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1.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332765

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
2.
Cell Res ; 30(4): 343-355, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32231345

RESUMO

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/prevenção & controle , Lipopeptídeos/farmacologia , Fusão de Membrana , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Administração Intranasal , Sequência de Aminoácidos , Animais , Betacoronavirus/efeitos dos fármacos , Fusão Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Vírus da SARS , Alinhamento de Sequência , Relação Estrutura-Atividade , Células Vero
3.
Nat Rev Neurol ; 16(4): 199-212, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203399

RESUMO

Most neurodegenerative diseases are characterized by the intracellular or extracellular aggregation of misfolded proteins such as amyloid-ß and tau in Alzheimer disease, α-synuclein in Parkinson disease, and TAR DNA-binding protein 43 in amyotrophic lateral sclerosis. Accumulating evidence from both human studies and disease models indicates that intercellular transmission and the subsequent templated amplification of these misfolded proteins are involved in the onset and progression of various neurodegenerative diseases. The misfolded proteins that are transferred between cells are referred to as 'pathological seeds'. Recent studies have made exciting progress in identifying the characteristics of different pathological seeds, particularly those isolated from diseased brains. Advances have also been made in our understanding of the molecular mechanisms that regulate the transmission process, and the influence of the host cell on the conformation and properties of pathological seeds. The aim of this Review is to summarize our current knowledge of the cell-to-cell transmission of pathological proteins and to identify key questions for future investigation.


Assuntos
Encéfalo/metabolismo , Doenças Neurodegenerativas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Transporte Proteico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Transporte Axonal , Encéfalo/patologia , Comunicação Celular , Proteínas de Ligação a DNA/metabolismo , Endocitose , Exossomos/metabolismo , Predisposição Genética para Doença , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Fusão de Membrana , Nanotubos , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
4.
PLoS Pathog ; 16(3): e1008392, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150576

RESUMO

Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to post-fusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses.


Assuntos
Antígeno Carcinoembrionário/química , Vírus da Hepatite Murina/química , Vírus da Hepatite Murina/fisiologia , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus , Animais , Antígeno Carcinoembrionário/metabolismo , Antígeno Carcinoembrionário/ultraestrutura , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Células HEK293 , Humanos , Fusão de Membrana , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Multimerização Proteica , Proteólise , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vírus da SARS/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Ligação Viral
5.
Emerg Microbes Infect ; 9(1): 457-468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32090689

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly emerging threat to the global porcine industry. PDCoV has been successfully isolated using various medium additives including trypsin, and although we know it is important for viral replication, the mechanism has not been fully elucidated. Here, we systematically investigated the role of trypsin in PDCoV replication including cell entry, cell-to-cell membrane fusion and virus release. Using pseudovirus entry assays, we demonstrated that PDCoV entry is not trypsin dependent. Furthermore, unlike porcine epidemic diarrhea virus (PEDV), in which trypsin is important for the release of virus from infected cells, PDCoV release was not affected by trypsin. We also demonstrated that trypsin promotes PDCoV replication by enhancing cell-to-cell membrane fusion. Most importantly, our study illustrates two distinct spreading patterns from infected cells to uninfected cells during PDCoV transmission, and the role of trypsin in PDCoV replication in cells with different virus spreading types. Overall, these results clarify that trypsin promotes PDCoV replication by mediating cell-to-cell fusion transmission but is not crucial for viral entry. This knowledge can potentially contribute to improvement of virus production efficiency in culture, not only for vaccine preparation but also to develop antiviral treatments.


Assuntos
Fusão Celular , Coronavirus/fisiologia , Fusão de Membrana , Tripsina/metabolismo , Animais , Linhagem Celular , Humanos , Suínos , Internalização do Vírus , Replicação Viral
6.
Nat Rev Mol Cell Biol ; 21(4): 204-224, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32071438

RESUMO

Owing to their ability to efficiently generate ATP required to sustain normal cell function, mitochondria are often considered the 'powerhouses of the cell'. However, our understanding of the role of mitochondria in cell biology recently expanded when we recognized that they are key platforms for a plethora of cell signalling cascades. This functional versatility is tightly coupled to constant reshaping of the cellular mitochondrial network in a series of processes, collectively referred to as mitochondrial membrane dynamics and involving organelle fusion and fission (division) as well as ultrastructural remodelling of the membrane. Accordingly, mitochondrial dynamics influence and often orchestrate not only metabolism but also complex cell signalling events, such as those involved in regulating cell pluripotency, division, differentiation, senescence and death. Reciprocally, mitochondrial membrane dynamics are extensively regulated by post-translational modifications of its machinery and by the formation of membrane contact sites between mitochondria and other organelles, both of which have the capacity to integrate inputs from various pathways. Here, we discuss mitochondrial membrane dynamics and their regulation and describe how bioenergetics and cellular signalling are linked to these dynamic changes of mitochondrial morphology.


Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Animais , Humanos , Fusão de Membrana/fisiologia , Membranas Mitocondriais/fisiologia , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais
7.
Sheng Wu Gong Cheng Xue Bao ; 36(1): 133-142, 2020 Jan 25.
Artigo em Chinês | MEDLINE | ID: mdl-32072788

RESUMO

Homotypic fusion and vacuole protein sorting(HOPS) is a protein complex consisting of VPS11, VPS16, VPS18, VPS33, VPS39, VPS41 and regulates membrane transport in vivo through membrane fusion mechanisms. The evidence suggests that HOPS complex as a fusion factor, facilitates autophagosome-lysosome fusion. To determine whether the HOPS complex directly interacts with the autophagic SNARE protein STX17 in vitro, the coding sequence of the six genes were amplified from the existing plasmids by PCR, and then ligated to the prokaryotic expression vector pGEX 4T-1-GST or pET-His-NusA. After identification through colony PCR and DNA sequencing, 6 recombinant plasmids were constructed and transferred into Escherichia coli BL21 (DE3). The recombinant proteins were purified by glutathione sepharose 4B and nickel column. We used the tobacco etch virus protease to cut off the GST-tag or His-NusA-tag, to obtain HA-VPS11 protein of about 105 kDa, Flag-VPS16 protein of about 97 kDa, HA-VPS18 protein of about 108 kDa, Flag-VPS33 protein of about 70 kDa, HA-VPS39 protein of about 97 kDa, and Flag-VPS41 protein of about 98 kDa. The function of the purified proteins was verified by in vitro glutathione S-transferases pull-down assay, confirming that autophagic SNARE protein STX17 interacted directly with HOPS components. Our findings provide experimental basis to further study the function and mechanism of HOPS complex in the process of autophagosome-lysosome fusion.


Assuntos
Autofagia , Vacúolos , Humanos , Fusão de Membrana , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão
8.
PLoS Biol ; 18(2): e3000626, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32040508

RESUMO

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.


Assuntos
Ebolavirus/fisiologia , Fusão de Membrana , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Regulação Alostérica , Cálcio/metabolismo , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas do Envelope Viral/genética , Internalização do Vírus
9.
J Phys Chem Lett ; 11(4): 1204-1208, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31944770

RESUMO

Biological transmission of vesicular content occurs by opening of a fusion pore. Recent experimental observations have illustrated that fusion pores between vesicles that are docked by an extended flat contact zone are located at the edge (vertex) of this zone. We modeled this experimentally observed scenario by coarse-grained molecular simulations and elastic theory. This revealed that fusion pores experience a direct attraction toward the vertex. The size adopted by the resulting vertex pore strongly depends on the apparent contact angle between the adhered vesicles even in the absence of membrane surface tension. Larger contact angles substantially increase the equilibrium size of the vertex pore. Because the cellular membrane fusion machinery actively docks membranes, it facilitates a collective expansion of the contact zone and increases the contact angle. In this way, the fusion machinery can drive expansion of the fusion pore by free energy equivalents of multiple tens of kBT from a distance and not only through the fusion proteins that reside within the fusion pore.


Assuntos
Bicamadas Lipídicas/metabolismo , Fusão de Membrana/fisiologia , Modelos Biológicos , Microscopia Crioeletrônica , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Termodinâmica , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
10.
Nat Commun ; 11(1): 231, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932584

RESUMO

The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.


Assuntos
Cálcio/metabolismo , Fusão de Membrana , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Animais , Exocitose , Cinética , Bicamadas Lipídicas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Multimerização Proteica , Ratos , Proteínas SNARE/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética
11.
Nat Commun ; 11(1): 230, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932647

RESUMO

Annexins are abundant cytoplasmic proteins, which bind to membranes that expose negatively charged phospholipids in a Ca2+-dependent manner. During cell injuries, the entry of extracellular Ca2+ activates the annexin membrane-binding ability, subsequently initiating membrane repair processes. However, the mechanistic action of annexins in membrane repair remains largely unknown. Here, we use high-speed atomic force microscopy (HS-AFM), fluorescence recovery after photobleaching (FRAP), confocal laser scanning microscopy (CLSM) and molecular dynamics simulations (MDSs) to analyze how annexin-V (A5) binds to phosphatidylserine (PS)-rich membranes leading to high Ca2+-concentrations at membrane, and then to changes in the dynamics and organization of lipids, eventually to a membrane phase transition. A5 self-assembly into lattices further stabilizes and likely structures the membrane into a gel phase. Our findings are compatible with the patch resealing through vesicle fusion mechanism in membrane repair and indicate that A5 retains negatively charged lipids in the inner leaflet in an injured cell.


