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1.
Nat Commun ; 11(1): 4958, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009395

RESUMO

Striatal dopamine (DA) is critical for action and learning. Recent data show that DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be determined by plasma membrane GABA uptake transporters (GATs) located on astrocytes and neurons. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs are downregulated, tonic GABAergic inhibition of DA release augmented, and nigrostriatal GABA co-release attenuated. These data define previously unappreciated and important roles for GATs and astrocytes in supporting DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions.


Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Regulação para Baixo , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Transtornos Parkinsonianos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Astrócitos/metabolismo , Membrana Celular/metabolismo , Modelos Animais de Doenças , Glutamato Descarboxilase/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Núcleo Accumbens/metabolismo
2.
Nat Commun ; 11(1): 4981, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020469

RESUMO

Antagonism or agonism of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) prevents weight gain and leads to dramatic weight loss in combination with glucagon-like peptide-1 receptor agonists in preclinical models. Based on the genetic evidence supporting GIPR antagonism, we previously developed a mouse anti-murine GIPR antibody (muGIPR-Ab) that protected diet-induced obese (DIO) mice against body weight gain and improved multiple metabolic parameters. This work reconciles the similar preclinical body weight effects of GIPR antagonists and agonists in vivo, and here we show that chronic GIPR agonism desensitizes GIPR activity in primary adipocytes, both differentiated in vitro and adipose tissue in vivo, and functions like a GIPR antagonist. Additionally, GIPR activity in adipocytes is partially responsible for muGIPR-Ab to prevent weight gain in DIO mice, demonstrating a role of adipocyte GIPR in the regulation of adiposity in vivo.


Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/uso terapêutico , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Peso Corporal/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Obesidade/patologia , Receptores dos Hormônios Gastrointestinais/deficiência , Receptores dos Hormônios Gastrointestinais/metabolismo
3.
Nat Commun ; 11(1): 4990, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020478

RESUMO

Neurons are highly compartmentalized cells with tightly controlled subcellular protein organization. While brain transcriptome, connectome and global proteome maps are being generated, system-wide analysis of temporal protein dynamics at the subcellular level are currently lacking. Here, we perform a temporally-resolved surfaceome analysis of primary neuron cultures and reveal dynamic surface protein clusters that reflect the functional requirements during distinct stages of neuronal development. Direct comparison of surface and total protein pools during development and homeostatic synaptic scaling demonstrates system-wide proteostasis-independent remodeling of the neuronal surface, illustrating widespread regulation on the level of surface trafficking. Finally, quantitative analysis of the neuronal surface during chemical long-term potentiation (cLTP) reveals fast externalization of diverse classes of surface proteins beyond the AMPA receptor, providing avenues to investigate the requirement of exocytosis for LTP. Our resource (neurosurfaceome.ethz.ch) highlights the importance of subcellular resolution for systems-level understanding of cellular processes.


Assuntos
Proteínas de Membrana/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores , Homeostase , Potenciação de Longa Duração , Mapas de Interação de Proteínas , Transporte Proteico , Proteostase , Ratos
4.
Nat Commun ; 11(1): 5065, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033237

RESUMO

The type VI protein secretion system (T6SS) is a powerful needle-like machinery found in Gram-negative bacteria that can penetrate the cytosol of receiving cells in milliseconds by physical force. Anchored by its membrane-spanning complex (MC) and a baseplate (BP), the T6SS sheath-tube is assembled in a stepwise process primed by TssA and terminated by TagA. However, the molecular details of its assembly remain elusive. Here, we systematically examined the initiation and termination of contractile and non-contractile T6SS sheaths in MC-BP, tssA and tagA mutants by fluorescence microscopy. We observe long pole-to-pole sheath-tube structures in the non-contractile MC-BP defective mutants but not in the Hcp tube or VgrG spike mutants. Combining overexpression and genetic mutation data, we demonstrate complex effects of TssM, TssA and TagA interactions on T6SS sheath-tube dynamics. We also report promiscuous interactions of TagA with multiple T6SS components, similar to TssA. Our results demonstrate that priming of the T6SS sheath-tube assembly is not dependent on TssA, nor is the assembly termination dependent on the distal end TssA-TagA interaction, and highlight the tripartite control of TssA-TssM-TagA on sheath-tube initiation and termination.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo VI/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/química , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/metabolismo , Viabilidade Microbiana , Modelos Biológicos , Mutação/genética , Ligação Proteica , Domínios Proteicos
5.
PLoS One ; 15(8): e0238452, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866191

RESUMO

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production.


