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1.
Gene ; 764: 145098, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32861881

RESUMO

Neocaridina denticulate sinensis is a small freshwater economic shrimp, as well as excellent laboratory model for their short life cycle and easy availability. However, the response of N. denticulate sinensis to pervasive copper pollution in aquatic environments has not been deeply investigated yet. Herein, we preformed Illumina sequencing technology to mine the alterations of cephalothorax transcriptome under 2.5 µmol/L of Cu2+ after 48 h. 122,512 unigenes were assembled and 219 unigenes were identified as significantly differentially expressed genes (DEGs) between control and Cu2+ treatment groups. Functional enrichment analysis revealed that DEGs were mostly associated with immune responses and molting, such as endocytosis, Fc gamma R-mediated phagocytosis and chitin metabolic process. Seven genes were chosen for qPCR verification, and the results showed that the transcriptome sequencing data were consistent with the qPCR results. This is the first report of transcriptome information about N. denticulate sinensis. These results provided a direction for the future research of resistance to Cu2+ in this shrimp, and simultaneously enriched gene information of N. denticulate sinensis.


Assuntos
Cobre/toxicidade , Decápodes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Frutos do Mar , Poluentes Químicos da Água/toxicidade , Animais , Quitina/metabolismo , Decápodes/efeitos dos fármacos , Decápodes/imunologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Muda/efeitos dos fármacos , Muda/genética , Fagocitose/efeitos dos fármacos , Fagocitose/genética , RNA-Seq , Transcriptoma/efeitos dos fármacos
2.
Proc Natl Acad Sci U S A ; 117(40): 25150-25158, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968023

RESUMO

The plasma membrane (PM) acts as the interface between intra- and extracellular environments and exhibits a tightly regulated molecular composition. The composition and amount of PM proteins are regulated by balancing endocytic and exocytic trafficking in a cargo-specific manner, according to the demands of specific cellular states and developmental processes. In plant cells, retrieval of membrane proteins from the PM depends largely on clathrin-mediated endocytosis (CME). However, the mechanisms for sorting PM proteins during CME remain ambiguous. In this study, we identified a homologous pair of ANTH domain-containing proteins, PICALM1a and PICALM1b, as adaptor proteins for CME of the secretory vesicle-associated longin-type R-SNARE VAMP72 group. PICALM1 interacted with the SNARE domain of VAMP72 and clathrin at the PM. The loss of function of PICALM1 resulted in faulty retrieval of VAMP72, whereas general endocytosis was not considerably affected by this mutation. The double mutant of PICALM1 exhibited impaired vegetative development, indicating the requirement of VAMP72 recycling for normal plant growth. In the mammalian system, VAMP7, which is homologous to plant VAMP72, is retrieved from the PM via the interaction with a clathrin adaptor HIV Rev-binding protein in the longin domain during CME, which is not functional in the plant system, whereas retrieval of brevin-type R-SNARE members is dependent on a PICALM1 homolog. These results indicate that ANTH domain-containing proteins have evolved to be recruited distinctly for recycling R-SNARE proteins and are critical to eukaryote physiology.


Assuntos
Endocitose/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Proteínas R-SNARE/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Membrana Celular/genética , Clatrina/metabolismo , Eucariotos/genética , Exocitose/genética , Regulação da Expressão Gênica de Plantas/genética , Células Vegetais/metabolismo , Domínios Proteicos/genética
3.
Arch Virol ; 165(10): 2165-2176, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32740830

RESUMO

The PI3K/Akt signalling pathway is a crucial signalling cascade that regulates transcription, protein translation, cell growth, proliferation, cell survival, and metabolism. During viral infection, viruses exploit a variety of cellular pathways, including the well-known PI3K/Akt signalling pathway. Conversely, cells rely on this pathway to stimulate an antiviral response. The PI3K/Akt pathway is manipulated by a number of viruses, including DNA and RNA viruses and retroviruses. The aim of this review is to provide up-to-date information about the role of the PI3K-Akt pathway in infection with members of five different families of negative-sense ssRNA viruses. This pathway is hijacked for viral entry, regulation of endocytosis, suppression of premature apoptosis, viral protein expression, and replication. Although less common, the PI3K/Akt pathway can be downregulated as an immunomodulatory strategy or as a mechanism for inducing autophagy. Moreover, the cell activates this pathway as an antiviral strategy for interferon and cytokine production, among other strategies. Here, we present new data concerning the role of this pathway in infection with the paramyxovirus Newcastle disease virus (NDV). Our data seem to indicate that NDV uses the PI3K/Akt pathway to delay cell death and increase cell survival as a means of improving its replication. The interference of negative-sense ssRNA viruses with this essential pathway might have implications for the development of antiviral therapies.


Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Infecções por Vírus de RNA/genética , Apoptose/genética , Autofagia/genética , Autofagia/imunologia , Citocinas/genética , Citocinas/imunologia , Endocitose/genética , Endocitose/imunologia , Filoviridae/genética , Filoviridae/metabolismo , Filoviridae/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Interferons/genética , Interferons/imunologia , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Orthomyxoviridae/patogenicidade , Paramyxoviridae/genética , Paramyxoviridae/metabolismo , Paramyxoviridae/patogenicidade , Fosfatidilinositol 3-Quinase/imunologia , Pneumovirinae/genética , Pneumovirinae/metabolismo , Pneumovirinae/patogenicidade , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Rhabdoviridae/genética , Rhabdoviridae/metabolismo , Rhabdoviridae/patogenicidade , Transdução de Sinais , Proteínas Virais/genética , Proteínas Virais/imunologia , Internalização do Vírus , Replicação Viral
4.
PLoS Genet ; 16(7): e1008484, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32673313

RESUMO

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. Missense alleles of the HXK2, REG1, GLC7 and SNF1 genes were shown to confer significant resistance to 2-deoxyglucose and all had the potential to alter the activity and or target selection of the Snf1 kinase signaling pathway. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Addition of 2DG promotes endocytosis of the glucose transporter Hxt3. All but one of the 2DG-resistant strains reduced the 2DG-mediated hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose but induction of these genes is not associated with 2DG-resistance. RNAseq analysis of the transcriptional response to 2DG showed large scale, genome-wide changes in mRNA abundance that were greatly reduced in the 2DG resistant strains. These findings suggest the common adaptive response to 2DG is to limit the magnitude of the response. Genetic studies of 2DG resistance using the dominant SNF1-G53R allele in cells that are genetically compromised in both the endocytosis and DOG pathways suggest that at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.


Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Hexoquinase/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Desoxiglucose/efeitos adversos , Desoxiglucose/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Mutação/genética , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Sequenciamento Completo do Genoma
5.
PLoS Genet ; 16(5): e1008763, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32384077

RESUMO

The ACK family tyrosine kinase SID-3 is involved in the endocytic uptake of double-stranded RNA. Here we identified SID-3 as a previously unappreciated recycling regulator in the Caenorhabditis elegans intestine. The RAB-10 effector EHBP-1 is required for the endosomal localization of SID-3. Accordingly, animals with loss of SID-3 phenocopied the recycling defects observed in ehbp-1 and rab-10 single mutants. Moreover, we detected sequential protein interactions between EHBP-1, SID-3, NCK-1, and DYN-1. In the absence of SID-3, DYN-1 failed to localize at tubular recycling endosomes, and membrane tubules breaking away from endosomes were mostly absent, suggesting that SID-3 acts synergistically with the downstream DYN-1 to promote endosomal tubule fission. In agreement with these observations, overexpression of DYN-1 significantly increased recycling transport in SID-3-deficient cells. Finally, we noticed that loss of RAB-10 or EHBP-1 compromised feeding RNAi efficiency in multiple tissues, implicating basolateral recycling in the transport of RNA silencing signals. Taken together, our study demonstrated that in C. elegans intestinal epithelia, SID-3 acts downstream of EHBP-1 to direct fission of recycling endosomal tubules in concert with NCK-1 and DYN-1.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Citocinese/genética , Dinaminas/fisiologia , Endocitose/fisiologia , Endossomos/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Animais Geneticamente Modificados , Transporte Biológico/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Endocitose/genética , Estudo de Associação Genômica Ampla , Transdução de Sinais/genética , Proteínas rab de Ligação ao GTP/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-32186931

