RESUMO
Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We found that glutamine and glucose also fuel an indispensable dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a substrate for cellular glycosyltransferases. Immune-activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-linked-ß-N-acetylglucosamine (O-GlcNAc) glycosyltransferase as compared with naive cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology.
Assuntos
Glucose/metabolismo , Glutamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/fisiologia , Uridina Difosfato N-Acetilglicosamina/metabolismo , Animais , Proliferação de Células/genética , Autorrenovação Celular/genética , Transformação Celular Neoplásica/genética , Células Clonais , Feminino , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Receptores Notch/metabolismoRESUMO
O-GlcNAcylation is a dynamic post-translational modification performed by two opposing enzymes: O-GlcNAc transferase and O-GlcNAcase. O-GlcNAcylation is generally believed to act as a metabolic integrator in numerous signalling pathways. The stoichiometry of this modification is tightly controlled throughout all stages of development, with both hypo/hyper O-GlcNAcylation resulting in broad defects. In this Primer, we discuss the role of O-GlcNAcylation in developmental processes from stem cell maintenance and differentiation to cell and tissue morphogenesis.
Assuntos
Processamento de Proteína Pós-Traducional , Células-Tronco , Glicosilação , Células-Tronco/metabolismo , Transdução de Sinais , N-Acetilglucosaminiltransferases/metabolismo , Diferenciação CelularRESUMO
The soil bacterium Bacillus subtilis is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give the B. subtilis biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in bslA deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.
Assuntos
Proteínas de Bactérias , Matriz Extracelular de Substâncias Poliméricas , Proteínas de Bactérias/metabolismo , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Biofilmes , Bacillus subtilis/metabolismo , Simulação de Dinâmica MolecularRESUMO
O-GlcNAc transferase (OGT) is the sole enzyme responsible for the post-translational modification of O-GlcNAc on thousands of target nucleocytoplasmic proteins. To date, nine variants of OGT that segregate with OGT Congenital Disorder of Glycosylation (OGT-CDG) have been reported and characterized. Numerous additional variants have been associated with OGT-CDG, some of which are currently undergoing investigation. This disorder primarily presents with global developmental delay and intellectual disability (ID), alongside other variable neurological features and subtle facial dysmorphisms in patients. Several hypotheses aim to explain the etiology of OGT-CDG, with a prominent hypothesis attributing the pathophysiology of OGT-CDG to mutations segregating with this disorder disrupting the OGT interactome. The OGT interactome consists of thousands of proteins, including substrates as well as interactors that require noncatalytic functions of OGT. A key aim in the field is to identify which interactors and substrates contribute to the primarily neural-specific phenotype of OGT-CDG. In this review, we will discuss the heterogenous phenotypic features of OGT-CDG seen clinically, the variable biochemical effects of mutations associated with OGT-CDG, and the use of animal models to understand this disorder. Furthermore, we will discuss how previously identified OGT interactors causal for ID provide mechanistic targets for investigation that could explain the dysregulated gene expression seen in OGT-CDG models. Identifying shared or unique altered pathways impacted in OGT-CDG patients will provide a better understanding of the disorder as well as potential therapeutic targets.
Assuntos
Defeitos Congênitos da Glicosilação , N-Acetilglucosaminiltransferases , Humanos , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/metabolismo , Animais , Mutação , Glicosilação , Processamento de Proteína Pós-TraducionalRESUMO
O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG.
Assuntos
Drosophila , Deficiência Intelectual , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Habituação Psicofisiológica/genética , Humanos , Hidrolases/genética , Deficiência Intelectual/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/genéticaRESUMO
Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Deficiência Intelectual , N-Acetilglucosaminiltransferases , Placa Neural , Fenótipo , Humanos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Placa Neural/metabolismo , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/patologia , Defeitos Congênitos da Glicosilação/metabolismo , Sistemas CRISPR-Cas , Glicosilação , Edição de Genes , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologiaRESUMO
Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) 1 . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates 2 . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate3,4. Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism 5 . Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biocatálise , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Cisteína/metabolismo , Esterificação , Células HEK293 , Humanos , Lisina/metabolismo , Modelos Moleculares , Domínios Proteicos , Proteômica , Serina/metabolismo , Especificidade por Substrato , Treonina/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Aspergillus fumigatus is a human opportunistic pathogen showing emerging resistance against a limited repertoire of antifungal agents available. The GTPase Rho1 has been identified as an important regulator of the cell wall integrity signaling pathway that regulates the composition of the cell wall, a structure that is unique to fungi and serves as a target for antifungal compounds. Rom2, the guanine nucleotide exchange factor to Rho1, contains a C-terminal citron homology (CNH) domain of unknown function that is found in many other eukaryotic genes. Here, we show that the Rom2 CNH domain interacts directly with Rho1 to modulate ß-glucan and chitin synthesis. We report the structure of the Rom2 CNH domain, revealing that it adopts a seven-bladed ß-propeller fold containing three unusual loops. A model of the Rho1-Rom2 CNH complex suggests that the Rom2 CNH domain interacts with the Rho1 Switch II motif. This work uncovers the role of the Rom2 CNH domain as a scaffold for Rho1 signaling in fungal cell wall biosynthesis.
