RESUMO
The nicotinic acetylcholine receptor has served, since its biochemical identification in the 1970s, as a model of an allosteric ligand-gated ion channel mediating signal transition at the synapse. In recent years, the application of X-ray crystallography and high-resolution cryo-electron microscopy, together with molecular dynamic simulations of nicotinic receptors and homologs, have opened a new era in the understanding of channel gating by the neurotransmitter. They reveal, at atomic resolution, the diversity and flexibility of the multiple ligand-binding sites, including recently discovered allosteric modulatory sites distinct from the neurotransmitter orthosteric site, and the conformational dynamics of the activation process as a molecular switch linking these multiple sites. The model emerging from these studies paves the way for a new pharmacology based, first, upon the occurrence of an original mode of indirect allosteric modulation, distinct from a steric competition for a single and rigid binding site, and second, the design of drugs that specifically interact with privileged conformations of the receptor such as agonists, antagonists, and desensitizers. Research on nicotinic receptors is still at the forefront of understanding the mode of action of drugs on the nervous system.
Assuntos
Sítio Alostérico , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Receptores Nicotínicos , Transdução de Sinais , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Regulação Alostérica , Humanos , Animais , Cristalografia por Raios X , Sítios de Ligação , Conformação Proteica , Ligantes , Modelos Moleculares , Multimerização Proteica , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacologia , Agonistas Nicotínicos/metabolismoRESUMO
Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
Assuntos
Sistemas CRISPR-Cas , Hexosiltransferases , Lipopolissacarídeos , Proteínas de Membrana , NF-kappa B , Transdução de Sinais , Receptor 4 Toll-Like , Hexosiltransferases/metabolismo , Hexosiltransferases/genética , NF-kappa B/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Humanos , Receptor 4 Toll-Like/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Células HEK293 , Inflamação/metabolismo , Inflamação/genética , Glicosilação , Microscopia Crioeletrônica , Domínio Catalítico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
The insulin receptor (IR) is a type II receptor tyrosine kinase that plays essential roles in metabolism, growth, and proliferation. Dysregulation of IR signaling is linked to many human diseases, such as diabetes and cancers. The resolution revolution in cryo-electron microscopy has led to the determination of several structures of IR with different numbers of bound insulin molecules in recent years, which have tremendously improved our understanding of how IR is activated by insulin. Here, we review the insulin-induced activation mechanism of IR, including (a) the detailed binding modes and functions of insulin at site 1 and site 2 and (b) the insulin-induced structural transitions that are required for IR activation. We highlight several other key aspects of the activation and regulation of IR signaling and discuss the remaining gaps in our understanding of the IR activation mechanism and potential avenues of future research.
Assuntos
Insulina , Receptor de Insulina , Humanos , Receptor de Insulina/genética , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Microscopia Crioeletrônica , Insulina/química , Insulina/metabolismo , Transdução de Sinais , Receptores Proteína Tirosina Quinases/metabolismo , FosforilaçãoRESUMO
Genomic DNA sequencing technologies have been one of the great advances of the 21st century, having decreased in cost by seven orders of magnitude and opening up new fields of investigation throughout research and clinical medicine. Genomics coupled with biochemical investigation has allowed the molecular definition of a growing number of new genetic diseases that reveal new concepts of immune regulation. Also, defining the genetic pathogenesis of these diseases has led to improved diagnosis, prognosis, genetic counseling, and, most importantly, new therapies. We highlight the investigational journey from patient phenotype to treatment using the newly defined XMEN disease, caused by the genetic loss of the MAGT1 magnesium transporter, as an example. This disease illustrates how genomics yields new fundamental immunoregulatory insights as well as how research genomics is integrated into clinical immunology. At the end, we discuss two other recently described diseases, CHAI/LATAIE (CTLA-4 deficiency) and PASLI (PI3K dysregulation), as additional examples of the journey from unknown immunological diseases to new precision medicine treatments using genomics.
Assuntos
Antígeno CTLA-4/genética , Proteínas de Transporte de Cátions/genética , Genômica , Doenças do Sistema Imunitário/genética , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Doenças do Sistema Imunitário/terapia , Masculino , Terapia de Alvo Molecular , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapiaRESUMO
Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.
Assuntos
Monoéster Fosfórico Hidrolases , Phytophthora , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/metabolismo , Phytophthora/química , Phytophthora/metabolismo , Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Fosfatase 2/metabolismo , Doenças das PlantasRESUMO
Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.
Assuntos
Antibacterianos , Bactérias , Microbiologia do Solo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bioensaio , DifosfatosRESUMO
Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.
Assuntos
Ativação do Canal Iônico , Receptores de Glicina/agonistas , Receptores de Glicina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Microscopia Crioeletrônica , Glicina , Células HEK293 , Humanos , Imageamento Tridimensional , Maleatos/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Neurotransmissores/metabolismo , Domínios Proteicos , Receptores de Glicina/genética , Receptores de Glicina/ultraestrutura , Estireno/química , Peixe-Zebra , Ácido gama-Aminobutírico/metabolismoRESUMO
The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.
Assuntos
Coenzimas/química , Manganês/química , Oxigênio/química , Complexo de Proteína do Fotossistema II/química , Água/química , Coenzimas/metabolismo , Cristalografia por Raios X , Expressão Gênica , Lasers , Manganês/metabolismo , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Teoria Quântica , Termodinâmica , Thermosynechococcus/química , Thermosynechococcus/enzimologia , Água/metabolismoRESUMO
The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
Assuntos
Dano ao DNA , Redes Reguladoras de Genes/fisiologia , Aminoquinolinas/farmacologia , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Camundongos , Ácidos Picolínicos/farmacologia , RNA Guia de Cinetoplastídeos/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.
Assuntos
DNA Glicosilases/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Endonucleases/química , Genoma , Ligases/química , Liases/química , DNA/metabolismo , DNA/ultraestrutura , Dano ao DNA , DNA Glicosilases/metabolismo , DNA Glicosilases/ultraestrutura , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/ultraestrutura , Endonucleases/metabolismo , Endonucleases/ultraestrutura , Eucariotos/genética , Eucariotos/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/enzimologia , Instabilidade Genômica , Humanos , Ligases/metabolismo , Ligases/ultraestrutura , Liases/metabolismo , Liases/ultraestrutura , Modelos Moleculares , Mutagênese , Conformação de Ácido Nucleico , Conformação ProteicaRESUMO
Energy-coupling factor (ECF)-type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Archaea/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Filogenia , Conformação ProteicaRESUMO
Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.
Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Translocases Mitocondriais de ADP e ATP/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Ácido Bongcréquico/metabolismo , Citoplasma/metabolismo , Mitocôndrias/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/ultraestrutura , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Lymphocyte-activation gene 3 (LAG-3) is an immune inhibitory receptor, with major histocompatibility complex class II (MHC-II) as a canonical ligand. However, it remains controversial whether MHC-II is solely responsible for the inhibitory function of LAG-3. Here, we demonstrate that fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand independent from MHC-II. FGL1 inhibits antigen-specific T cell activation, and ablation of FGL1 in mice promotes T cell immunity. Blockade of the FGL1-LAG-3 interaction by monoclonal antibodies stimulates tumor immunity and is therapeutic against established mouse tumors in a receptor-ligand inter-dependent manner. FGL1 is highly produced by human cancer cells, and elevated FGL1 in the plasma of cancer patients is associated with a poor prognosis and resistance to anti-PD-1/B7-H1 therapy. Our findings reveal an immune evasion mechanism and have implications for the design of cancer immunotherapy.
Assuntos
Antígenos CD/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Animais , Antígenos CD/imunologia , Linhagem Celular , Fibrinogênio/imunologia , Fibrinogênio/metabolismo , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunoterapia , Ligantes , Fígado/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
In response to viral infection, how cells balance translational shutdown to limit viral replication and the induction of antiviral components like interferons (IFNs) is not well understood. Moreover, how distinct isoforms of IFN-induced oligoadenylate synthetase 1 (OAS1) contribute to this antiviral response also requires further elucidation. Here, we show that human, but not mouse, OAS1 inhibits SARS-CoV-2 replication through its canonical enzyme activity via RNase L. In contrast, both mouse and human OAS1 protect against West Nile virus infection by a mechanism distinct from canonical RNase L activation. OAS1 binds AU-rich elements (AREs) of specific mRNAs, including IFNß. This binding leads to the sequestration of IFNß mRNA to the endomembrane regions, resulting in prolonged half-life and continued translation. Thus, OAS1 is an ARE-binding protein with two mechanisms of antiviral activity: driving inhibition of translation but also a broader, non-canonical function of protecting IFN expression from translational shutdown.
Assuntos
2',5'-Oligoadenilato Sintetase , Interferons , Oligorribonucleotídeos , Viroses , Febre do Nilo Ocidental , Animais , Humanos , Camundongos , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina , Antivirais/farmacologia , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/metabolismo , Vírus do Nilo Ocidental/patogenicidadeRESUMO
Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteoma/análise , Proteômica/métodos , Azepinas/química , Azepinas/metabolismo , Azepinas/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Estradiol/farmacologia , Humanos , Marcação por Isótopo , Células Jurkat , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Espectrometria de Massas em Tandem , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.
Assuntos
Proteínas de Algas/metabolismo , Ciclo do Carbono , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Proteínas de Algas/química , Dióxido de Carbono/metabolismo , Anidrases Carbônicas/metabolismo , Chlamydomonas reinhardtii/química , Cloroplastos/química , Proteínas Luminescentes/análise , Microscopia Confocal , Fotossíntese , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Approximately 30%-40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.
Assuntos
Chlamydomonas reinhardtii/citologia , Cloroplastos/ultraestrutura , Proteínas de Algas/metabolismo , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/química , Cloroplastos/metabolismo , Microscopia Crioeletrônica , Biogênese de Organelas , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains â¼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. VIDEO ABSTRACT.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Basigina/metabolismo , Membrana Celular/metabolismo , Cromossomos Humanos Par 17/metabolismo , Técnicas de Silenciamento de Genes , Haplótipos , Hepatócitos/metabolismo , Heterozigoto , Código das Histonas , Humanos , Fígado/metabolismo , Modelos Moleculares , Transportadores de Ácidos Monocarboxílicos/químicaRESUMO
The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na+-activated K+ channel, as a function of the Na+ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na+ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na+-dependent manner, without evidence of Na+-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.
Assuntos
Proteínas Aviárias/química , Galinhas/metabolismo , Proteínas do Tecido Nervoso/química , Canais de Potássio/química , Animais , Proteínas Aviárias/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Conformação Proteica , Sódio/químicaRESUMO
O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding ß-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.