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1.
Nat Commun ; 10(1): 2370, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147549

RESUMO

FAM134B/RETREG1 is a selective ER-phagy receptor that regulates the size and shape of the endoplasmic reticulum. The structure of its reticulon-homology domain (RHD), an element shared with other ER-shaping proteins, and the mechanism of membrane shaping remain poorly understood. Using molecular modeling and molecular dynamics (MD) simulations, we assemble a structural model for the RHD of FAM134B. Through MD simulations of FAM134B in flat and curved membranes, we relate the dynamic RHD structure with its two wedge-shaped transmembrane helical hairpins and two amphipathic helices to FAM134B functions in membrane-curvature induction and curvature-mediated protein sorting. FAM134B clustering, as expected to occur in autophagic puncta, amplifies the membrane-shaping effects. Electron microscopy of in vitro liposome remodeling experiments support the membrane remodeling functions of the different RHD structural elements. Disruption of the RHD structure affects selective autophagy flux and leads to disease states.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/genética , Forma das Organelas/genética , Autofagia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Humanos , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Proteínas de Membrana/genética , Microscopia Eletrônica , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico/genética
2.
Plant Cell Rep ; 38(7): 825-833, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139894

RESUMO

Recently, plants have emerged as a lucrative alternative system for the production of recombinant proteins, as recombinant proteins produced in plants are safer and cheaper than those produced in bacteria and animal cell-based production systems. To obtain high yields in plants, recombinant proteins are produced in chloroplasts using different strategies. The first strategy is based on chloroplast transformation, followed by gene expression and translation in chloroplasts. This has proven to be a powerful approach for the production of proteins at high levels. The second approach is based on nuclear transformation, followed by post-translational import of proteins from the cytosol into chloroplasts. In the nuclear transformation approach, foreign genes are stably integrated into the nuclear genome or transiently expressed in the nucleus by non-integrating T-DNA. Although this approach also has great potential for protein production at high levels, it has not been thoroughly investigated. In this review, we focus on nuclear transformation-based protein expression and its subsequent sequestration in chloroplasts, and summarize the different strategies used for high-level production of recombinant proteins. We also discuss future directions for further improvements in protein production in chloroplasts through nuclear transformation-based gene expression.


Assuntos
Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Cloroplastos/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Genética/genética
3.
Methods Mol Biol ; 1964: 119-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929239

RESUMO

Aptamers are nucleic acid molecules that bind to a target molecule with high affinity and specificity, which are generated by a process known as systematic evolution of ligands by exponential enrichment (SELEX). Because of their high affinity and specificity, aptamers were developed as therapeutic agents. Although aptamers are investigated as promising therapeutic agents, the mechanism of their high affinity and specificity is not clear. Therefore, structural and biophysical studies are important to know that. To date, ITC is increasingly being used to study the thermodynamic basis of aptamer-target protein interactions. Understanding the mechanism of aptamer binding would contribute to their development for therapeutic applications. In this chapter, we describe the protocol to study the thermodynamics of aptamer-protein interactions.


Assuntos
Calorimetria/métodos , Proteínas/química , Técnica de Seleção de Aptâmeros/métodos , Fenômenos Biofísicos , Humanos , Ligantes , Transporte Proteico/genética , Termodinâmica
4.
Methods Cell Biol ; 151: 353-376, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948018

RESUMO

Single-domain antibodies, also known as nanobodies, are small antigen-binding fragments (~15kDa) that are derived from heavy chain only antibodies present in camelids (VHH, from camels and llamas), and cartilaginous fishes (VNAR, from sharks). Nanobody V-like domains are useful alternatives to conventional antibodies due to their small size, and high solubility and stability across many applications. In addition, phage display, ribosome display, and mRNA/cDNA display methods can be used for the efficient generation and optimization of binders in vitro. The resulting nanobodies can be genetically encoded, tagged, and expressed in cells for in vivo localization and functional studies of target proteins. Collectively, these properties make nanobodies ideal for use within echinoderm embryos. This chapter describes the optimization and imaging of genetically encoded nanobodies in the sea urchin embryo. Examples of live-cell antigen tagging (LCAT) and the manipulation of green fluorescent protein (GFP) are shown. We discuss the potentially transformative applications of nanobody technology for probing membrane protein trafficking, cytoskeleton re-organization, receptor signaling events, and gene regulation during echinoderm development.


