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1.
Cell ; 186(5): 906-922, 2023 03 02.
Article in English | MEDLINE | ID: mdl-36787743

ABSTRACT

ACE2 is the indispensable entry receptor for SARS-CoV and SARS-CoV-2. Because of the COVID-19 pandemic, it has become one of the most therapeutically targeted human molecules in biomedicine. ACE2 serves two fundamental physiological roles: as an enzyme, it alters peptide cascade balance; as a chaperone, it controls intestinal amino acid uptake. ACE2's tissue distribution, affected by co-morbidities and sex, explains the broad tropism of coronaviruses and the clinical manifestations of SARS and COVID-19. ACE2-based therapeutics provide a universal strategy to prevent and treat SARS-CoV-2 infections, applicable to all SARS-CoV-2 variants and other emerging zoonotic coronaviruses exploiting ACE2 as their cellular receptor.


Subject(s)
COVID-19 , Severe acute respiratory syndrome-related coronavirus , Humans , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2 , Peptidyl-Dipeptidase A/metabolism , Pandemics
2.
Cell ; 181(4): 905-913.e7, 2020 05 14.
Article in English | MEDLINE | ID: mdl-32333836

ABSTRACT

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Peptidyl-Dipeptidase A/pharmacology , Pneumonia, Viral/drug therapy , Recombinant Proteins/pharmacology , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Betacoronavirus/ultrastructure , Blood Vessels/virology , COVID-19 , Chlorocebus aethiops , Humans , Kidney/cytology , Kidney/virology , Mice , Organoids/virology , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
3.
Cell ; 181(6): 1246-1262.e22, 2020 06 11.
Article in English | MEDLINE | ID: mdl-32442405

ABSTRACT

There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.


Subject(s)
Anaplastic Lymphoma Kinase/genetics , Thinness/genetics , Adipose Tissue/metabolism , Adult , Animals , Cell Line , Cohort Studies , Drosophila/genetics , Estonia , Female , Humans , Leptin/genetics , Lipolysis/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/genetics , RNA Interference/physiology , Young Adult
4.
Nat Immunol ; 21(10): 1172-1180, 2020 10.
Article in English | MEDLINE | ID: mdl-32839611

ABSTRACT

Fibroblasts are one of the most common but also neglected types of stromal cells, the heterogeneity of which underlies the specific function of tissue microenvironments in development and regeneration. In the thymus, autoreactive T cells are thought to be negatively selected by reference to the self-antigens expressed in medullary epithelial cells, but the contribution of other stromal cells to tolerance induction has been poorly examined. In the present study, we report a PDGFR+ gp38+ DPP4- thymic fibroblast subset that is required for T cell tolerance induction. The deletion of the lymphotoxin ß-receptor in thymic fibroblasts caused an autoimmune phenotype with decreased expression of tissue-restricted and fibroblast-specific antigens, offering insight into the long-sought target of lymphotoxin signaling in the context of the regulation of autoimmunity. Thus, thymic medullary fibroblasts play an essential role in the establishment of central tolerance by producing a diverse array of self-antigens.


Subject(s)
Fibroblasts/immunology , T-Lymphocytes/immunology , Thymus Gland/metabolism , Animals , Autoantigens/immunology , Autoimmunity , Cells, Cultured , Cellular Microenvironment , Clonal Selection, Antigen-Mediated , Dipeptidyl Peptidase 4/metabolism , Immune Tolerance , Lymphotoxin beta Receptor/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction , Thymus Gland/cytology
6.
Cell ; 164(3): 353-64, 2016 Jan 28.
Article in English | MEDLINE | ID: mdl-26824653

ABSTRACT

More than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, "on/off" manner. Trim28(+/D9) mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-"on" state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine.


Subject(s)
Epigenesis, Genetic , Haploinsufficiency , Nuclear Proteins/genetics , Obesity/genetics , Repressor Proteins/genetics , Thinness/genetics , Adolescent , Animals , Body Mass Index , Child , Child, Preschool , Humans , Mice , Nutrition Surveys , Polymorphism, Genetic , Tripartite Motif-Containing Protein 28
7.
Nat Immunol ; 18(6): 675-682, 2017 06.
Article in English | MEDLINE | ID: mdl-28436956

ABSTRACT

Immunoglobulin A (IgA) maintains a symbiotic equilibrium with intestinal microbes. IgA induction in the gut-associated lymphoid tissues (GALTs) is dependent on microbial sampling and cellular interaction in the subepithelial dome (SED). However it is unclear how IgA induction is predominantly initiated in the SED. Here we show that previously unrecognized mesenchymal cells in the SED of GALTs regulate bacteria-specific IgA production and diversify the gut microbiota. Mesenchymal cells expressing the cytokine RANKL directly interact with the gut epithelium to control CCL20 expression and microfold (M) cell differentiation. The deletion of mesenchymal RANKL impairs M cell-dependent antigen sampling and B cell-dendritic cell interaction in the SED, which results in a reduction in IgA production and a decrease in microbial diversity. Thus, the subepithelial mesenchymal cells that serve as M cell inducers have a fundamental role in the maintenance of intestinal immune homeostasis.


