ABSTRACT
TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.
Subject(s)
Arthritis, Rheumatoid , Autoimmune Diseases , Psoriasis , Humans , TYK2 Kinase , Genome-Wide Association Study , Autoimmune Diseases/drug therapy , Psoriasis/drug therapyABSTRACT
TYK2 is a member of the JAK family of kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, TYK2 inhibition is an attractive therapeutic strategy for these diseases. Herein, we report the discovery of a series of highly selective catalytic site TYK2 inhibitors designed using FEP+ and structurally enabled design starting from a virtual screen hit. We highlight the structure-based optimization to identify a lead candidate 30, a potent cellular TYK2 inhibitor with excellent selectivity, pharmacokinetic properties, and in vivo efficacy in a mouse psoriasis model.
Subject(s)
Psoriasis , TYK2 Kinase , Animals , Humans , Janus Kinases , Mice , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Psoriasis/drug therapy , RodentiaABSTRACT
Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.
Subject(s)
Carbon-Nitrogen Ligases/metabolism , Protein Isoforms/metabolism , Cell Proliferation/physiology , Humans , Immunologic Deficiency Syndromes/metabolism , T-Lymphocytes/metabolismABSTRACT
Interleukin-2-inducible T cell kinase (ITK) is critical for T cell signaling and cytotoxicity, and control of Epstein-Barr virus (EBV). We identified a patient with a novel homozygous missense mutation (D540N) in a highly conserved residue in the kinase domain of ITK who presented with EBV-positive lymphomatoid granulomatosis. She was treated with interferon and chemotherapy and her disease went into remission; however, she has persistent elevation of EBV DNA in the blood, low CD4 T cells, low NK cells, and nearly absent iNKT cells. Molecular modeling predicts that the mutation increases the flexibility of the ITK kinase domain impairing phosphorylation of the protein. Stimulation of her T cells resulted in reduced phosphorylation of ITK, PLCγ, and PKC. The CD8 T cells were moderately impaired for cytotoxicity and degranulation. Importantly, addition of magnesium to her CD8 T cells in vitro restored cytotoxicity and degranulation to levels similar to controls. Supplemental magnesium in patients with mutations in another protein important for T cell signaling, MAGT1, was reported to restore EBV-specific cytotoxicity. Our findings highlight the critical role of ITK for T cell activation and suggest the potential for supplemental magnesium to treat patients with ITK deficiency.
Subject(s)
Blood Cells/immunology , Blood Cells/metabolism , Disease Susceptibility , Magnesium/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Adult , DNA Mutational Analysis , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/virology , Female , Homozygote , Humans , Lymphomatoid Granulomatosis/diagnosis , Lymphomatoid Granulomatosis/etiology , Mutation, Missense , Protein Interaction Domains and Motifs/genetics , Protein-Tyrosine Kinases/chemistry , Structure-Activity Relationship , Exome SequencingABSTRACT
Phosphatidylinositol 3-kinase-gamma (PI3Kγ) is highly expressed in leukocytes and is an attractive drug target for immune modulation. Different experimental systems have led to conflicting conclusions regarding inflammatory and anti-inflammatory functions of PI3Kγ. Here, we report a human patient with bi-allelic, loss-of-function mutations in PIK3CG resulting in absence of the p110γ catalytic subunit of PI3Kγ. She has a history of childhood-onset antibody defects, cytopenias, and T lymphocytic pneumonitis and colitis, with reduced peripheral blood memory B, memory CD8+ T, and regulatory T cells and increased CXCR3+ tissue-homing CD4 T cells. PI3Kγ-deficient macrophages and monocytes produce elevated inflammatory IL-12 and IL-23 in a GSK3α/ß-dependent manner upon TLR stimulation. Pik3cg-deficient mice recapitulate major features of human disease after exposure to natural microbiota through co-housing with pet-store mice. Together, our results emphasize the physiological importance of PI3Kγ in restraining inflammation and promoting appropriate adaptive immune responses in both humans and mice.