Assuntos
Biologia Molecular/métodos , Proteínas/isolamento & purificação , Ouriços-do-Mar/ultraestrutura , Anticorpos de Domínio Único/biossíntese , Animais , Camelídeos Americanos/imunologia , Camelus/imunologia , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário/genética , Cadeias Pesadas de Imunoglobulinas/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteínas/genética , Ouriços-do-Mar/crescimento & desenvolvimento , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia
5.
Genes Cells ; 24(6): 408-421, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963674

RESUMO

The flagellar protein export apparatus switches its substrate specificity when hook length has reached approximately 55 nm in Salmonella. The C-terminal cytoplasmic domain of FlhB (FlhBC ) is involved in this switching process. FlhBC consists of FlhBCN and FlhBCC polypeptides. FlhBCC has a flexible C-terminal tail (FlhBCCT ). FlhBCC is involved in substrate recognition, and conformational rearrangements of FlhBCN -FlhBCC boundary are postulated to be required for the export switching. However, it remains unknown how it occurs. To clarify this question, we carried out mutational analysis of highly conserved residues in FlhBC . The flhB(E230A) mutation reduced the FlhB function. The flhB(E11S) mutation restored the protein transport activity of the flhB(E230A) mutant to the wild-type level, suggesting that the interaction of FlhBCN with the extreme N-terminal region of FlhB is required for flagellar protein export. The flhB(R320A) mutation affected hydrophobic interaction networks in FlhBCC , thereby increasing insolubility of FlhBC . The R320A mutation also affected the export switching, thereby producing longer hooks with the filament attached. C-terminal truncations of FlhBCCT induced a conformational change of FlhBCN -FlhBCC boundary, resulting in a loose hook length control. We propose that FlhBCCT may control conformational arrangements of FlhBCN -FlhBCC boundary through the hydrophobic interaction networks of FlhBCC .


Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Salmonella typhi/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Análise Mutacional de DNA/métodos , Flagelos/genética , Flagelos/fisiologia , Proteínas de Membrana/metabolismo , Mutação , Domínios Proteicos , Transporte Proteico/genética , Salmonella/genética , Salmonella/metabolismo , Salmonella typhi/metabolismo , Especificidade por Substrato
6.
J Biotechnol ; 296: 22-31, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30878516

RESUMO

In previous studies of Lactococcus lactis, the levels of proteins secreted using heterologous signal peptides were observed to be lower than those obtained using the signal peptide from Usp45, the major secreted lactococcal protein. In this study, G1 (the native signal peptide of CGTase) and the signal peptide M5 (mutant of the G1 signal peptide) were introduced into L. lactis to investigate the effect of signal peptides on lactococcal protein secretion to improve secretion efficiency. The effectiveness of these signal peptides were compared to the Usp45 signal peptide. The highest secretion levels were obtained using the G1 signal peptide. Sequence analysis of signal peptide amino acids revealed that a basic N-terminal signal peptide is not absolutely required for efficient protein export in L. lactis. Moreover, the introduction of a helix-breaking residue in the H-region of the M5 signal peptide caused a reduction in the signal peptide hydrophobicity and decreased protein secretion. In addition, the optimization of cultivation conditions for recombinant G1-CGTase production via response surface methodology (RSM) showed that CGTase activity increased approximately 2.92-fold from 5.01 to 16.89 U/ml compared to the unoptimized conditions.


Assuntos
Proteínas de Bactérias/genética , Glucosiltransferases/efeitos dos fármacos , Lactococcus lactis/enzimologia , Sinais Direcionadores de Proteínas/genética , Proteínas de Bactérias/química , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Transporte Proteico/genética
7.
Orphanet J Rare Dis ; 14(1): 63, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30832686

RESUMO

BACKGROUND: y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein. RESULTS: System y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants. CONCLUSIONS: The three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Cadeias Leves da Proteína-1 Reguladora de Fusão/genética , Cadeias Leves da Proteína-1 Reguladora de Fusão/metabolismo , Mutação , Transporte Proteico/genética , Adulto , Animais , Arginina/metabolismo , Células CHO , Células Cultivadas , Criança , Pré-Escolar , Cricetulus , Citosol/metabolismo , Feminino , Humanos , Masculino , Monócitos
8.
Genes Dev ; 33(9-10): 536-549, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30842217

RESUMO

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.


Assuntos
Exossomos/genética , Exossomos/metabolismo , Proteínas Nucleares/fisiologia , RNA Helicases/metabolismo , Animais , Núcleo Celular/metabolismo , Células-Tronco Embrionárias , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Transporte Proteico/genética , Estabilidade de RNA/genética
9.
World J Microbiol Biotechnol ; 35(4): 54, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30900052

RESUMO

Filamentous fungi are important microorganisms used in industrial production of proteins and enzymes. Among these organisms, Trichoderma reesei, Aspergilli, and more recently Myceliophthora thermophile are the most widely used and promising ones which have powerful protein secretion capability. In recent years, there have been tremendous achievements in understanding the molecular mechanisms of the secretory pathways in filamentous fungi. The acquired pieces of knowledge can be harnessed to enhance protein production in filamentous fungi with assistance of state-of-the-art genetic engineering techniques.


Assuntos
Proteínas Fúngicas/biossíntese , Fungos/metabolismo , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Aspergillus/metabolismo , Códon , Proteínas Fúngicas/genética , Fungos/genética , Fungos/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Glicosilação , Peptídeos/metabolismo , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Saccharomycetales/metabolismo , Via Secretória/genética , Trichoderma/metabolismo
10.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837368

RESUMO

Autophagy is a highly conserved intracellular degradation pathway in eukaryotic cells that responds to environmental changes. Genetic analyses have shown that more than 40 autophagy-related genes (ATG) are directly involved in this process in fungi. In addition to Atg proteins, most vesicle transport regulators are also essential for each step of autophagy. The present study showed that one Endoplasmic Reticulum protein in Saccharomyces cerevisiae, Tip20, which controls Golgi-to-ER retrograde transport, was also required for starvation-induced autophagy under high temperature stress. In tip20 conditional mutant yeast, the transport of Atg8 was impaired during starvation, resulting in multiple Atg8 puncta dispersed outside the vacuole that could not be transported to the pre-autophagosomal structure/phagophore assembly site (PAS). Several Atg8 puncta were trapped in ER exit sites (ERES). Moreover, the GFP-Atg8 protease protection assay indicated that Tip20 functions before autophagosome closure. Furthermore, genetic studies showed that Tip20 functions downstream of Atg5 and upstream of Atg1, Atg9 and Atg14 in the autophagy pathway. The present data show that Tip20, as a vesicle transport regulator, has novel roles in autophagy.


Assuntos
Autofagia/genética , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteína 5 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Proteínas de Membrana/genética , Mutação , Fagossomos/genética , Proteínas Quinases/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo
11.
Biomed Res Int ; 2019: 2436924, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834257

RESUMO

The prediction of apoptosis protein subcellular localization plays an important role in understanding the progress in cell proliferation and death. Recently computational approaches to this issue have become very popular, since the traditional biological experiments are so costly and time-consuming that they cannot catch up with the growth rate of sequence data anymore. In order to improve the prediction accuracy of apoptosis protein subcellular localization, we proposed a sparse coding method combined with traditional feature extraction algorithm to complete the sparse representation of apoptosis protein sequences, using multilayer pooling based on different sizes of dictionaries to integrate the processed features, as well as oversampling approach to decrease the influences caused by unbalanced data sets. Then the extracted features were input to a support vector machine to predict the subcellular localization of the apoptosis protein. The experiment results obtained by Jackknife test on two benchmark data sets indicate that our method can significantly improve the accuracy of the apoptosis protein subcellular localization prediction.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Biologia Computacional , Algoritmos , Sequência de Aminoácidos/genética , Proteínas Reguladoras de Apoptose/ultraestrutura , Proliferação de Células/genética , Bases de Dados de Proteínas , Humanos , Transporte Proteico/genética , Máquina de Vetores de Suporte
12.
Neurochem Res ; 44(6): 1367-1374, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30796750

RESUMO

Cell surface ß-Amyloid precursor protein (APP) is known to have a functional role in iron homeostasis through stabilising the iron export protein ferroportin (FPN). Mechanistic evidence of this role has previously only been provided through transcriptional or translational depletion of total APP levels. However, numerous post-translational modifications of APP are reported to regulate the location and trafficking of this protein to the cell surface. Stable overexpressing cell lines were generated that overexpressed APP with disrupted N-glycosylation (APPN467K and APPN496K) or ectodomain phosphorylation (APPS206A); sites selected for their proximity to the FPN binding site on the E2 domain of APP. We hypothesise that impaired N-glycosylation or phosphorylation of APP disrupts the functional location on the cell surface or binding to FPN to consequentially alter intracellular iron levels through impaired cell surface FPN stability. Outcomes confirm that these post-translational modifications are essential for the correct location of APP on the cell surface and highlight a novel mechanism by which the cell can modulate iron homeostasis. Further interrogation of other post-translational processes to APP is warranted in order to fully understand how each modification plays a role on regulating intracellular iron levels in health and disease.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Glicosilação , Camundongos , Fosforilação/genética , Mutação Puntual , Processamento de Proteína Pós-Traducional/genética , Transporte Proteico/genética
13.
Rev. neurol. (Ed. impr.) ; 68(4): 155-159, 16 feb., 2019. tab, ilus, graf
Artigo em Espanhol | IBECS | ID: ibc-180706

RESUMO

Introducción. La enfermedad CLN8 es uno de los 13 tipos genéticos reconocidos de lipofuscinosis neuronal ceroidea, un grupo de trastornos neurodegenerativos de acumulación lisosómica, los más frecuentes en la infancia. La causan mutaciones en la proteína transmembrana CLN8 de 286 aminoácidos, cuya función se desconoce. Las variantes patológicas en el gen CLN8 se asociaron con dos fenotipos diferentes: la variante infantil tardía en individuos de diversos países alrededor del mundo, y la epilepsia progresiva con retraso mental, que aparece en pacientes finlandeses y turcos. Caso clínico. Niña que mostró retraso psicomotor y demencia desde el nacimiento, convulsiones tonicoclónicas, mioclonía, ataxia con atrofia cerebelosa y muerte temprana a los 12 años. La microscopia electrónica de la piel mostró una mezcla de citosomas con patrones de depósitos osmiofílicos granulares, curvilíneos y de «huella digital», y mitocondrias hipertrofiadas. Se encontraron dos variantes patológicas de ADN en el gen CLN8 (exón 2 c.1A>G; p.?/ exón 3 c.792C>G; p.Asn264Lys), lo que confirmó un genotipo heterocigoto compuesto. Conclusión. Éste es el caso índice en América Latina para el nuevo fenotipo congénito de la enfermedad CLN8. La sospecha de esta patología debería sustentarse genéticamente en casos de síndrome neurodegenerativo con retraso psicomotor desde el nacimiento, dificultad del habla y convulsiones. El curso clínico incluye ataxia, atrofia cerebelosa y muerte temprana


Introduction. CLN8 disease is one of the thirteen recognized genetic types of neuronal ceroid lipofuscinosis, a group of neurodegenerative lysosomal storage disorders, most frequent in childhood. A putative 286 amino acids transmembrane CLN8 protein with unknown function is affected. Pathological variants in the CLN8 gene were associated with two different phenotypes: variant late-infantile in individuals from many countries worldwide, and epilepsy progressive with mental retardation, appearing in Finnish and Turkish subjects. Case report. The girl showed psychomotor delay and dementia since birth, tonic-clonic seizures, myoclonus, ataxia with cerebellar atrophy, and early death at 12 years old. Electron microscopy of the skin showed mixed GROD, curvilinear, fingerprint cytosomes and mitochondrial hypertrophy. Two pathological DNA variants in the CLN8 gene (exon 2 c.1A>G; p.?/ exon 3 c.792C>G; p.Asn264Lys) were found confirming a compound heterozygous genotype. Conclusion. This case is the Latin American index for a new congenital phenotype of the CLN8 disease. The congenital phenotype has to be added to the clinical spectrum of the CLN8 disease. The suspicion of CLN8 disease should be genetically sustained in challenging cases of a neurodegenerative syndrome with psychomotor delay since birth, speech difficulty and seizures. The course includes ataxia, cerebellar atrophy, and early death