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP; ADCYAP1) is a pleiotropic neuropeptide widely distributed in both the peripheral and central nervous systems. PACAP and its specific cognate PAC1 receptor (ADCYAP1R1) play critical roles in the homeostatic maintenance of multiple physiological and behavioral systems. Notably, maladaptations in the PACAPergic system have been associated with several psychopathologies related to fear and anxiety. PAC1 receptor transcripts are highly expressed in granule cells of the dentate gyrus (DG). Here, we examined the direct effects of PACAP on DG granule cells in brain slices using whole cell patch recordings in current clamp mode. PACAP significantly increased the intrinsic excitability of DG granule cells via PAC1 receptor activation. This increased excitability was not mediated by adenylyl cyclase/cAMP or phospholipase C/PKC activation, but instead via activation of an extracellular signal-regulated kinase (ERK) signaling pathway initiated through PAC1 receptor endocytosis/endosomal signaling. PACAP failed to increase excitability in DG granule cells pretreated with the persistent sodium current blocker riluzole, suggesting that the observed PACAP effects required this component of the inward sodium current.


Assuntos
Ansiedade/genética , Encéfalo/metabolismo , Giro Denteado/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Animais , Ansiedade/patologia , Encéfalo/patologia , Proliferação de Células/efeitos dos fármacos , Giro Denteado/patologia , Endocitose/genética , Endossomos/genética , Medo/psicologia , Regulação da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Neurônios/metabolismo , Neurônios/patologia , Técnicas de Patch-Clamp , Ratos , Riluzol/farmacologia
7.
Genes Cells ; 25(6): 391-401, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32167217

RESUMO

Vesicular transport serves as an important mechanism for cell shape regulation during development. Although the semaphorin signaling molecule, a well-known regulator of axon guidance, induces endocytosis in the growth cone and the axonal transport of vertebrate neurons, the underlying molecular mechanisms remain largely unclear. Here, we show that the Caenorhabditis elegans SNT-1/synaptotagmin-UNC-41/stonin2 system, whose role in synaptic vesicle recycling in neurons has been studied extensively, is involved in semaphorin-regulated vesicular transport in larval epidermal cells. Mutations in the snt-1/unc-41 genes strongly suppressed the cell shape defects of semaphorin mutants. The null mutation in the semaphorin receptor gene, plx-1, altered the expression and localization pattern of endocytic and exocytic markers in the epidermal cells while repressing the transport of SNT-1-containing vesicles toward late endosome/lysosome pathways. Our findings suggest that the nematode semaphorins regulate the vesicular transport in epidermal cells in a manner distinct from that of vertebrate semaphorins in neurons.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Epidérmicas/metabolismo , Semaforinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagminas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico Ativo/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Endocitose/genética , Endossomos/genética , Endossomos/metabolismo , Exocitose/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Semaforinas/genética , Transdução de Sinais/genética , Sinaptotagminas/genética , Proteínas de Transporte Vesicular/genética
8.
Nat Commun ; 11(1): 1113, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111843

RESUMO

Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.


Assuntos
Sistemas CRISPR-Cas , Vesículas Extracelulares/metabolismo , Pequeno RNA não Traduzido/metabolismo , Transporte Biológico , Comunicação Celular , Linhagem Celular , Endocitose/genética , Vesículas Extracelulares/genética , Fluorescência , Genes Reporter/genética , Células HEK293 , Humanos , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/genética
9.
Sci Rep ; 10(1): 2794, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066816

RESUMO

Cholesterol, a necessary component of animal cell membranes, is also needed by the lethal human malaria parasite Plasmodium falciparum. Because P. falciparum lacks a cholesterol synthesis pathway and malaria patients have low blood cholesterol, we speculated that it scavenges cholesterol from them in some way. We used time-lapse holotomographic microscopy to observe cholesterol transport in live P. falciparum parasites and structurally investigate erythrocyte membranes, both during and after P. falciparum invasion of human erythrocytes. After P. falciparum initially acquired free cholesterol or inner erythrocytic membrane-derived cholesterol, we observed budding lipid membranes elongating into the cytosol and/or membrane segments migrating there and eventually fusing with the parasite membranes, presumably at the parasitophorous vacuole membrane (PVM). Finally, the cholesterol-containing segments were seen to surround the parasite nucleus. Our imaging data suggest that a novel membrane transport system operates in the cytosol of P. falciparum-infected erythrocytes as a cholesterol import system, likely between the PVM and the erythrocyte membrane, and that this transportation process occurs during the live erythrocyte stages of P. falciparum.