Assuntos
Acremonium/metabolismo , Membrana Celular/metabolismo , Cefalosporinas/biossíntese , Cefalosporinas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Adenosina Trifosfatases/metabolismo , Meios de Cultura/metabolismo , Fermentação/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , beta-Lactamas/metabolismo
6.
PLoS Biol ; 18(8): e3000820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866173

RESUMO

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.


Assuntos
Ácido Glutâmico/metabolismo , Cinesina/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Neurônios/metabolismo , Espastina/deficiência , Tubulina (Proteína)/metabolismo , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Atividade Motora , Neurônios/patologia , Neurônios/ultraestrutura , Transporte Proteico , Espastina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo
7.
Adv Exp Med Biol ; 1274: 5-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894505

RESUMO

Biophysical properties of membranes are dependent on their glycerophospholipid compositions. Lysophospholipid acyltransferases (LPLATs) selectively incorporate fatty chains into lysophospholipids to affect the fatty acid composition of membrane glycerophospholipids. Lysophosphatidic acid acyltransferases (LPAATs) of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family incorporate fatty chains into phosphatidic acid during the de novo glycerophospholipid synthesis in the Kennedy pathway. Other LPLATs of both the AGPAT and the membrane bound O-acyltransferase (MBOAT) families further modify the fatty chain compositions of membrane glycerophospholipids in the remodeling pathway known as the Lands' cycle. The LPLATs functioning in these pathways possess unique characteristics in terms of their biochemical activities, regulation of expressions, and functions in various biological contexts. Essential physiological functions for LPLATs have been revealed in studies using gene-deficient mice, and important roles for several enzymes are also indicated in human diseases where their mutation or dysregulation causes or contributes to the pathological condition. Now several LPLATs are emerging as attractive therapeutic targets, and further understanding of the mechanisms underlying their physiological and pathological roles will aid in the development of novel therapies to treat several diseases that involve altered glycerophospholipid metabolism.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/antagonistas & inibidores , Aciltransferases/antagonistas & inibidores , Membrana Celular/metabolismo , Desenvolvimento de Medicamentos , Glicerofosfolipídeos/biossíntese , Glicerofosfolipídeos/química , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Animais , Membrana Celular/química , Membrana Celular/enzimologia , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos
8.
Nat Commun ; 11(1): 4512, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908147

RESUMO

Hydrogen peroxide (H2O2) is recognized to act as a signaling molecule. Peroxiredoxins (Prxs) have the ability to transfer H2O2-derived oxidizing equivalents to redox-regulated target proteins, thus facilitating the transmission of H2O2 signals. It has remained unclear how Prxs and their target proteins are brought together to allow for target-specific protein thiol oxidation. Addressing the specific case of Prx2-dependent STAT3 oxidation, we here show that the association of the two proteins occurs prior to Prx oxidation and depends on a scaffolding protein, the membrane chaperone annexin A2. Deletion or depletion of annexin A2 interrupts the transfer of oxidizing equivalents from Prx2 to STAT3, which is observed to take place on membranes. These findings support the notion that the Prx2-STAT3 redox relay is part of a highly organized membrane signaling domain.