Assuntos
Humanos , Feminino , Criança , Lipofuscinoses Ceroides Neuronais/diagnóstico por imagem , Lipofuscinoses Ceroides Neuronais/genética , Transporte Proteico/genética , Insuficiência de Crescimento/genética , Imagem por Ressonância Magnética/métodos , Microscopia Eletrônica/métodos
14.
Biochem J ; 476(1): 25-37, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30617163

RESUMO

The mitochondrial proteome is estimated to contain ∼1100 proteins, the vast majority of which are nuclear-encoded, with only 13 proteins encoded by the mitochondrial genome. The import of these nuclear-encoded proteins into mitochondria was widely believed to be unidirectional, but recent discoveries have revealed that many these 'mitochondrial' proteins are exported, and have extra-mitochondrial activities divergent from their mitochondrial function. Surprisingly, three of the exported proteins discovered thus far are mitochondrially encoded and have significantly different extra-mitochondrial roles than those performed within the mitochondrion. In this review, we will detail the wide variety of proteins once thought to only reside within mitochondria, but now known to 'emigrate' from mitochondria in order to attain 'dual citizenship', present both within mitochondria and elsewhere.


Assuntos
Núcleo Celular , DNA Mitocondrial , Genoma Mitocondrial , Mitocôndrias , Proteínas Mitocondriais , Proteoma , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transporte Proteico/genética , Proteoma/genética , Proteoma/metabolismo
15.
PLoS Genet ; 15(1): e1007955, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30699121

RESUMO

The Drosophila protocadherins Dachsous and Fat regulate growth and tissue polarity by modulating the levels, membrane localization and polarity of the atypical myosin Dachs. Localization to the apical junctional membrane is critical for Dachs function, and the adapter protein Vamana/Dlish and palmitoyl transferase Approximated are required for Dachs membrane localization. However, how Dachs levels are regulated is poorly understood. Here we identify the early girl gene as playing an essential role in Fat signaling by limiting the levels of Dachs protein. early girl mutants display overgrowth of the wings and reduced cross vein spacing, hallmark features of mutations affecting Fat signaling. Genetic experiments reveal that it functions in parallel with Fat to regulate Dachs. early girl encodes an E3 ubiquitin ligase, physically interacts with Dachs, and regulates its protein stability. Concomitant loss of early girl and approximated results in accumulation of Dachs and Vamana in cytoplasmic punctae, suggesting that it also regulates their trafficking to the apical membrane. Our findings establish a crucial role for early girl in Fat signaling, involving regulation of Dachs and Vamana, two key downstream effectors of this pathway.


Assuntos
Aciltransferases/genética , Moléculas de Adesão Celular/genética , Proteínas de Drosophila/genética , Ubiquitina-Proteína Ligases/genética , Animais , Polaridade Celular/genética , Drosophila melanogaster/genética , Humanos , Proteínas de Membrana/genética , Mutação , Miosinas/genética , Transporte Proteico/genética , Transdução de Sinais , Asas de Animais/crescimento & desenvolvimento
16.
Genes Dev ; 33(1-2): 75-89, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30567999

RESUMO

Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins.