Subject(s)
Gastrointestinal Microbiome/immunology , Immunoglobulin A/immunology , Lymphoid Tissue/immunology , Mesenchymal Stem Cells/immunology , RANK Ligand/immunology , Animals , B-Lymphocytes/immunology , Biodiversity , Cell Differentiation/immunology , Chemokine CCL20/immunology , Dendritic Cells/immunology , Flow Cytometry , Gastrointestinal Microbiome/genetics , Germinal Center , Lymphoid Tissue/cytology , Mesenchymal Stem Cells/ultrastructure , Mice , Microscopy, Electron , RANK Ligand/genetics , RNA, Ribosomal, 16S/genetics , Reverse Transcriptase Polymerase Chain Reaction
8.
Cell ; 157(3): 636-50, 2014 Apr 24.
Article in English | MEDLINE | ID: mdl-24766809

ABSTRACT

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.


Subject(s)
Central Nervous System Diseases/genetics , Mutation, Missense , Nuclear Proteins/metabolism , Peripheral Nervous System Diseases/genetics , Phosphotransferases/metabolism , RNA, Transfer/metabolism , Transcription Factors/metabolism , Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Animals , Central Nervous System Diseases/pathology , Cerebrum/pathology , Child, Preschool , Endoribonucleases/metabolism , Female , Fibroblasts/metabolism , Humans , Infant , Male , Mice , Mice, Inbred CBA , Microcephaly/genetics , Peripheral Nervous System Diseases/pathology , RNA, Transfer/genetics , RNA-Binding Proteins
9.
Cell ; 153(1): 112-25, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-23477864

ABSTRACT

Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.


Subject(s)
Active Transport, Cell Nucleus , Docosahexaenoic Acids/immunology , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H5N1 Subtype/physiology , Orthomyxoviridae Infections/immunology , Virus Replication , Active Transport, Cell Nucleus/drug effects , Animals , Cell Line , Docosahexaenoic Acids/analysis , Docosahexaenoic Acids/pharmacology , Humans , Mice , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/virology , Virus Replication/drug effects
10.
Nat Methods ; 21(5): 868-881, 2024 May.
Article in English | MEDLINE | ID: mdl-38374263

ABSTRACT

The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.


Subject(s)
Cell Differentiation , Hematopoiesis , Induced Pluripotent Stem Cells , Organoids , Humans , Organoids/cytology , Organoids/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Animals , Mice , Hematopoietic Stem Cells/cytology , Bone Marrow/metabolism , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cell Culture Techniques/methods , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism
11.
Cell ; 151(2): 414-26, 2012 Oct 12.
Article in English | MEDLINE | ID: mdl-23063129

ABSTRACT

Diabetes, obesity, and cancer affect upward of 15% of the world's population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca(2+)-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of "selective partial agonists," capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes.


Subject(s)
Adipose Tissue, Brown/metabolism , Glycolysis , Hedgehog Proteins/metabolism , Muscle Cells/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , AMP-Activated Protein Kinase Kinases , Adipocytes/metabolism , Animals , Cell Line , Cells, Cultured , Cilia/metabolism , Diabetes Mellitus/metabolism , Humans , Mice , Neoplasms/metabolism , Obesity/metabolism , Protein Kinases/metabolism , Smoothened Receptor
12.
Nature ; 589(7842): 442-447, 2021 01.
Article in English | MEDLINE | ID: mdl-33361811

ABSTRACT

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.


Subject(s)
Diabetes, Gestational/immunology , Fetal Death/etiology , Receptor Activator of Nuclear Factor-kappa B/metabolism , T-Lymphocytes, Regulatory/immunology , Thymus Gland/immunology , Adipocytes/pathology , Animals , Cell Proliferation , Diabetes, Gestational/etiology , Diabetes, Gestational/metabolism , Diabetes, Gestational/pathology , Epithelial Cells/immunology , Female , Fetus/immunology , Fetus/metabolism , Fetus/pathology , Glucose/metabolism , Glucose Intolerance/genetics , Humans , Intra-Abdominal Fat/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Placenta/immunology , Placenta/pathology , Pregnancy , Receptor Activator of Nuclear Factor-kappa B/deficiency , Receptor Activator of Nuclear Factor-kappa B/genetics , T-Lymphocytes, Regulatory/cytology , Thymus Gland/cytology , Transcription Factors/metabolism , AIRE Protein
13.
EMBO J ; 40(19): e108375, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34375000

ABSTRACT

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.