Assuntos
Anexina A2/metabolismo , Peroxirredoxinas/metabolismo , Fator de Transcrição STAT3/metabolismo , Anexina A2/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Dissulfetos/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Ligação Proteica , Domínios Proteicos , Transdução de Sinais
9.
Adv Exp Med Biol ; 1267: 101-115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894479

RESUMO

Pathogenic bacteria colonize or disseminate into cells and tissues by inducing large-scale remodeling of host membranes. The physical phenomena underpinning these massive membrane extension and deformation are poorly understood. Invasive strategies of pathogens have been recently enriched by the description of a spectacular mode of opening of large transendothelial cell macroaperture (TEM) tunnels correlated to the dissemination of EDIN-producing strains of Staphylococcus aureus via a hematogenous route or to the induction of gelatinous edema triggered by the edema toxin from Bacillus anthracis. Remarkably, these highly dynamic tunnels close rapidly after they reach a maximal size. Opening and closure of TEMs in cells lasts for hours without inducing endothelial cell death. Multidisciplinary studies have started to provide a broader perspective of both the molecular determinants controlling cytoskeleton organization at newly curved membranes generated by the opening of TEMs and the physical processes controlling the dynamics of these tunnels. Here we discuss the analogy between the opening of TEM tunnels and the physical principles of dewetting, stemming from a parallel between membrane tension and surface tension. This analogy provides a broad framework to investigate biophysical constraints in cell membrane dynamics and their diversion by certain invasive microbial agents.


Assuntos
Bactérias/patogenicidade , Membrana Celular/microbiologia , Membrana Celular/patologia , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Molhabilidade , Membrana Celular/metabolismo , Edema/metabolismo , Edema/microbiologia , Edema/patologia , Células Endoteliais/metabolismo , Humanos , Tensão Superficial
10.
Anticancer Res ; 40(9): 4857-4867, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878773

RESUMO

BACKGROUND/AIM: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. MATERIALS AND METHODS: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. RESULTS: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. CONCLUSION: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.


Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/farmacologia , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , L-Lactato Desidrogenase/metabolismo , Leucemia Mieloide/metabolismo , Necrose , Proteína Supressora de Tumor p53/metabolismo
11.
PLoS Pathog ; 16(9): e1008878, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946535

RESUMO

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.


Assuntos
Proteínas de Bactérias , Membrana Celular/metabolismo , Chlamydia trachomatis , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/patologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidade , Células HeLa , Humanos , Mutação , Domínios Proteicos , Pseudópodes/genética , Pseudópodes/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética
12.
PLoS Pathog ; 16(9): e1008883, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956394

RESUMO

Infection by human parainfluenza viruses (HPIVs) causes widespread lower respiratory diseases, including croup, bronchiolitis, and pneumonia, and there are no vaccines or effective treatments for these viruses. HPIV3 is a member of the Respirovirus species of the Paramyxoviridae family. These viruses are pleomorphic, enveloped viruses with genomes composed of single-stranded negative-sense RNA. During viral entry, the first step of infection, the viral fusion complex, comprised of the receptor-binding glycoprotein hemagglutinin-neuraminidase (HN) and the fusion glycoprotein (F), mediates fusion upon receptor binding. The HPIV3 transmembrane protein HN, like the receptor-binding proteins of other related viruses that enter host cells using membrane fusion, binds to a receptor molecule on the host cell plasma membrane, which triggers the F glycoprotein to undergo major conformational rearrangements, promoting viral entry. Subsequent fusion of the viral and host membranes allows delivery of the viral genetic material into the host cell. The intermediate states in viral entry are transient and thermodynamically unstable, making it impossible to understand these transitions using standard methods, yet understanding these transition states is important for expanding our knowledge of the viral entry process. In this study, we use cryo-electron tomography (cryo-ET) to dissect the stepwise process by which the receptor-binding protein triggers F-mediated fusion, when forming a complex with receptor-bearing membranes. Using an on-grid antibody capture method that facilitates examination of fresh, biologically active strains of virus directly from supernatant fluids and a series of biological tools that permit the capture of intermediate states in the fusion process, we visualize the series of events that occur when a pristine, authentic viral particle interacts with target receptors and proceeds from the viral entry steps of receptor engagement to membrane fusion.


Assuntos
Membrana Celular/metabolismo , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Animais , Membrana Celular/ultraestrutura , Chlorocebus aethiops , Humanos , Vírus da Parainfluenza 3 Humana/ultraestrutura , Células Vero
13.
Proc Natl Acad Sci U S A ; 117(37): 23073-23084, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32873638

RESUMO

The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Culina/metabolismo , Hipocampo/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Morfogênese/fisiologia , Animais , Membrana Celular/metabolismo , Movimento Celular/fisiologia , Dendritos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Camundongos , Neurogênese/fisiologia , Células Piramidais/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo
14.
Open Biol ; 10(9): 200209, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898469

RESUMO

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.