Subject(s)
Adaptive Immunity/immunology , Class Ib Phosphatidylinositol 3-Kinase/immunology , Immunologic Deficiency Syndromes/immunology , Inflammation/immunology , Microbiota/immunology , Adaptive Immunity/genetics , Animals , Cells, Cultured , Class Ib Phosphatidylinositol 3-Kinase/deficiency , Class Ib Phosphatidylinositol 3-Kinase/genetics , Disease Models, Animal , Female , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/metabolism , Inflammation/genetics , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
BACKGROUND: Advances in genomics have facilitated the discovery of monogenic disorders in patients with unique gastro-intestinal phenotypes. Syndromic diarrhea, also called tricho-hepato-enteric (THE) syndrome, results from deleterious mutations in SKIV2L or TTC37 genes. The main features of this disorder are intractable diarrhea, abnormal hair, facial dysmorphism, immunodeficiency and liver disease. AIM: To report on a patient with THE syndrome and present the genetic analysis that facilitated diagnosis. METHODS: Whole-exome sequencing (WES) was performed in a 4-month-old female with history of congenital diarrhea and severe failure to thrive but without hair anomalies or dysmorphism. Since the parents were first-degree cousins, the analysis focused on an autosomal recessive model. Sanger sequencing was used to validate suspected variants. Mutated protein structure was modeled to assess the effect of the mutation on protein function. RESULTS: We identified an autosomal recessive C.1891G > A missense mutation (NM_006929) in SKIV2L gene that was previously described only in a compound heterozygous state as causing THE syndrome. The mutation was determined to be deleterious in multiple prediction models. Protein modeling suggested that the mutation has the potential to cause structural destabilization of SKIV2L, either through conformational changes, interference with the protein's packing, or changes at the protein's interface. CONCLUSIONS: THE syndrome can present with a broad range of clinical features in the neonatal period. WES is an important diagnostic tool in patients with congenital diarrhea and can facilitate diagnosis of various diseases presenting with atypical features.
Subject(s)
DNA Helicases/genetics , Diarrhea, Infantile/genetics , Fetal Growth Retardation/genetics , Hair Diseases/genetics , Mutation, Missense , Diarrhea, Infantile/diagnosis , Facies , Female , Fetal Growth Retardation/diagnosis , Genetic Markers , Hair Diseases/diagnosis , Humans , Infant , Exome SequencingABSTRACT
This corrects the article DOI: 10.1038/ng.3898.
ABSTRACT
BACKGROUND: Studies of monogenic gastrointestinal diseases have revealed molecular pathways critical to gut homeostasis and enabled the development of targeted therapies. METHODS: We studied 11 patients with abdominal pain and diarrhea caused by early-onset protein-losing enteropathy with primary intestinal lymphangiectasia, edema due to hypoproteinemia, malabsorption, and less frequently, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease; the disorder followed an autosomal recessive pattern of inheritance. Whole-exome sequencing was performed to identify gene variants. We evaluated the function of CD55 in patients' cells, which we confirmed by means of exogenous induction of expression of CD55. RESULTS: We identified homozygous loss-of-function mutations in the gene encoding CD55 (decay-accelerating factor), which lead to loss of protein expression. Patients' T lymphocytes showed increased complement activation causing surface deposition of complement and the generation of soluble C5a. Costimulatory function and cytokine modulation by CD55 were defective. Genetic reconstitution of CD55 or treatment with a complement-inhibitory therapeutic antibody reversed abnormal complement activation. CONCLUSIONS: CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (the CHAPLE syndrome) is caused by abnormal complement activation due to biallelic loss-of-function mutations in CD55. (Funded by the National Institute of Allergy and Infectious Diseases and others.).
Subject(s)
CD55 Antigens/genetics , Complement Activation/genetics , Complement System Proteins/metabolism , Mutation , Protein-Losing Enteropathies/genetics , Thrombosis/genetics , CD55 Antigens/blood , Child , Child, Preschool , Complement Activation/drug effects , Complement Inactivating Agents/pharmacology , Female , Homozygote , Humans , Immunoglobulin A/blood , Infant , Intestine, Small/pathology , Male , Pedigree , Protein-Losing Enteropathies/complications , Statistics, Nonparametric , Syndrome , T-Lymphocytes/metabolismABSTRACT
Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.
Subject(s)
CARD Signaling Adaptor Proteins/genetics , Dermatitis, Atopic/genetics , Germ-Line Mutation , Guanylate Cyclase/genetics , Amino Acid Transport System ASC/metabolism , Cohort Studies , DNA Mutational Analysis , Dermatitis, Atopic/immunology , Female , Genes, Dominant , Glutamine/metabolism , Humans , Jurkat Cells , Lymphocyte Activation , Male , Mechanistic Target of Rapamycin Complex 1 , Minor Histocompatibility Antigens/metabolism , Multiprotein Complexes/metabolism , NF-kappa B/metabolism , Pedigree , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
MDA5 is a cytosolic sensor of double-stranded RNA (ds)RNA including viral byproducts and intermediates. We studied a child with life-threatening, recurrent respiratory tract infections, caused by viruses including human rhinovirus (HRV), influenza virus, and respiratory syncytial virus (RSV). We identified in her a homozygous missense mutation in IFIH1 that encodes MDA5. Mutant MDA5 was expressed but did not recognize the synthetic MDA5 agonist/(ds)RNA mimic polyinosinic-polycytidylic acid. When overexpressed, mutant MDA5 failed to drive luciferase activity from the IFNB1 promoter or promoters containing ISRE or NF-κB sequence motifs. In respiratory epithelial cells or fibroblasts, wild-type but not knockdown of MDA5 restricted HRV infection while increasing IFN-stimulated gene expression and IFN-ß/λ. However, wild-type MDA5 did not restrict influenza virus or RSV replication. Moreover, nasal epithelial cells from the patient, or fibroblasts gene-edited to express mutant MDA5, showed increased replication of HRV but not influenza or RSV. Thus, human MDA5 deficiency is a novel inborn error of innate and/or intrinsic immunity that causes impaired (ds)RNA sensing, reduced IFN induction, and susceptibility to the common cold virus.
Subject(s)
Interferon-Induced Helicase, IFIH1/genetics , Mutation , Picornaviridae Infections/genetics , Picornaviridae Infections/virology , Rhinovirus/physiology , Antiviral Agents/pharmacology , Base Sequence , Cells, Cultured , Child, Preschool , DNA Mutational Analysis/methods , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/virology , Gene Expression/drug effects , Genes, Recessive/genetics , Heterozygote , Homozygote , Host-Pathogen Interactions , Humans , Interferon-Induced Helicase, IFIH1/deficiency , Interferons/pharmacology , Male , PedigreeSubject(s)
Class I Phosphatidylinositol 3-Kinases/genetics , Immunologic Deficiency Syndromes/genetics , Adolescent , Adult , Child , Female , Humans , Male , MutationABSTRACT
In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)-related diseases. Three patients presented with EBV-associated Hodgkin's lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro-generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70-CD27 interactions therefore play a nonredundant role in T and B cell-mediated immunity, especially for protection against EBV and humoral immunity.
Subject(s)
B-Lymphocytes/immunology , CD27 Ligand/deficiency , Epstein-Barr Virus Infections/complications , Hodgkin Disease/etiology , Immunologic Deficiency Syndromes/complications , Adolescent , Adult , CD27 Ligand/genetics , CD8-Positive T-Lymphocytes/immunology , Child , Cytotoxicity, Immunologic , Female , Herpesvirus 4, Human/immunology , Humans , Immunologic Memory , Male , Mutation , Tumor Necrosis Factor Receptor Superfamily, Member 7/physiologyABSTRACT
Elevated basal serum tryptase levels are present in 4-6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase-encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.