Assuntos
Aspergillus fumigatus/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Parede Celular/fisiologia , Proteínas Fúngicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genéticaRESUMO
In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT.
Assuntos
Fator C1 de Célula Hospedeira/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteólise , Motivos de Aminoácidos , Animais , Domínio Catalítico , Simulação por Computador , Evolução Molecular , Humanos , Invertebrados/enzimologia , Modelos Moleculares , Mutação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
Protein O-GlcNAcylation is a dynamic posttranslational modification that is catalyzed by the enzyme O-GlcNAc transferase (OGT) and is essential for neurodevelopment and postnatal neuronal function. Missense mutations in OGT segregate with a novel X-linked intellectual disability syndrome, the OGT congenital disorder of glycosylation (OGT-CDG). One hypothesis for the etiology of OGT-CDG is that loss of OGT activity leads to hypo-O-GlcNAcylation of as yet unidentified, specific neuronal proteins, affecting essential embryonic, and postnatal neurodevelopmental processes; however, the identity of these O-GlcNAcylated proteins is not known. Here, we used bioinformatic techniques to integrate sequence conservation, structural data, clinical data, and the available literature to identify 22 candidate proteins that convey OGT-CDG. We found using gene ontology and PANTHER database data that these candidate proteins are involved in diverse processes including Ras/MAPK signaling, translational repression, cytoskeletal dynamics, and chromatin remodeling. We also identify pathogenic missense variants at O-GlcNAcylation sites that segregate with intellectual disability. This work establishes a preliminary platform for the mechanistic dissection of the links between protein O-GlcNAcylation and neurodevelopment in OGT-CDG.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X , N-Acetilglucosaminiltransferases , Processamento de Proteína Pós-Traducional , Biologia Computacional , Deficiências do Desenvolvimento/genética , Glicosilação , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação de Sentido Incorreto , N-Acetilglucosaminiltransferases/genética , Processamento de Proteína Pós-Traducional/genética , SíndromeRESUMO
Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC50 of 2 µM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.
Assuntos
Aspergilose , Aspergillus fumigatus , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Glucanos/metabolismo , Humanos , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismoRESUMO
Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas , Proteínas/metabolismo , Glicosilação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosaminidase/metabolismo , Acetilglucosamina/metabolismoRESUMO
O-GlcNAcylation is an essential post-translational modification that has been implicated in neurodevelopmental and neurodegenerative disorders. O-GlcNAcase (OGA), the sole enzyme catalyzing the removal of O-GlcNAc from proteins, has emerged as a potential drug target. OGA consists of an N-terminal OGA catalytic domain and a C-terminal pseudo histone acetyltransferase (HAT) domain with unknown function. To investigate phenotypes specific to loss of OGA catalytic activity and dissect the role of the HAT domain, we generated a constitutive knock-in mouse line, carrying a mutation of a catalytic aspartic acid to alanine. These mice showed perinatal lethality and abnormal embryonic growth with skewed Mendelian ratios after day E18.5. We observed tissue-specific changes in O-GlcNAc homeostasis regulation to compensate for loss of OGA activity. Using X-ray microcomputed tomography on late gestation embryos, we identified defects in the kidney, brain, liver, and stomach. Taken together, our data suggest that developmental defects during gestation may arise upon prolonged OGA inhibition specifically because of loss of OGA catalytic activity and independent of the function of the HAT domain.