Assuntos
Betacoronavirus/metabolismo , Eucariotos/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Membrana Celular/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Eucariotos/citologia , Humanos , Microssomos/metabolismo , Mutação , Pandemias , Filogenia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/classificação , Proteínas do Envelope Viral/genética
15.
Nat Commun ; 11(1): 4514, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908139

RESUMO

The velocity of nerve conduction is moderately enhanced by larger axonal diameters and potently sped up by myelination of axons. Myelination thus allows rapid impulse propagation with reduced axonal diameters; however, no myelin-dependent mechanism has been reported that restricts radial growth of axons. By label-free proteomics, STED-microscopy and cryo-immuno electron-microscopy we here identify CMTM6 (chemokine-like factor-like MARVEL-transmembrane domain-containing family member-6) as a myelin protein specifically localized to the Schwann cell membrane exposed to the axon. We find that disruption of Cmtm6-expression in Schwann cells causes a substantial increase of axonal diameters but does not impair myelin biogenesis, radial sorting or integrity of axons. Increased axonal diameters correlate with accelerated sensory nerve conduction and sensory responses and perturbed motor performance. These data show that Schwann cells utilize CMTM6 to restrict the radial growth of axons, which optimizes nerve function.


Assuntos
Axônios/metabolismo , Proteínas da Mielina/metabolismo , Nervos Periféricos/citologia , Células de Schwann/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Axônios/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Masculino , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Condução Nervosa , Nervos Periféricos/metabolismo , Nervos Periféricos/ultraestrutura , Proteômica , Células de Schwann/citologia , Células de Schwann/ultraestrutura , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/ultraestrutura
16.
Nat Commun ; 11(1): 4285, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855390

RESUMO

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.


Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Citocininas/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brefeldina A/farmacologia , Citocininas/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Meristema/citologia , Meristema/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Nat Commun ; 11(1): 3914, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764676

RESUMO

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Polaridade Celular/fisiologia , Ácidos Indolacéticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Membrana Celular/metabolismo , Polaridade Celular/genética , Metabolismo dos Lipídeos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Reguladores de Crescimento de Planta/metabolismo , Plantas Geneticamente Modificadas , Agregados Proteicos , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais
18.
Nat Commun ; 11(1): 3944, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769983

RESUMO

Triacylglycerols (TG) are synthesized at the endoplasmic reticulum (ER) bilayer and packaged into organelles called lipid droplets (LDs). LDs are covered by a single phospholipid monolayer contiguous with the ER bilayer. This connection is used by several monotopic integral membrane proteins, with hydrophobic membrane association domains (HDs), to diffuse between the organelles. However, how proteins partition between ER and LDs is not understood. Here, we employed synthetic model systems and found that HD-containing proteins strongly prefer monolayers and returning to the bilayer is unfavorable. This preference for monolayers is due to a higher affinity of HDs for TG over membrane phospholipids. Protein distribution is regulated by PC/PE ratio via alterations in monolayer packing and HD-TG interaction. Thus, HD-containing proteins appear to non-specifically accumulate to the LD surface. In cells, protein editing mechanisms at the ER membrane would be necessary to prevent unspecific relocation of HD-containing proteins to LDs.


Assuntos
Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Triglicerídeos/metabolismo , Dicroísmo Circular , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico , Triglicerídeos/química
19.
PLoS Genet ; 16(8): e1008975, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750056

RESUMO

The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Receptores de Adiponectina/genética , Alelos , Animais , Caenorhabditis elegans/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Mutação com Ganho de Função/genética , Células HEK293 , Humanos , Fluidez de Membrana/genética , Fenótipo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo
20.
Nat Commun ; 11(1): 3825, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732874

RESUMO

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.


Assuntos
Lipídeos , Malária Falciparum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/parasitologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Transporte Proteico , Vacúolos/metabolismo , Vacúolos/parasitologia
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