Subject(s)
Chronic Pain/genetics , Connective Tissue Diseases/genetics , DNA Copy Number Variations/genetics , Dysautonomia, Familial/genetics , Gastrointestinal Diseases/genetics , Pruritus/genetics , Skin Diseases/genetics , Tryptases/blood , Tryptases/genetics , Adolescent , Adult , Aged , Child , Chronic Pain/blood , Chronic Pain/enzymology , Connective Tissue Diseases/blood , Connective Tissue Diseases/enzymology , Dysautonomia, Familial/blood , Dysautonomia, Familial/enzymology , Female , Gastrointestinal Diseases/blood , Gastrointestinal Diseases/enzymology , Humans , Male , Middle Aged , Pruritus/blood , Pruritus/enzymology , Skin Diseases/blood , Skin Diseases/enzymology , Young AdultABSTRACT
The cytokine interleukin-12 (IL-12) was thought to have a central role in T cell-mediated responses in inflammation for more than a decade after it was first identified. Discovery of the cytokine IL-23, which shares a common p40 subunit with IL-12, prompted efforts to clarify the relative contribution of these two cytokines in immune regulation. Ustekinumab, a therapeutic agent targeting both cytokines, was recently approved to treat psoriasis and psoriatic arthritis, and related agents are in clinical testing for a variety of inflammatory disorders. Here we discuss the therapeutic rationale for targeting these cytokines, the unintended consequences for host defense and tumor surveillance and potential ways in which these therapies can be applied to treat additional immune disorders.
Subject(s)
Immunity , Inflammation/immunology , Interleukin-12/metabolism , Interleukin-23/metabolism , Molecular Targeted Therapy , Animals , Humans , Immunologic Surveillance , Inflammation/pathology , Interleukin-12/antagonists & inhibitors , Interleukin-23/antagonists & inhibitorsABSTRACT
Overexpression of sirtuins (NAD(+)-dependent protein deacetylases) has been reported to increase lifespan in budding yeast (Saccharomyces cerevisiae), Caenorhabditis elegans and Drosophila melanogaster. Studies of the effects of genes on ageing are vulnerable to confounding effects of genetic background. Here we re-examined the reported effects of sirtuin overexpression on ageing and found that standardization of genetic background and the use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high-level sir-2.1 overexpression abrogated the longevity increase, but did not abrogate sir-2.1 overexpression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low-copy-number sir-2.1 overexpression also abrogated longevity. A Drosophila strain with ubiquitous overexpression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported, but was not long-lived relative to the appropriate transgenic controls, and nor was a new line with stronger overexpression of dSir2. These findings underscore the importance of controlling for genetic background and for the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life-extending effect of dietary restriction on ageing in Drosophila has also been reported to be dSir2 dependent. We found that dietary restriction increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects of sirtuins on lifespan in C. elegans and Drosophila.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/physiology , Histone Deacetylases/genetics , Longevity/physiology , Sirtuins/genetics , Aging/genetics , Aging/physiology , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Caloric Restriction , Crosses, Genetic , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , Gene Expression , Histone Deacetylases/metabolism , Longevity/genetics , Male , RNA, Messenger/analysis , RNA, Messenger/genetics , Sirtuins/metabolismABSTRACT
Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e(-11)). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16::DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF 'parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/physiology , Forkhead Transcription Factors/metabolism , Gene Expression Profiling/methods , Longevity/physiology , Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism , Transcription Factors/metabolism , Animals , Binding Sites , Chromatin/metabolism , DNA Methylation , Gene Expression Regulation, Developmental , Longevity/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The superoxide radical (O(2)(-)) has long been considered a major cause of aging. O(2)(-) in cytosolic, extracellular, and mitochondrial pools is detoxified by dedicated superoxide dismutase (SOD) isoforms. We tested the impact of each SOD isoform in Caenorhabditis elegans by manipulating its five sod genes and saw no major effects on life span. sod genes are not required for daf-2 insulin/IGF-1 receptor mutant longevity. However, loss of the extracellular Cu/ZnSOD sod-4 enhances daf-2 longevity and constitutive diapause, suggesting a signaling role for sod-4. Overall, these findings imply that O(2)(-) is not a major determinant of aging in C. elegans.