Assuntos
Colesterol/isolamento & purificação , Malária Falciparum/diagnóstico por imagem , Imagem Molecular , Plasmodium falciparum/patogenicidade , Animais , Colesterol/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/genética , Eritrócitos/ultraestrutura , Humanos , Malária Falciparum/parasitologia , Malária Falciparum/patologia
10.
Cell Mol Life Sci ; 77(24): 5223-5242, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32065241

RESUMO

Endocytosis of the amyloid precursor protein (APP) is critical for generation of ß-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.


Assuntos
Doença de Alzheimer/genética , Motivos de Aminoácidos/genética , Precursor de Proteína beta-Amiloide/genética , Proteínas rab5 de Ligação ao GTP/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Proteínas de Transporte/genética , Endocitose/genética , Endossomos/genética , Humanos , Camundongos , Transporte Proteico/genética , Vesículas Transportadoras/genética
11.
Cell Mol Biol Lett ; 25: 1, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31988639

RESUMO

The ubiquitin system, known as a common feature in eukaryotes, participates in multiple cellular processes, such as signal transduction, cell-cycle progression, receptor trafficking and endocytosis, and even the immune response. In lung cancer, evidence has revealed that aberrant events in ubiquitin-mediated processes can cause a variety of pathological outcomes including tumorigenesis and metastasis. Likewise, ubiquitination on the core components contributing to the activity of cell signaling controls bio-signal turnover and cell final destination. Given this, inhibitors targeting the ubiquitin system have been developed for lung cancer therapies and have shown great prospects for clinical application. However, the exact biological effects and physiological role of the drugs used in lung cancer therapies are still not clearly elucidated, which might seriously impede the progress of treatment. In this work, we summarize current research advances in cell signal regulation processes mediated through the ubiquitin system during the development of lung cancer, with the hope of improving the therapeutic effects by means of aiming at efficient targets.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas ras/metabolismo , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Endocitose/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Sistema de Sinalização das MAP Quinases/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina/química , Ubiquitinação/genética , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética
12.
Science ; 367(6473): 51-59, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31896710

RESUMO

Artemisinin and its derivatives (ARTs) are the frontline drugs against malaria, but resistance is jeopardizing their effectiveness. ART resistance is mediated by mutations in the parasite's Kelch13 protein, but Kelch13 function and its role in resistance remain unclear. In this study, we identified proteins located at a Kelch13-defined compartment. Inactivation of eight of these proteins, including Kelch13, rendered parasites resistant to ART, revealing a pathway critical for resistance. Functional analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell. Parasites with inactivated Kelch13 or a resistance-conferring Kelch13 mutation displayed reduced hemoglobin endocytosis. ARTs are activated by degradation products of hemoglobin. Hence, reduced activity of Kelch13 and its interactors diminishes hemoglobin endocytosis and thereby ART activation, resulting in parasite resistance.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos/genética , Endocitose/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Hemoglobinas/metabolismo , Humanos , Malária Falciparum/tratamento farmacológico , Mutação
13.
Nat Cell Biol ; 22(2): 213-224, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31988382

RESUMO

Endosomal transport is essential for cellular organization and compartmentalization and cell-cell communication. Sorting endosomes provide a crossroads for various trafficking pathways and determine recycling, secretion or degradation of proteins. The organization of these processes requires membrane-tethering factors to coordinate Rab GTPase function with membrane fusion. Here, we report a conserved tethering platform that acts in the Rab11 recycling pathways at sorting endosomes, which we name factors for endosome recycling and Rab interactions (FERARI). The Rab-binding module of FERARI consists of Rab11FIP5 and rabenosyn-5/RABS-5, while the SNARE-interacting module comprises VPS45 and VIPAS39. Unexpectedly, the membrane fission protein EHD1 is also a FERARI component. Thus, FERARI appears to combine fusion activity through the SM protein VPS45 with pinching activity through EHD1 on SNX-1-positive endosomal membranes. We propose that coordination of fusion and pinching through a kiss-and-run mechanism drives cargo at endosomes into recycling pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Endocitose/genética , Endossomos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Intestinos/citologia , Imagem Molecular , Oócitos/citologia , Oócitos/metabolismo , Transporte Proteico , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/metabolismo
14.
Nucleic Acids Res ; 48(3): 1372-1391, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31840180