Assuntos
Biotinilação/métodos , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Fatores de Transcrição/metabolismo , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Técnicas de Silenciamento de Genes , Humanos , Proteína Homóloga a MRE11/metabolismo , Ligação Proteica , Transporte Proteico/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
17.
Oncogene ; 38(16): 3019-3032, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30575817

RESUMO

Long noncoding RNAs (lncRNAs) are pervasive transcripts that play pivotal roles in regulating chromatin dynamics, gene and protein expression. Aberrant expression and mutations of lncRNAs represent a driving force behind tumor invasion and metastasis, making them attractive cancer targets. However, most of the lncRNAs are still being discovered and conclusive experimental evidence for their functional relevance is still lacking for most malignancies. In this study, a differentially expressed lncRNA, designated as lnc-CRCMSL, is identified by microarray-based screenings on non-metastatic and metastatic CRC specimens. Lnc-CRCMSL is verified as an anti-metastatic gene and negatively correlated with the poor prognosis of CRC patients. Lnc-CRCMSL overexpression restricts tumor growth and metastasis in vivo and in vitro. Instead, lnc-CRCMSL silencing accelerates CRC cell proliferation and migration. RNA-pulldown assay identifies high mobility group box 2 (HMGB2) as a downstream protein of lnc-CRCMSL. Mechanically, lnc-CRCMSL physically binds to HMGB2 and stabilizes the localization of HMGB2 in the cytoplasm. Notably, lnc-CRCMSL knockdown lead to the shift of HMGB2 into nuclear, in which it triggers epithelial to mesenchymal transition (EMT) programming. Importantly, lnc-CRCMSL controls the cytoplasmic retention of HMGB2 and attenuates the interaction between HMGB2 and OCT4 to suppress EMT. Treatment of leptomycin B (LMB), a potent and specific nuclear export inhibitor, counteracts lnc-CRCMSL-mediated suppression of aggressive phenotypes and EMT process by accumulating the nuclear HMGB2. CONCLUSION: Our data highlight the anti-metastatic role of lnc-CRCMSL in stabilizing HMGB2 through lncRNA-protein interactions in the cytoplasm, and suggest that targeting lnc-CRCMSL may represent a therapeutic opportunity for managing metastatic CRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteína HMGB2/genética , Transporte Proteico/genética , RNA Longo não Codificante/genética , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/fisiologia , Proliferação de Células/genética , Citoplasma/fisiologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
18.
Microbiol Res ; 218: 97-107, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30454663

RESUMO

In Escherichia coli, the Twin-arginine translocation (Tat) pathway secretes a set of folded proteins with important physiological functions to the periplasm and outer membrane. The loss of Tat secretion impairs outer membrane integrity and leads to decreased cell growth. Only recently, the Tat pathway has gained more attention due to its essential role in bacterial virulence and applications in the production of fully folded heterologous proteins. In this study, we investigated the influence of the deletion of all active Tat pathway components on the E. coli cells. The comprehensive proteomic analysis revealed activation of several stress responses and experimentally confirmed the dependence of certain proteins on the Tat system for export. We observed that a tat deletion triggers protein aggregation, membrane vesiculation, synthesis of colanic acid and biofilm formation. Furthermore, the mislocalization of Tat-dependent proteins disturbs iron and molybdenum homeostasis and impairs the cell envelope integrity. The results show that the functional Tat pathway is important for the physiological stability and that its dysfunction leads to a series of severe changes in E. coli cells.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Transporte Proteico/genética , Estresse Fisiológico/fisiologia , Sistema de Translocação de Argininas Geminadas/genética , Membrana Celular/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Periplasma/metabolismo , Transporte Proteico/fisiologia , Proteoma/metabolismo
19.
Microb Cell Fact ; 17(1): 199, 2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30577801

RESUMO

BACKGROUND: The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity. RESULTS: Here we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres. CONCLUSIONS: The methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.


Assuntos
Expressão Gênica/genética , Transporte Proteico/genética , Proteínas Recombinantes/metabolismo
20.
J Vis Exp ; (137)2018 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-30102267

RESUMO

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and γ-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.


Assuntos
Caenorhabditis elegans/metabolismo , Indóis/uso terapêutico , Transporte Proteico/genética , Coloração e Rotulagem/métodos , Animais , Proteínas de Fluorescência Verde/farmacologia , Indóis/farmacologia
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