Subject(s)
Aging , Caenorhabditis elegans/metabolism , Oxidative Stress , Superoxide Dismutase/genetics , Superoxides/metabolism , Animals , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/physiology , Gene Deletion , Isoenzymes/physiology , Life Expectancy , Models, Biological , Receptor, Insulin/physiology , Superoxide Dismutase/physiologyABSTRACT
The DAF-2 insulin/IGF-1 receptor regulates development, metabolism, and aging in the nematode Caenorhabditis elegans. However, complex differences among daf-2 alleles complicate analysis of this gene. We have employed epistasis analysis, transcript profile analysis, mutant sequence analysis, and homology modeling of mutant receptors to understand this complexity. We define an allelic series of nonconditional daf-2 mutants, including nonsense and deletion alleles, and a putative null allele, m65. The most severe daf-2 alleles show incomplete suppression by daf-18(0) and daf-16(0) and have a range of effects on early development. Among weaker daf-2 alleles there exist distinct mutant classes that differ in epistatic interactions with mutations in other genes. Mutant sequence analysis (including 11 newly sequenced alleles) reveals that class 1 mutant lesions lie only in certain extracellular regions of the receptor, while class 2 (pleiotropic) and nonconditional missense mutants have lesions only in the ligand-binding pocket of the receptor ectodomain or the tyrosine kinase domain. Effects of equivalent mutations on the human insulin receptor suggest an altered balance of intracellular signaling in class 2 alleles. These studies consolidate and extend our understanding of the complex genetics of daf-2 and its underlying molecular biology.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Insulin/genetics , Receptor, IGF Type 1/genetics , Receptor, Insulin/genetics , Amino Acid Sequence , Animals , Base Sequence , Molecular Sequence Data , Multigene Family , Mutation , PhylogenyABSTRACT
BACKGROUND: To what extent are the determinants of aging in animal species universal? Insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an evolutionarily conserved (public) regulator of longevity; yet it remains unclear whether the genes and biochemical processes through which IIS acts on aging are public or private (that is, lineage specific). To address this, we have applied a novel, multi-level cross-species comparative analysis to compare gene expression changes accompanying increased longevity in mutant nematodes, fruitflies and mice with reduced IIS. RESULTS: Surprisingly, there is little evolutionary conservation at the level of individual, orthologous genes or paralogous genes under IIS regulation. However, a number of gene categories are significantly enriched for genes whose expression changes in long-lived animals of all three species. Down-regulated categories include protein biosynthesis-associated genes. Up-regulated categories include sugar catabolism, energy generation, glutathione-S-transferases (GSTs) and several other categories linked to cellular detoxification (that is, phase 1 and phase 2 metabolism of xenobiotic and endobiotic toxins). Protein biosynthesis and GST activity have recently been linked to aging and longevity assurance, respectively. CONCLUSION: These processes represent candidate, regulated mechanisms of longevity-control that are conserved across animal species. The longevity assurance mechanisms via which IIS acts appear to be lineage-specific at the gene level (private), but conserved at the process level (or semi-public). In the case of GSTs, and cellular detoxification generally, this suggests that the mechanisms of aging against which longevity assurance mechanisms act are, to some extent, lineage specific.
Subject(s)
Evolution, Molecular , Gene Expression Profiling , Insulin-Like Growth Factor I/genetics , Insulin/genetics , Longevity/genetics , Aging/genetics , Animals , Caenorhabditis elegans/genetics , Drosophila/genetics , Glutathione Transferase/genetics , Mice , Protein Biosynthesis/genetics , Signal TransductionABSTRACT
The longevity of the Caenorhabditis elegans diapausal dauer larva greatly exceeds that of the adult. Dauer formation and adult ageing are both regulated by insulin/IGF-1 signaling (IIS). Reduced IIS, e.g. by mutation of the daf-2 insulin/IGF-1 receptor gene, increases adult lifespan. This may reflect mis-expression in the adult of dauer longevity-assurance processes. Since IIS plays a central role in the regulation of metabolism, metabolic alterations shared by dauer larvae and daf-2 adults represent candidate mechanisms for lifespan determination. We have conducted a detailed comparison of transcript profile data from dauers and daf-2 mutant adults, focusing on expression of metabolic pathway genes. Our results imply up-regulation in both dauers and daf-2 mutant adults of gluconeogenesis, glyoxylate pathway activity, and trehalose biosynthesis. Down-regulation of the citric acid cycle and mitochondrial respiratory chain occurs in dauers, but not daf-2 adults. However, the F(1) ATPase inhibitor was up-regulated in both, implying enhanced homeostasis in conditions where mitochondria are stressed. Overall, the data implies increased conversion of fat to carbohydrate, and conservation of ATP stocks in daf-2 mutant adults, suggesting a state of increased energy availability. We postulate that this fuels increased somatic maintenance activity, as suggested by the disposable soma theory.