RESUMO

Release of phosphorothioate antisense oligonucleotides (PS-ASOs) from late endosomes (LEs) is a rate-limiting step and a poorly defined process for productive intracellular ASO drug delivery. Here, we examined the role of Golgi-endosome transport, specifically M6PR shuttling mediated by GCC2, in PS-ASO trafficking and activity. We found that reduction in cellular levels of GCC2 or M6PR impaired PS-ASO release from endosomes and decreased PS-ASO activity in human cells. GCC2 relocated to LEs upon PS-ASO treatment, and M6PR also co-localized with PS-ASOs in LEs or on LE membranes. These proteins act through the same pathway to influence PS-ASO activity, with GCC2 action preceding that of M6PR. Our data indicate that M6PR binds PS-ASOs and facilitates their vesicular escape. The co-localization of M6PR and of GCC2 with ASOs is influenced by the PS modifications, which have been shown to enhance the affinity of ASOs for proteins, suggesting that localization of these proteins to LEs is mediated by ASO-protein interactions. Reduction of M6PR levels also decreased PS-ASO activity in mouse cells and in livers of mice treated subcutaneously with PS-ASO, indicating a conserved mechanism. Together, these results demonstrate that the transport machinery between LE and Golgi facilitates PS-ASO release.


Assuntos
Endossomos/genética , Proteínas da Matriz do Complexo de Golgi/genética , Oligonucleotídeos Antissenso/genética , Receptor IGF Tipo 2/genética , Animais , Endocitose/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Camundongos , Oligonucleotídeos Fosforotioatos/genética , Transporte Proteico/genética , Receptor IGF Tipo 2/metabolismo
15.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31727776

RESUMO

Axon initial segments (AISs) initiate action potentials and regulate the trafficking of vesicles between somatodendritic and axonal compartments. However, the mechanisms controlling AIS assembly remain poorly defined. We performed differential proteomics and found nuclear mitotic apparatus protein 1 (NuMA1) is downregulated in AIS-deficient neonatal mouse brains and neurons. NuMA1 is transiently located at the AIS during development where it interacts with the scaffolding protein 4.1B and the dynein regulator lissencephaly 1 (Lis1). Silencing NuMA1 or protein 4.1B by shRNA disrupts AIS assembly, but not maintenance. Silencing Lis1 or overexpressing NuMA1 during AIS assembly increased the density of AIS proteins, including ankyrinG and neurofascin-186 (NF186). NuMA1 inhibits the endocytosis of AIS NF186 by impeding Lis1's interaction with doublecortin, a potent facilitator of NF186 endocytosis. Our results indicate the transient expression and AIS localization of NuMA1 stabilizes the developing AIS by inhibiting endocytosis and removal of AIS proteins.


Assuntos
Segmento Inicial do Axônio/metabolismo , Proteínas de Ciclo Celular/genética , Dineínas/genética , Endocitose/genética , Proteômica , 1-Alquil-2-acetilglicerofosfocolina Esterase/antagonistas & inibidores , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Anquirinas/genética , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Citoesqueleto/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Fatores de Crescimento Neural/genética , Neurônios/metabolismo , Transporte Proteico/genética , RNA Interferente Pequeno/farmacologia
16.
Sci Rep ; 9(1): 19368, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852969

RESUMO

Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, the Drosophila homolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound to dap160 and endophilin B transcription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition, kismet mutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored at kismet mutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.


Assuntos
Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Proteínas de Drosophila/genética , Endocitose/genética , Proteínas de Homeodomínio/genética , Vesículas Sinápticas/genética , Aciltransferases/genética , Animais , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Histona Desacetilase 1/genética , Humanos , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Glutamato/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética
17.
Elife ; 82019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31855180

RESUMO

Actin dynamics generate forces to deform the membrane and overcome the cell's high turgor pressure during clathrin-mediated endocytosis (CME) in yeast, but precise molecular details are still unresolved. Our previous models predicted that actin filaments of the endocytic meshwork continually polymerize and disassemble, turning over multiple times during an endocytic event, similar to other actin systems. We applied single-molecule speckle tracking in live fission yeast to directly measure molecular turnover within CME sites for the first time. In contrast with the overall ~20 s lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 s, and similarly high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins' motions. These results indicate that endocytic proteins turn over up to five times during the formation of an endocytic vesicle, and suggest revising quantitative models of force production.


Assuntos
Actinas/genética , Clatrina/genética , Endocitose/genética , Vesículas Transportadoras/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas do Capsídeo/genética , Membrana Celular/genética , Clatrina/metabolismo , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Schizosaccharomyces/genética , Imagem Individual de Molécula , Vesículas Transportadoras/metabolismo
18.
ACS Appl Mater Interfaces ; 11(49): 46101-46111, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31729219

RESUMO

The theranostic ability of a new fluorescently labeled cationic cyclodextrin-graphene nanoplatform (GCD@Ada-Rhod) was investigated by studying its intracellular trafficking and its ability to deliver plasmid DNA and microRNA. The nanoplatform was synthesized by both covalent and supramolecular approaches, and its chemical structure, morphology, and colloidal behavior were investigated by TGA, TEM, spectroscopic analysis such as UV-vis, fluorescence emission, DLS, and ζ-potential measurements. The cellular internalization of GCD@Ada-Rhod and its perinuclear localization were assessed by FLIM, Raman imaging, and fluorescence microscopy. Biological experiments with pCMS-EGFP and miRNA-15a evidenced the excellent capability of GCD@Ada-Rhod to deliver both pDNA and microRNA without significant cytotoxicity. The biological results evidenced an unforeseen caveolae-mediated endocytosis internalization pathway (generally expected for particles <200 nm), despite the fact that the GCD@Ada-Rhod size is about 400 nm (by DLS and TEM data). We supposed that the internalization pathway was driven by physical-chemical features of GCD@Ada-Rhod, and the caveolae-mediated uptake enhanced the transfection efficiency, avoiding the lysosomal acid degradation. The cellular effects of internalized miRNA-15a on the oncogene protein BCL-2 were investigated at two different concentrations (N/P = 10 and 5), and a reduction of the BCL-2 level was detected at a low concentration (i.e., N/P = 10). miRNA-15a is considered an ideal cancer therapy molecule due to its activity on multiple transcription factors, and the elucidation of the correlation between the concentration of delivered miRNA-15a and the down-/up-regulation of the BCL-2 level, documented for the first time in this work, could be an important contribution to guide its clinical application.


Assuntos
Transporte Biológico , Técnicas de Transferência de Genes , MicroRNAs/farmacologia , Plasmídeos/farmacologia , Endocitose/efeitos dos fármacos , Endocitose/genética , Grafite/química , Humanos , Lisossomos/química , Lisossomos/genética , MicroRNAs/química , MicroRNAs/genética , Plasmídeos/química , Plasmídeos/genética , Transfecção , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacologia
19.
PLoS Biol ; 17(10): e3000500, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31652255

RESUMO

Clathrin-mediated endocytosis involves the sequential assembly of more than 60 proteins at the plasma membrane. An important fraction of these proteins regulates the assembly of an actin-related protein 2/3 (Arp2/3)-branched actin network, which is essential to generate the force during membrane invagination. We performed, on wild-type (WT) yeast and mutant strains lacking putative actin crosslinkers, a side-by-side comparison of in vivo endocytic phenotypes and in vitro rigidity measurements of reconstituted actin patches. We found a clear correlation between softer actin networks and a decreased efficiency of endocytosis. Our observations support a chain-of-consequences model in which loss of actin crosslinking softens Arp2/3-branched actin networks, directly limiting the transmission of the force. Additionally, the lifetime of failed endocytic patches increases, leading to a larger number of patches and a reduced pool of polymerizable actin, which slows down actin assembly and further impairs endocytosis.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Endocitose/genética , Regulação Fúngica da Expressão Gênica , Mecanotransdução Celular , Saccharomyces cerevisiae/genética , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/deficiência , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Fenômenos Biomecânicos , Clatrina/deficiência , Clatrina/genética , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética
20.
PLoS Biol ; 17(10): e3000512, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658248

RESUMO

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.


Assuntos
Arrestinas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Nucleosídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tiamina/metabolismo , Arrestinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Endocitose/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Proteínas de Transporte de Nucleosídeos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Secundária de Proteína , Proteômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Tiamina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação
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