RESUMO
Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.
Assuntos
Tecido Adiposo Marrom/metabolismo , Receptor Constitutivo de Androstano/metabolismo , Lipólise , Receptores Acoplados a Proteínas G/metabolismo , Termogênese , Adipócitos/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Temperatura Baixa , Gorduras na Dieta/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Sistema Nervoso Simpático/metabolismo , Transcrição GênicaRESUMO
Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.
Assuntos
Retículo Endoplasmático/metabolismo , Ácido Láctico/metabolismo , Magnésio/metabolismo , Animais , Células COS , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Chlorocebus aethiops , Retículo Endoplasmático/fisiologia , Feminino , Células HeLa , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismoRESUMO
Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.
Assuntos
Arabidopsis/química , Arabidopsis/citologia , Polaridade Celular/fisiologia , Células Vegetais/fisiologia , Polimerização , Domínios Proteicos , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteína Axina/química , Proteína Axina/metabolismo , Bryopsida/química , Bryopsida/citologia , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Células COS , Chlorocebus aethiops , Proteínas Desgrenhadas/metabolismo , Células HEK293 , Humanos , Marchantia/química , Marchantia/citologia , Marchantia/genética , Marchantia/crescimento & desenvolvimento , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Plantas Geneticamente Modificadas , Proteínas Repressoras/metabolismo , Via de Sinalização WntRESUMO
Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from "off" to "on" states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.
Assuntos
Receptores Imunológicos/metabolismo , Receptores Imunológicos/ultraestrutura , Animais , Axônios/metabolismo , Células COS , Caenorhabditis elegans/metabolismo , Proteínas de Transporte , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Cultura Primária de Células , Transdução de Sinais , Proteínas RoundaboutRESUMO
In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.
Assuntos
Imunidade Adaptativa , Linfócitos B/metabolismo , Diferenciação Celular , Fator de Transcrição Ikaros/metabolismo , Doenças da Imunodeficiência Primária/metabolismo , Linfócitos T/metabolismo , Animais , Linfócitos B/imunologia , Células COS , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Fator de Transcrição Ikaros/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação de Sentido Incorreto , Células NIH 3T3 , Doenças da Imunodeficiência Primária/genética , Doenças da Imunodeficiência Primária/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion.
Assuntos
Retículo Endoplasmático/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Microscopia de FluorescênciaRESUMO
Endoplasmic reticulum (ER) membrane contact sites (MCSs) mark positions where endosomes undergo fission for cargo sorting. To define the role of ER at this unique MCS, we targeted a promiscuous biotin ligase to cargo-sorting domains on endosome buds. This strategy identified the ER membrane protein TMCC1, a member of a conserved protein family. TMCC1 concentrates at the ER-endosome MCSs that are spatially and temporally linked to endosome fission. When TMCC1 is depleted, endosome morphology is normal, buds still form, but ER-associated bud fission and subsequent cargo sorting to the Golgi are impaired. We find that the endosome-localized actin regulator Coronin 1C is required for ER-associated fission of actin-dependent cargo-sorting domains. Coronin 1C is recruited to endosome buds independently of TMCC1, while TMCC1/ER recruitment requires Coronin 1C. This link between TMCC1 and Coronin 1C suggests that the timing of TMCC1-dependent ER recruitment is tightly regulated to occur after cargo has been properly sequestered into the bud.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células COS , Canais de Cálcio , Chlorocebus aethiops , Retículo Endoplasmático/fisiologia , Endossomos/fisiologia , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/fisiologia , Microtúbulos/metabolismo , Transporte Proteico/fisiologiaRESUMO
Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.
Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Receptores de Hialuronatos/metabolismo , Receptores Imunológicos/metabolismo , Adulto , Animais , Sítios de Ligação , Células COS , Células Cultivadas , Chlorocebus aethiops , Feminino , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Terapia Genética/métodos , Oligonucleotídeos Antissenso/farmacologia , Animais , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Repetições de Microssatélites , Splicing de RNA , Expansão das Repetições de TrinucleotídeosRESUMO
G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.
Assuntos
Proteínas de Ligação ao GTP/química , Guanosina Trifosfato/farmacologia , Receptores da Calcitonina/agonistas , Receptores da Calcitonina/química , Difosfato de Adenosina/biossíntese , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ligantes , Conformação Proteica , Multimerização Proteica , Receptores da Calcitonina/metabolismoRESUMO
Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.
Assuntos
Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Tálamo/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dominância Ocular , Humanos , Camundongos , Camundongos Knockout , Doenças do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Sinapses/metabolismoRESUMO
Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading and disassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions.
Assuntos
Canais de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Ataxia/genética , Células COS , Cálcio/metabolismo , Canais de Cálcio/genética , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Deficiência Intelectual/genética , Membranas Intracelulares/metabolismo , Camundongos , Camundongos Knockout , Osteogênese/genética , Alinhamento de SequênciaRESUMO
Understanding how membrane nanoscale organization controls transmembrane receptors signaling activity remains a challenge. We studied interferon-γ receptor (IFN-γR) signaling in fibroblasts from homozygous patients with a T168N mutation in IFNGR2. By adding a neo-N-glycan on IFN-γR2 subunit, this mutation blocks IFN-γ activity by unknown mechanisms. We show that the lateral diffusion of IFN-γR2 is confined by sphingolipid/cholesterol nanodomains. In contrast, the IFN-γR2 T168N mutant diffusion is confined by distinct actin nanodomains where conformational changes required for Janus-activated tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) activation by IFN-γ could not occur. Removing IFN-γR2 T168N-bound galectins restored lateral diffusion in lipid nanodomains and JAK/STAT signaling in patient cells, whereas adding galectins impaired these processes in control cells. These experiments prove the critical role of dynamic receptor interactions with actin and lipid nanodomains and reveal a new function for receptor glycosylation and galectins. Our study establishes the physiological relevance of membrane nanodomains in the control of transmembrane receptor signaling in vivo. VIDEO ABSTRACT.
Assuntos
Fibroblastos/metabolismo , Mutação de Sentido Incorreto , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais , Actinas/química , Actinas/metabolismo , Animais , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Difusão , Endocitose , Ativação Enzimática , Glicosilação , Humanos , Interferon gama/metabolismo , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Receptores de Interferon/químicaRESUMO
Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.
Assuntos
Glicosaminoglicanos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/metabolismo , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Camundongos , Polimerização , Transdução de Sinais/fisiologia , Transportadores de Sulfato/metabolismo , Vacínia/metabolismo , Vaccinia virus/patogenicidadeRESUMO
Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.
Assuntos
Homeostase/fisiologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Monócitos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Apoptose/fisiologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Células COS , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Chlorocebus aethiops , Feminino , Humanos , Pulmão/patologia , Macrófagos Alveolares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Metástase Neoplásica/patologia , Proteômica/métodos , Transdução de Sinais/fisiologiaRESUMO
Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, whereas their close relative B. thailandensis is non-pathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion, and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate, and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection.
Assuntos
Actinas/metabolismo , Infecções por Burkholderia/microbiologia , Burkholderia/fisiologia , Burkholderia/patogenicidade , Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Burkholderia/classificação , Burkholderia/enzimologia , Células COS , Fusão Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HEK293 , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.
Assuntos
Antígeno CD47/metabolismo , Cromossomos Humanos Par 10/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Degeneração Macular/genética , Osteopontina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação/fisiologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Olho/patologia , Predisposição Genética para Doença/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Transdução de Sinais/genéticaRESUMO
Endocytic cargo and Rab GTPases are segregated to distinct domains of an endosome. These domains maintain their identity until they undergo fission to traffic cargo. It is not fully understood how segregation of cargo or Rab proteins is maintained along the continuous endosomal membrane or what machinery is required for fission. Endosomes form contact sites with the endoplasmic reticulum (ER) that are maintained during trafficking. Here, we show that stable contacts form between the ER and endosome at constricted sorting domains, and free diffusion of cargo is limited at these positions. We demonstrate that the site of constriction and fission for early and late endosomes is spatially and temporally linked to contact sites with the ER. Lastly, we show that altering ER structure and dynamics reduces the efficiency of endosome fission. Together, these data reveal a surprising role for ER contact in defining the timing and position of endosome fission.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , Microtúbulos/metabolismo , Proteínas da Mielina/metabolismo , Proteínas Nogo , Fatores de TempoRESUMO
Transporting epithelial cells build apical microvilli to increase membrane surface area and enhance absorptive capacity. The intestinal brush border provides an elaborate example with tightly packed microvilli that function in nutrient absorption and host defense. Although the brush border is essential for physiological homeostasis, its assembly is poorly understood. We found that brush border assembly is driven by the formation of Ca(2+)-dependent adhesion links between adjacent microvilli. Intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips and interact to form a trans-heterophilic complex. The cytoplasmic domains of microvillar protocadherins interact with the scaffolding protein, harmonin, and myosin-7b, which promote localization to microvillar tips. Finally, a mouse model of Usher syndrome lacking harmonin exhibits microvillar protocadherin mislocalization and severe defects in brush border morphology. These data reveal an adhesion-based mechanism for brush border assembly and illuminate the basis of intestinal pathology in patients with Usher syndrome. PAPERFLICK:
Assuntos
Caderinas/metabolismo , Enterócitos/metabolismo , Microvilosidades/metabolismo , Animais , Células COS , Células CACO-2 , Proteínas Relacionadas a Caderinas , Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Chlorocebus aethiops , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Enterócitos/citologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Microvilosidades/ultraestrutura , Miosinas/metabolismo , Síndromes de Usher/patologiaRESUMO
Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.