Subject(s)
Receptors, Mitogen/metabolism , SARS-CoV-2/metabolism , Animals , Binding Sites/physiology , COVID-19/virology , Cell Line , Chlorocebus aethiops , Glycosylation , HEK293 Cells , Humans , Mice , Molecular Dynamics Simulation , Protein Binding/physiology , Vero Cells , Virus Internalization
14.
EMBO J ; 40(19): e108863, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34459017

ABSTRACT

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.


Subject(s)
Autophagy , Disease Susceptibility , Animals , Autophagy/drug effects , Autophagy/genetics , Autophagy/immunology , Biomarkers , Gene Expression Regulation , Genetic Predisposition to Disease , Homeostasis , Host-Pathogen Interactions , Humans , Organ Specificity , Signal Transduction
15.
Int Immunol ; 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38916145

ABSTRACT

The thymus is an organ required for T cell development and is also an eosinophil-rich organ; however, the nature and function of thymic eosinophils remain unclear. Here, we characterized the gene expression and differentiation mechanism of thymic eosinophils in mice. Thymic eosinophils showed a distinct gene expression profile compared with other organ-resident eosinophils. The number of thymic eosinophils was controlled by medullary thymic epithelial cells. In Rag-deficient mice, the unique gene expression signature of thymic eosinophils was lost but restored by pre-T cell receptor signaling, which induces CD4+ CD8+ thymocyte differentiation, indicating that T cell differentiation beyond the CD4- CD8- stage is necessary and sufficient for the induction of thymic eosinophils. These results demonstrate that thymic eosinophils are quantitatively and qualitatively regulated by medullary thymic epithelial cells and developing thymocytes, respectively, suggesting that thymic eosinophils are a distinct, thymus-specific cell subset, induced by interactions with thymic cells.

16.
Circ Res ; 132(4): 498-510, 2023 02 17.
Article in English | MEDLINE | ID: mdl-36795852

ABSTRACT

Despite enormous advances, cardiovascular disorders are still a major threat to global health and are responsible for one-third of deaths worldwide. Research for new therapeutics and the investigation of their effects on vascular parameters is often limited by species-specific pathways and a lack of high-throughput methods. The complex 3-dimensional environment of blood vessels, intricate cellular crosstalks, and organ-specific architectures further complicate the quest for a faithful human in vitro model. The development of novel organoid models of various tissues such as brain, gut, and kidney signified a leap for the field of personalized medicine and disease research. By utilizing either embryonic- or patient-derived stem cells, different developmental and pathological mechanisms can be modeled and investigated in a controlled in vitro environment. We have recently developed self-organizing human capillary blood vessel organoids that recapitulate key processes of vasculogenesis, angiogenesis, and diabetic vasculopathy. Since then, this organoid system has been utilized as a model for other disease processes, refined, and adapted for organ specificity. In this review, we will discuss novel and alternative approaches to blood vessel engineering and explore the cellular identity of engineered blood vessels in comparison to in vivo vasculature. Future perspectives and the therapeutic potential of blood vessel organoids will be discussed.


Subject(s)
Organoids , Pluripotent Stem Cells , Humans , Organoids/metabolism , Pluripotent Stem Cells/metabolism , Brain
17.
EMBO Rep ; 24(12): e56815, 2023 Dec 06.
Article in English | MEDLINE | ID: mdl-37846480

ABSTRACT

HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.


Subject(s)
Ubiquitin-Protein Ligases , Animals , Mice , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , TOR Serine-Threonine Kinases , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism
18.
Cell ; 140(1): 148-60, 2010 Jan 08.
Article in English | MEDLINE | ID: mdl-20074523

ABSTRACT

Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.


Subject(s)
Drosophila Proteins/metabolism , Hedgehog Proteins/metabolism , Obesity/genetics , Adipocytes, Brown/metabolism , Adipocytes, White/metabolism , Adipogenesis , Animals , Cyclic AMP/metabolism , Glucocorticoids/metabolism , Humans , Mice , Mice, Knockout , Muscle Cells/metabolism , Repressor Proteins/genetics
19.
Cell ; 143(4): 628-38, 2010 Nov 12.
Article in English | MEDLINE | ID: mdl-21074052

ABSTRACT

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.


Subject(s)
Calcium Channels/genetics , Drosophila Proteins/genetics , Drosophila/genetics , Pain/genetics , Adult , Animals , Back Pain/genetics , Calcium Channels/metabolism , Drosophila Proteins/metabolism , Gene Knockdown Techniques , Genome-Wide Association Study , Hot Temperature , Humans , Mice , Polymorphism, Single Nucleotide , RNA Interference
20.
Cell ; 141(1): 142-53, 2010 Apr 02.
Article in English | MEDLINE | ID: mdl-20371351

ABSTRACT

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.


Subject(s)
Drosophila melanogaster/physiology , Models, Animal , Animals , Cardiomyopathies/genetics , Cardiomyopathies/physiopathology , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Female , Genome-Wide Association Study , Heart/embryology , Heart/physiology , Humans , Male , Mice , Mice, Knockout , Promoter Regions, Genetic , RNA Interference
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