Assuntos
Desenvolvimento Embrionário/fisiologia , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Domínio Catalítico , Desenvolvimento Embrionário/genética , Feminino , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/fisiologia , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/metabolismo , Gravidez , Processamento de Proteína Pós-Traducional , Microtomografia por Raio-X/métodos , beta-N-Acetil-Hexosaminidases/genética , beta-N-Acetil-Hexosaminidases/fisiologiaRESUMO
Aspergillus fumigatus is an opportunistic mold responsible for severe life-threatening fungal infections in immunocompromised patients. The cell wall, an essential structure composed of glucan, chitin, and galactomannan, is considered to be a target for the development of antifungal drugs. The nucleotide sugar donor GDP-mannose (GDP-Man) is required for the biosynthesis of galactomannan, glycosylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting from fructose-6-phosphate, GDP-Man is produced by the sequential action of the enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP-mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown approaches we demonstrate that the phosphomannomutase encoding gene in A. fumigatus (pmmA) is essential for survival. Reduced expression of pmmA is associated with significant morphological defects including retarded germination, growth, reduced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target.
Assuntos
Aspergillus fumigatus/genética , Guanosina Difosfato Manose/metabolismo , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergilose/patologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Parede Celular/metabolismo , Deleção de Genes , Humanos , Virulência/genéticaRESUMO
O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein-protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.
Assuntos
Domínio Catalítico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Deficiência Intelectual/genética , Mutação com Perda de Função , N-Acetilglucosaminiltransferases/genética , Animais , Linhagem Celular , Drosophila , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Deficiência Intelectual/patologia , Camundongos , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/metabolismo , Neurogênese , Mutação Puntual , Gêmeos MonozigóticosRESUMO
O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, we generated catalytically inactive and precise knockout Oga alleles (OgaD133N and OgaKO , respectively) in Drosophila melanogaster Adult OgaD133N and OgaKO flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionarily conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected a number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, we report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.
Assuntos
Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , beta-N-Acetil-Hexosaminidases/metabolismo , Acilação , Animais , Cognição , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Técnicas de Inativação de Genes , Locomoção , Longevidade , Sinapses/fisiologia , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment including host defenses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. As this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective submicromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/enzimologia , Vias Biossintéticas/efeitos dos fármacos , Glucosamina 6-Fosfato N-Acetiltransferase/antagonistas & inibidores , Hexosaminas/metabolismo , Animais , Antifúngicos/química , Aspergilose/tratamento farmacológico , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Domínio Catalítico/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitina/metabolismo , Cristalografia por Raios X , Glucosamina 6-Fosfato N-Acetiltransferase/química , Glucosamina 6-Fosfato N-Acetiltransferase/metabolismo , Masculino , Camundongos , Modelos Moleculares , Terapia de Alvo Molecular , Conformação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
O-GlcNAcylation is an abundant and dynamic protein posttranslational modification (PTM), with crucial roles in metazoans. Studies of this modification are hampered by the lack of convenient methods for detecting native O-GlcNAcylation. Here, we describe a novel gel-based approach, Separation of O-GlcNAcylated Proteins by Polyacrylamide Gel Electrophoresis (SOPAGE), which enables detection of O-GlcNAc levels and dynamics.
Assuntos
Acetilglucosamina , Proteínas , Eletroforese em Gel de Poliacrilamida , Glicosilação , Processamento de Proteína Pós-Traducional , Proteínas/metabolismoRESUMO
Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc (O-GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila, null mutants of the Polycomb gene O-GlcNAc transferase (OGT; also known as super sex combs (sxc)) display homeotic phenotypes. To dissect the requirement for O-GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxcK872M , was recessive lethal, whereas a second mutant, the hypomorphic sxcH537A , was homozygous viable. We observed that reduced total protein O-GlcNAcylation in the sxcH537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxcH537A and a null allele of Drosophila host cell factor (dHcf), encoding an extensively O-GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxcH537A flies lacking a copy of skuld (skd), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxcH537A enabled us to identify pleiotropic effects of globally reduced protein O-GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development.
Assuntos
Acetilglucosamina/metabolismo , Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , N-Acetilglucosaminiltransferases/genética , Acilação , Alelos , Animais , Sistemas CRISPR-Cas , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Olho/genética , Edição de Genes , Genes de Insetos , Genes Letais , Homozigoto , Masculino , Mutação , N-Acetilglucosaminiltransferases/metabolismo , Fenótipo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Asas de Animais/irrigação sanguíneaRESUMO
O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase-protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate-the so-called HCF-1PRO repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1PRO repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1PRO-repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor Rp-αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1PRO-repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1PRO-repeat sequences, we show that proposed OGT-based or UDP-GlcNAc-based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies.