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1.
PLoS One ; 15(8): e0233247, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857759

RESUMO

Poly(glycine-alanine) (polyGA) is one of the polydipeptides expressed in Frontotemporal Dementia and/or Amyotrophic Lateral Sclerosis 1 caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ)-expanded Huntingtin exon 1 (Httex1) in Huntington's disease. Both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms and therefore recruit the same repertoire of endogenous proteins. When co-expressed in the same cell, polyGA101 and Httex1(Q97) inclusions adopted immiscible phases suggesting different endogenous proteins would be enriched. Proteomic analyses identified 822 proteins in the inclusions. Only 7 were specific to polyGA and 4 specific to Httex1(Q97). Quantitation demonstrated distinct enrichment patterns for the proteins not specific to each inclusion type (up to ~8-fold normalized to total mass). The proteasome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in Httex1(Q97) aggregates. Both structures revealed a collection of folding and degradation machinery including proteins in the Httex1(Q97) aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.


Assuntos
Corpos de Inclusão/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Éxons , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Corpos de Inclusão/genética , Corpos de Inclusão/patologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Proteínas/genética , Proteólise , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Risco , Solubilidade
2.
PLoS One ; 15(7): e0233583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735619

RESUMO

Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.


Assuntos
Éxons/genética , Proteína Huntingtina/genética , Elongação Traducional da Cadeia Peptídica , Agregados Proteicos/genética , RNA Mensageiro/genética , Ribossomos/metabolismo , Sequência de Bases , Células Epiteliais , Genes Reporter , Células HEK293 , Humanos , Proteína Huntingtina/biossíntese , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Repetições Minissatélites , Peptídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
3.
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
4.
Proc Natl Acad Sci U S A ; 117(28): 16313-16323, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601209

RESUMO

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.


Assuntos
Peroxirredoxinas/química , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
PLoS One ; 15(7): e0236185, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730344

RESUMO

Fluorescent markers are a powerful tool and have been widely applied in biology for different purposes. The genome sequence of Xanthomonas citri subsp. citri (X. citri) revealed that approximately 30% of the genes encoded hypothetical proteins, some of which could play an important role in the success of plant-pathogen interaction and disease triggering. Therefore, revealing their functions is an important strategy to understand the bacterium pathways and mechanisms involved in plant-host interaction. The elucidation of protein function is not a trivial task, but the identification of the subcellular localization of a protein is key to understanding its function. We have constructed an integrative vector, pMAJIIc, under the control of the arabinose promoter, which allows the inducible expression of red fluorescent protein (mCherry) fusions in X. citri, suitable for subcellular localization of target proteins. Fluorescence microscopy was used to track the localization of VrpA protein, which was visualized surrounding the bacterial outer membrane, and the GyrB protein, which showed a diffused cytoplasmic localization, sometimes with dots accumulated near the cellular poles. The integration of the vector into the amy locus of X. citri did not affect bacterial virulence. The vector could be stably maintained in X. citri, and the disruption of the α-amylase gene provided an ease screening method for the selection of the transformant colonies. The results demonstrate that the mCherry-containing vector here described is a powerful tool for bacterial protein localization in cytoplasmic and periplasmic environments.


Assuntos
Proteínas de Bactérias/metabolismo , Citoplasma/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Xanthomonas/metabolismo , Arabinose/farmacologia , Cromossomos Bacterianos/genética , Vetores Genéticos/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Amido/metabolismo , Frações Subcelulares/efeitos dos fármacos , Xanthomonas/patogenicidade
6.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
7.
Nature ; 584(7820): 291-297, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728216

RESUMO

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.


Assuntos
Espaço Extracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Solubilidade , Especificidade por Substrato
8.
Proc Natl Acad Sci U S A ; 117(31): 18832-18839, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32709746

RESUMO

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.


Assuntos
Proteínas NLR , Imunidade Vegetal , Proteínas de Plantas , Proteínas Recombinantes de Fusão , Transdução de Sinais , Animais , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Mamíferos , Proteínas NLR/química , Proteínas NLR/genética , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Imunidade Vegetal/genética , Imunidade Vegetal/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
9.
PLoS One ; 15(6): e0234430, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511274

RESUMO

Excess presence of the human epidermal growth factor receptor 2 (HER2) as well as of the focal adhesion protein complexes are associated with increased proliferation, migratory, and invasive behavior of cancer cells. A cross-regulation between HER2 and integrin signaling pathways has been found, but the exact mechanism remains elusive. Here, we investigated whether HER2 colocalizes with focal adhesion complexes on breast cancer cells overexpressing HER2. For this purpose, vinculin or talin green fluorescent protein (GFP) fusion proteins, both key constituents of focal adhesions, were expressed in breast cancer cells. HER2 was either extracellularly or intracellularly labeled with fluorescent quantum dots nanoparticles (QDs). The cell-substrate interface was analyzed at the location of the focal adhesions by means of total internal reflection fluorescent microscopy or correlative fluorescence- and scanning transmission electron microscopy. Expression of HER2 at the cell-substrate interface was only observed upon intracellular labeling, and was heterogeneous with both HER2-enriched and -low regions. In contrast to an expected enrichment of HER2 at focal adhesions, an anti-correlated expression pattern was observed for talin and HER2. Our findings suggest a spatial anti-correlation between HER2 and focal adhesion complexes for adherent cells.


Assuntos
Membrana Celular/metabolismo , Adesões Focais/metabolismo , Receptor ErbB-2/metabolismo , Análise Espacial , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Adesões Focais/ultraestrutura , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Eletrônica de Transmissão e Varredura , Microscopia de Fluorescência , Receptor ErbB-2/análise , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Talina/análise , Talina/genética , Talina/metabolismo , Vinculina/análise , Vinculina/genética , Vinculina/metabolismo
10.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 969-978, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567280

RESUMO

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.


Assuntos
Neoplasias Pulmonares , Proteínas Recombinantes de Fusão , Animais , Antígeno CTLA-4/genética , Proliferação de Células , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Receptor de Morte Celular Programada 1/genética , Ligação Proteica , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
11.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
12.
Mol Cell ; 79(1): 167-179.e11, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32497496

RESUMO

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.


Assuntos
Proteínas Argonauta/fisiologia , Células-Tronco Embrionárias/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Feminino , Regulação da Expressão Gênica , Glioma/genética , Glioma/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Hidrolases/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , Ligação Proteica , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética
13.
Nat Protoc ; 15(6): 1922-1953, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32350457

RESUMO

Protein-DNA interactions are essential for establishing cell type-specific chromatin architecture and gene expression. We recently developed scDam&T-seq, a multi-omics method that can simultaneously quantify protein-DNA interactions and the transcriptome in single cells. The method effectively combines two existing methods: DNA adenine methyltransferase identification (DamID) and CEL-Seq2. DamID works through the tethering of a protein of interest (POI) to the Escherichia coli DNA adenine methyltransferase (Dam). Upon expression of this fusion protein, DNA in proximity to the POI is methylated by Dam and can be selectively digested and amplified. CEL-Seq2, in contrast, makes use of poly-dT primers to reverse transcribe mRNA, followed by linear amplification through in vitro transcription. scDam&T-seq is the first technique capable of providing a combined readout of protein-DNA contact and transcription from single-cell samples. Once suitable cell lines have been established, the protocol can be completed in 5 d, with a throughput of hundreds to thousands of cells. The processing of raw sequencing data takes an additional 1-2 d. Our method can be used to understand the transcriptional changes a cell undergoes upon the DNA binding of a POI. It can be performed in any laboratory with access to FACS, robotic and high-throughput-sequencing facilities.


Assuntos
DNA/metabolismo , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Proteínas/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , DNA/genética , Metilação de DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Camundongos , Ligação Proteica , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Transcriptoma
14.
Prog Biophys Mol Biol ; 155: 29-35, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32360608

RESUMO

In December 2019, an atypical pneumonia invaded the city of Wuhan, China, and the causative agent of this disease turned out to be a new coronavirus. In January 2020, the World Health Organization named the new coronavirus 2019-nCoV and subsequently it is referred to as SARS-CoV2 and the related disease as CoViD-19 (Lai et al., 2020). Very quickly, the epidemic led to a pandemic and it is now a worldwide emergency requiring the creation of new antiviral therapies and a related vaccine. The purpose of this article is to review and investigate further the molecular mechanism by which the SARS-CoV2 virus infection proceeds via the formation of a hetero-trimer between its protein S, the ACE2 receptor and the B0AT1 protein, which is the "entry receptor" for the infection process involving membrane fusion (Li et al., 2003). A reverse engineering process uses the formalism of the Hill function to represent the functions related to the dynamics of the biochemical interactions of the viral infection process. Then, using a logical evaluation of viral density that measures the rate at which the cells are hijacked by the virus (and they provide a place for the virus to replicate) and considering the "time delay" given by the interaction between cell and virus, the expected duration of the incubation period is predicted. The conclusion is that the density of the virus varies from the "exposure time" to the "interaction time" (virus-cells). This model can be used both to evaluate the infectious condition and to analyze the incubation period. BACKGROUND: The ongoing threat of the new coronavirus SARS-CoV2 pandemic is alarming and strategies for combating infection are highly desired. This RNA virus belongs to the ß-coronavirus genus and is similar in some features to SARS-CoV. Currently, no vaccine or approved medical treatment is available. The complex dynamics of the rapid spread of this virus can be demonstrated with the aid of a computational framework. METHODS: A mathematical model based on the principles of cell-virus interaction is developed in this manuscript. The amino acid sequence of S proein and its interaction with the ACE-2 protein is mimicked with the aid of Hill function. The mathematical model with delay is solved with the aid of numerical solvers and the parametric values are obtained with the help of MCMC algorithm. RESULTS: A delay differential equation model is developed to demonstrate the dynamics of target cells, infected cells and the SARS-CoV2. The important parameters and coefficients are demonstrated with the aid of numerical computations. The resulting thresholds and forecasting may prove to be useful tools for future experimental studies and control strategies. CONCLUSIONS: From the analysis, I is concluded that control strategy via delay is a promising technique and the role of Hill function formalism in control strategies can be better interpreted in an inexpensive manner with the aid of a theoretical framework.


Assuntos
Betacoronavirus/metabolismo , Membrana Celular/metabolismo , Infecções por Coronavirus/metabolismo , Simulação de Dinâmica Molecular , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Permeabilidade da Membrana Celular , Humanos , Proteínas de Membrana/metabolismo , Pandemias , Ligação Proteica , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Vírus da SARS/metabolismo
15.
PLoS One ; 15(5): e0232532, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357161

RESUMO

Early endosomes, regarded as the main sorting station on endocytic pathway, are characterized by high frequency of homotypic fusions mediated by tethering protein EEA1. Despite intensive investigations, biogenesis of endosomes, boundaries between early and late endosomes, and process of cargo transition though them remain obscure. Here, using EGF/EGFR endocytosis as a model and confocal microscopy of fixed and live cells, we provide evidence favoring EEA1-vesicles being pre-existed vesicular compartment, that maintains its resident proteins' level and is sensitive to biosynthetic, but not endocytic pathway disturbance. EEA1-vesicles directly fuse with incoming EGF/EGFR-vesicles into hybrid endosomes with separated EEA1- and EGFR-domains, thus providing a platform for rapid achievement of an excess of surface-derived membrane that is used to form intraluminal vesicles (ILVs). Thus, multivesicular structures colocalized with EEA1 are still early endosomes. "EEA1-cycle" ends by exclusion of EGFR-containing domains with ILVs inside that turns into MVE and restoration of initial EEA1-vesicles population.


Assuntos
Endossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vias Biossintéticas , Meios de Cultura Livres de Soro , Vesículas Citoplasmáticas/metabolismo , Endocitose , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Modelos Biológicos , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
16.
PLoS One ; 15(5): e0226472, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379828

RESUMO

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.


Assuntos
Proteínas de Bactérias/metabolismo , Centrômero/química , Centrômero/metabolismo , DNA Bacteriano/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/genética , Simportadores/metabolismo
17.
Parasitol Res ; 119(6): 1753-1765, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32363442

RESUMO

RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure. RbAp48 is the essential subunit of the chromatin assembly factor-1 (CAF-1) complex, therefore also named as CAF-1C. A detailed in silico sequence and structure analysis of homologs of RbAp46/48 in Plasmodium falciparum (PF3D7_0110700 and PF3D7_1433300) exhibited conservation of characteristic features in both the protein-seven-bladed WD40 ß-propeller conformation and different binding interfaces. A comparative structural analysis highlighted species-specific features of the parasite, yeast, drosophila, and human RbAp46/48. In the present study, we report cloning, expression, and characterization of P. falciparum PF3D7_0110700, a putative RbAp46/48 (PfRbAp46/48). PfRbAp46/48 was cloned into pTEM11 vector in fusion with 6xHistidine tag and over-expressed in Escherichia coli B834 cells. The protein was purified by Ni-NTA followed by gel permeation chromatography. The protein expressed in all the three asexual blood stages and exhibited nuclear localization. We showed direct interaction of the purified rPfRbAp46/48 with the histone H4. These findings further our understanding of RbAp46/48 proteins and role of these proteins in the parasite biology.


Assuntos
Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Expressão Gênica , Chaperonas de Histonas/genética , Histonas/metabolismo , Estágios do Ciclo de Vida/genética , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
18.
Mol Immunol ; 123: 88-96, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32447084

RESUMO

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.


Assuntos
Bacillus subtilis/metabolismo , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Vacinas Bacterianas/uso terapêutico , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/imunologia , Animais , Bacillus subtilis/genética , Toxinas Bacterianas/metabolismo , Vacinas Bacterianas/química , Vacinas Bacterianas/genética , Infecções por Clostridium/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Vacinação/métodos , Vacinas Sintéticas/química , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêutico
19.
Arch Biochem Biophys ; 688: 108401, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32376316

RESUMO

HIV-1 glycoprotein 41 (gp41) mediates fusion between virus and target cells by folding into a fusion active state, in which the C-terminal heptad repeat (CHR) regions associate externally to the N-terminal heptad repeat (NHR) trimer and form a very stable six-helix bundle coiled-coil structure. Therefore, interfering with the NHR-CHR interaction of gp41 is a promising therapeutic approach against HIV-1. However, a full understanding of the molecular and mechanistic details of this interaction is still incomplete. Here, we use single-chain, chimeric proteins (named covNHR) that reproduce accurately the CHR-NHR interactions to analyze the binding thermodynamics of several peptides with different length from the CHR region. The results indicate that cooperative binding involving two or more pockets of the NHR groove is necessary to obtain relevant affinities and that the binding energy is broadly distributed along the interface, underlining a crucial role of a middle pocket to achieve tight binding. In contrast, targeting only the deep hydrophobic pocket is insufficient to achieve significant affinity. Moreover, calorimetry experiments in combination with limited proteolysis performed using a mutant with occluded binding in the N-terminal pocket reveal the existence of an allosteric communication between the different regions. This study is the first detailed thermodynamic dissection of the NHR-CHR interaction in gp41 and contributes therefore to a better understanding of HIV fusion. These results are relevant for the development of potential fusion inhibitors.


Assuntos
Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/química , Fragmentos de Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Calorimetria , Proteína gp41 do Envelope de HIV/química , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Termodinâmica
20.
Nucleic Acids Res ; 48(10): 5684-5694, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356894

RESUMO

Studies on biological functions of N6-methyladenosine (m6A) modification in mRNA have drawn significant attention in recent years. Here we describe the construction and characterization of a CRISPR-Cas13b-based tool for targeted demethylation of specific mRNA. A fusion protein, named dm6ACRISPR, was created by linking a catalytically inactive Type VI-B Cas13 enzyme from Prevotella sp. P5-125 (dPspCas13b) to m6A demethylase AlkB homolog 5 (ALKBH5). dm6ACRISPR specifically demethylates m6A of targeted mRNA such as cytochrome b5 form A (CYB5A) to increase its mRNA stability. It can also demethylate ß-catenin-encoding CTNNB1 mRNA that contains multiple m6A sites to trigger its translation. In addition, the dm6ACRISPR system incurs efficient demethylation of targeted epitranscriptome transcripts with limited off-target effects. Targeted demethylation of transcripts coding for oncoproteins such as epidermal growth factor receptor (EGFR) and MYC can suppress proliferation of cancer cells. Together, we provide a programmable and in vivo manipulation tool to study mRNA modification of specific genes and their related biological functions.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Adenosina/análogos & derivados , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Proliferação de Células , Desmetilação , Células HEK293 , Células HeLa , Humanos , Oncogenes , Prevotella/enzimologia , Engenharia de Proteínas , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/metabolismo
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