Assuntos
Anexina A5/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Anexina A5/química , Cálcio/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Fusão de Membrana , Microscopia de Força Atômica , Microscopia Confocal , Simulação de Dinâmica Molecular , Transição de Fase , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Agregados Proteicos
12.
Proc Natl Acad Sci U S A ; 117(2): 1036-1041, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888993

RESUMO

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.


Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/metabolismo , Exocitose/fisiologia , Cinética , Fusão de Membrana/fisiologia , Chaperonas Moleculares/química , Proteínas Munc18/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
13.
Biosci Biotechnol Biochem ; 84(4): 686-694, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31852366

RESUMO

Budded viruses (BVs) of baculovirus such as Autographa californica nucleopolyhedrovirus (AcNPV) have recently been studied as biological nanomaterials, and methods for their longer-term storage without deterioration would be desirable. The cryopreservation of virions with a naturally occurring saccharide like trehalose as a cryoprotectant is known to be useful for maintaining the viral structure and function. In this study, we examined how useful trehalose is as protectant for BV cryopreservation during repeated freeze-thaw cycles: 1) membrane fusion between liposomes (multilamellar vesicles, MLVs) and BVs, 2) infection of insect culture cells (Sf9 cells) by RFP-expressing BVs, and 3) morphologies of these BVs were investigated by fluorescent dequenching assay, fluorescence microscopy, and transmission electron microscopy (TEM), respectively. The results suggest that the BVs deteriorate in quality with each freeze-thaw cycle, and this deterioration can be diminished with the use of trehalose to an extent similar to that seen with storage on ice.


Assuntos
Crioprotetores/farmacologia , Congelamento , Fusão de Membrana , Nucleopoliedrovírus/patogenicidade , Trealose/farmacologia , Vírion/fisiologia , Animais , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Células Sf9
14.
Toxicol Lett ; 321: 32-43, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31862506

RESUMO

Cadmium (Cd) is an important environmental pollutant. Previous studies have shown that Cd can induce liver cell injury; however, the toxicity mechanisms of Cd have not been fully elucidated. This study aimed to further confirm the hepatotoxic effects of Cd in mouse liver cells by various methods both in vivo and in vitro. In addition, it found that Cd induced autophagy but also caused autophagy blockade, and autophagy blockade intensified Cd-induced injury in liver cells. Subsequently, the study investigated the effects of Cd on lysosomes and found that Cd induced lysosomal acidification, promoted the expression of lysosomal-associated membrane protein 2 and lysosomal hydrolase cathepsin B both in vivo and in vitro, and enhanced the lysosomal degradation capacity. It indicated that Cd triggered lysosomal activation. However, the fusion of autophagosomes with lysosomes was inhibited by Cd both in vivo and in vitro. Next, the expression of Rab7, a key protein that regulates autophagosome-lysosome fusion, was examined. Cd was found to inhibit Rab7 expression both in vivo and in vitro. In conclusion, the results indicated that Cd obstructed the autophagic flux by inhibiting the fusion of autophagosomes with lysosomes, thus exacerbating the Cd-induced hepatotoxicity. Moreover, the molecular mechanism of Cd-induced inhibition of autophagosome-lysosome fusion is probably related to the Cd-induced downregulation of Rab7.


Assuntos
Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Animais , Autofagossomos/metabolismo , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/metabolismo , Catepsina B/metabolismo , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Concentração de Íons de Hidrogênio , Fígado/metabolismo , Fígado/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Proteólise , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo
16.
Nat Commun ; 10(1): 4492, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582802

RESUMO

Drug delivery with nanocarriers relies on the interaction of individual nanocarriers with the cell surface. For lipid-based NCs, this interaction uniquely involves a process of membrane fusion between the lipid bilayer that makes up the NC and the cell membrane. Cubosomes have emerged as promising fusogenic NCs, however their individual interactions had not yet been directly observed due to difficulties in achieving adequate resolution or disentangling multiple interactions with common characterization techniques. Moreover, many studies on these interactions have been performed under static conditions which may not mimic the actual transport of NCs. Herein we have observed fusion of lipid cubosome NCs with lipid bilayers under flow. Total internal reflection microscopy has allowed visualisation of the fusion event which was sensitive to the lipid compositions and rationalized by lipid diffusion. The fusion event in supported lipid bilayers has been compared with those in cells, revealing a distinct similarity in kinetics.


Assuntos
Membrana Celular/metabolismo , Portadores de Fármacos/farmacologia , Microscopia Intravital/métodos , Fusão de Membrana , Animais , Linhagem Celular , Membrana Celular/ultraestrutura , Portadores de Fármacos/química , Células Epiteliais , Fibroblastos , Humanos , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Camundongos , Microscopia de Força Atômica , Microscopia de Interferência , Nanopartículas/química , Células-Tronco , Imagem com Lapso de Tempo
17.
Adv Exp Med Biol ; 1175: 93-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31583585

RESUMO

Astrocytes are secretory cells, actively participating in cell-to-cell communication in the central nervous system (CNS). They sense signaling molecules in the extracellular space, around the nearby synapses and also those released at much farther locations in the CNS, by their cell surface receptors, get excited to then release their own signaling molecules. This contributes to the brain information processing, based on diffusion within the extracellular space around the synapses and on convection when locales relatively far away from the release sites are involved. These functions resemble secretion from endocrine cells, therefore astrocytes were termed to be a part of the gliocrine system in 2015. An important mechanism, by which astrocytes release signaling molecules is the merger of the vesicle membrane with the plasmalemma, i.e., exocytosis. Signaling molecules stored in astroglial secretory vesicles can be discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This leads to a fusion pore formation, a channel that must widen to allow the exit of the Vesiclal cargo. Upon complete vesicle membrane fusion, this process also integrates other proteins, such as receptors, transporters and channels into the plasma membrane, determining astroglial surface signaling landscape. Vesiclal cargo, together with the whole vesicle can also exit astrocytes by the fusion of multivesicular bodies with the plasma membrane (exosomes) or by budding of vesicles (ectosomes) from the plasma membrane into the extracellular space. These astroglia-derived extracellular vesicles can later interact with various target cells. Here, the characteristics of four types of astroglial secretory vesicles: synaptic-like microvesicles, dense-core vesicles, secretory lysosomes, and extracellular vesicles, are discussed. Then machinery for vesicle-based exocytosis, second messenger regulation and the kinetics of exocytotic vesicle content discharge or release of extracellular vesicles are considered. In comparison to rapidly responsive, electrically excitable neurons, the receptor-mediated cytosolic excitability-mediated astroglial exocytotic vesicle-based transmitter release is a relatively slow process.


Assuntos
Astrócitos/citologia , Sistema Nervoso Central/citologia , Exocitose , Vesículas Secretórias/fisiologia , Humanos , Fusão de Membrana
18.
Nat Commun ; 10(1): 4326, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31548544

RESUMO

Munc18-1 and Munc13-1 orchestrate assembly of the SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, allowing exquisite regulation of neurotransmitter release. Non-regulated neurotransmitter release might be prevented by αSNAP, which inhibits exocytosis and SNARE-dependent liposome fusion. However, distinct mechanisms of inhibition by αSNAP were suggested, and it is unknown how such inhibition is overcome. Using liposome fusion assays, FRET and NMR spectroscopy, here we provide a comprehensive view of the mechanisms underlying the inhibitory functions of αSNAP, showing that αSNAP potently inhibits liposome fusion by: binding to syntaxin-1, hindering Munc18-1 binding; binding to syntaxin-1-SNAP-25 heterodimers, precluding SNARE complex formation; and binding to trans-SNARE complexes, preventing fusion. Importantly, inhibition by αSNAP is avoided only when Munc18-1 binds first to syntaxin-1, leading to Munc18-1-Munc13-1-dependent liposome fusion. We propose that at least some of the inhibitory activities of αSNAP ensure that neurotransmitter release occurs through the highly-regulated Munc18-1-Munc13-1 pathway at the active zone.


Assuntos
Proteínas Munc18/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Bovinos , Cricetulus , Escherichia coli/genética , Fusão de Membrana , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Conformação Proteica , Ratos , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo
19.
Nat Commun ; 10(1): 4307, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541088

RESUMO

To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-ß and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).


Assuntos
Autoantígenos/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Metáfase/fisiologia , Proteínas de Transporte Vesicular/metabolismo , alfa Carioferinas/metabolismo , Autoantígenos/genética , Cristalografia por Raios X , Pontos de Checagem da Fase G2 do Ciclo Celular , Células HEK293 , Humanos , Fusão de Membrana , Proteínas de Membrana/genética , Mitose/fisiologia , Fosforilação , Ligação Proteica , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo
20.
Nat Commun ; 10(1): 4076, 2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501440

RESUMO

Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Mamíferos/metabolismo , Fusão de Membrana , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/metabolismo , Animais , Cálcio/metabolismo , Camundongos Knockout , Mutagênese , Domínios Proteicos , Proteínas SNARE/metabolismo , Transmissão Sináptica , Sinaptotagmina I/química
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