacroix, Mark P. Burns, Brendan P. Lucey, Esther M. Blessing, Emmanuel Planel
Abstract
Protein aggregates are emerging therapeutic targets in rare monogenic causes of cardiomyopathy and amyloid heart disease, but their role in more prevalent heart-failure syndromes remains mechanistically unexamined. We observed mislocalization of desmin and sarcomeric proteins to aggregates in human myocardium with ischemic cardiomyopathy and in mouse hearts with post–myocardial infarction ventricular remodeling, mimicking findings of autosomal-dominant cardiomyopathy induced by the R120G mutation in the cognate chaperone protein CRYAB. In both syndromes, we demonstrate increased partitioning of CRYAB phosphorylated on serine 59 to NP40-insoluble aggregate-rich biochemical fraction. While CRYAB undergoes phase separation to form condensates, the phosphomimetic mutation of serine 59 to aspartate (S59D) in CRYAB mimics R120G-CRYAB mutants with reduced condensate fluidity, formation of protein aggregates, and increased cell death. Conversely, changing serine to alanine (phosphorylation-deficient mutation) at position 59 (S59A) restored condensate fluidity and reduced both R120G-CRYAB aggregates and cell death. In mice, S59D CRYAB knockin was sufficient to induce desmin mislocalization and myocardial protein aggregates, while S59A CRYAB knockin rescued left ventricular systolic dysfunction after myocardial infarction and preserved desmin localization with reduced myocardial protein aggregates. 25-Hydroxycholesterol attenuated CRYAB serine 59 phosphorylation and rescued post–myocardial infarction adverse remodeling. Thus, targeting CRYAB phosphorylation-induced condensatopathy is an attractive strategy to counter ischemic cardiomyopathy.
Authors
Moydul Islam, David R. Rawnsley, Xiucui Ma, Walter Navid, Chen Zhao, Xumin Guan, Layla Foroughi, John T. Murphy, Honora Navid, Carla J. Weinheimer, Attila Kovacs, Jessica Nigro, Aaradhya Diwan, Ryan P. Chang, Minu Kumari, Martin E. Young, Babak Razani, Kenneth B. Margulies, Mahmoud Abdellatif, Simon Sedej, Ali Javaheri, Douglas F. Covey, Kartik Mani, Abhinav Diwan
Abstract
Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the dystrophia myotonica protein kinase (DMPK) gene. The expanded CUG repeat RNA (CUGexp RNA) transcribed from the mutant allele sequesters the muscleblind-like (MBNL) family of RNA-binding proteins, causing their loss of function and disrupting regulated pre-mRNA processing. We used a DM1 heart mouse model that inducibly expresses CUGexp RNA to test the contribution of MBNL loss to DM1 cardiac abnormalities and explored MBNL restoration as a potential therapy. AAV9-mediated overexpression of MBNL1 and/or MBNL2 significantly rescued DM1 cardiac phenotypes including conduction delays, contractile dysfunction, hypertrophy, and misregulated alternative splicing and gene expression. While robust, the rescue was partial compared with reduced CUGexp RNA and plateaued with increased exogenous MBNL expression. These findings demonstrate that MBNL loss is a major contributor to DM1 cardiac manifestations and suggest that additional mechanisms play a role, highlighting the complex nature of DM1 pathogenesis.
Authors
Rong-Chi Hu, Yi Zhang, Larissa Nitschke, Sara J. Johnson, Ayrea E. Hurley, William R. Lagor, Zheng Xia, Thomas A. Cooper
Abstract
Polymorphisms in Nos3 increase risk for glaucoma, the leading cause of irreversible blindness worldwide. A key modifiable risk factor for glaucoma is intraocular pressure (IOP), which is regulated by NO — a product of nitric oxide synthase 3 (encoded by Nos3) — in Schlemm’s canal of the conventional outflow pathway. We studied the effects of a conditional, endothelial cell–specific postnatal deletion of Nos3 (Endo-SclCre-ERT;Nos3fl/fl) on tissues of the outflow pathway. We observed that Cre-ERT expression spontaneously and gradually increased with time in vascular endothelia including in Schlemm’s canal, beginning at P10, with complete Nos3 deletion occurring around P90. Whereas outflow resistance was reduced in global Nos3-KO mice, outflow resistance and IOP in Endo-SclCre-ERT;Nos3fl/fl mice were normal. We observed — coincident with Nos3 deletion — recruitment of macrophages to and induction of both ELAM1 and NOS2 expression by endothelia in the distal portion of the outflow pathway, which increased vessel diameter. These adjustments reduced outflow resistance to maintain IOP in these Endo-SclCre-ERT;Nos3fl/fl mice. Selective inhibition of iNOS by 1400W resulted in narrowing of distal vessels and IOP elevation. Together, the results emphasize the pliability of the outflow system and the importance of NO signaling in IOP control, and imply an substantial role for macrophages in IOP homeostasis.
Authors
Ruth A. Kelly, Megan S. Kuhn, Ester Reina-Torres, Revathi Balasubramanian, Kristin M. Perkumas, Guorong Li, Takamune Takahashi, Simon W.M. John, Michael H. Elliott, Darryl R. Overby, W. Daniel Stamer
Abstract
Neuroretinal degenerations including retinitis pigmentosa (RP) comprise a heterogeneous collection of pathogenic mutations that ultimately result in blindness. Despite recent advances in precision medicine, therapies for rarer mutations are hindered by burdensome developmental costs. To this end, Von Hippel-Lindau (VHL) is an attractive therapeutic target to treat RP. By ablating VHL in rod photoreceptors and elevating hypoxia-inducible factor (HIF) levels, we demonstrate a path to therapeutically enhancing glycolysis independent of the underlying genetic variant that slows degeneration of both rod and cone photoreceptors in a preclinical model of retinitis pigmentosa. This rod-specific intervention also resulted in reciprocal, decreased glycolytic activity within the retinal pigment epithelium (RPE) cells despite no direct genetic modifications to the RPE. Suppressing glycolysis in the RPE provided notable, noncell-autonomous therapeutic benefits to the photoreceptors, indicative of metabolically sensitive crosstalk between different cellular compartments of the retina. Surprisingly, targeting HIF2A in RPE cells did not impact RPE glycolysis, potentially implicating HIF1A as a major regulator in mouse RPE and providing a rationale for future therapeutic efforts aimed at modulating RPE metabolism.
Authors
Salvatore Marco Caruso, Xuan Cui, Brian M. Robbings, Noah Heapes, Aykut Demikrol, Bruna Lopes Da Costa, Daniel T. Hass, Peter M.J. Quinn, Jianhai Du, James B. Hurley, Stephen H. Tsang
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