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[PMID]:29187139
[Au] Autor:Calyseva J; Vihinen M
[Ad] Endereço:Protein Structure and Bioinformatics, Department of Experimental Medical Science, Lund University, BMC B13, SE-22 184, Lund, Sweden.
[Ti] Título:PON-SC - program for identifying steric clashes caused by amino acid substitutions.
[So] Source:BMC Bioinformatics;18(1):531, 2017 Nov 29.
[Is] ISSN:1471-2105
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Amino acid substitutions due to DNA nucleotide replacements are frequently disease-causing because of affecting functionally important sites. If the substituting amino acid does not fit into the protein, it causes structural alterations that are often harmful. Clashes of amino acids cause local or global structural changes. Testing structural compatibility of variations has been difficult due to the lack of a dedicated method that could handle vast amounts of variation data produced by next generation sequencing technologies. RESULTS: We developed a method, PON-SC, for detecting protein structural clashes due to amino acid substitutions. The method utilizes side chain rotamer library and tests whether any of the common rotamers can be fitted into the protein structure. The tool was tested both with variants that cause and do not cause clashes and found to have accuracy of 0.71 over five test datasets. CONCLUSIONS: We developed a fast method for residue side chain clash detection. The method provides in addition to the prediction also visualization of the variant in three dimensional structure.
[Mh] Termos MeSH primário: Aminoácidos/química
Proteínas/química
Software
[Mh] Termos MeSH secundário: Algoritmos
Substituição de Aminoácidos
Aminoácidos/metabolismo
Bases de Dados de Proteínas
Conformação Proteica
Engenharia de Proteínas/métodos
Proteínas/genética
Proteínas/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Amino Acids); 0 (Proteins)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180306
[Lr] Data última revisão:
180306
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171201
[St] Status:MEDLINE
[do] DOI:10.1186/s12859-017-1947-7


  2 / 15265 MEDLINE  
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[PMID]:28466278
[Au] Autor:Yu X; Marshall MJE; Cragg MS; Crispin M
[Ad] Endereço:The Antibody and Vaccine Group, Cancer Sciences Unit, University of Southampton, Faculty of Medicine, General Hospital, Southampton, SO16 6YD, UK. x.yu@soton.ac.uk.
[Ti] Título:Improving Antibody-Based Cancer Therapeutics Through Glycan Engineering.
[So] Source:BioDrugs;31(3):151-166, 2017 Jun.
[Is] ISSN:1179-190X
[Cp] País de publicação:New Zealand
[La] Idioma:eng
[Ab] Resumo:Antibody-based therapeutics has emerged as a major tool in cancer treatment. Guided by the superb specificity of the antibody variable domain, it allows the precise targeting of tumour markers. Recently, eliciting cellular effector functions, mediated by the Fc domain, has gained traction as a means by which to generate more potent antibody therapeutics. Extensive mutagenesis studies of the Fc protein backbone has enabled the generation of Fc variants that more optimally engage the Fcγ receptors known to mediate cellular effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and cellular phagocytosis. In addition to the protein backbone, the homodimeric Fc domain contains two opposing N-linked glycans, which represent a further point of potential immunomodulation, independent of the Fc protein backbone. For example, a lack of core fucose usually attached to the IgG Fc glycan leads to enhanced ADCC activity, whereas a high level of terminal sialylation is associated with reduced inflammation. Significant growth in knowledge of Fc glycosylation over the last decade, combined with advancement in genetic engineering, has empowered glyco-engineering to fine-tune antibody therapeutics. This has culminated in the approval of two glyco-engineered antibodies for cancer therapy: the anti-CCR4 mogamulizumab approved in 2012 and the anti-CD20 obinutuzumab in 2013. We discuss here the technological platforms for antibody glyco-engineering and review the current clinical landscape of glyco-engineered antibodies.
[Mh] Termos MeSH primário: Anticorpos Monoclonais/farmacologia
Anticorpos Monoclonais/uso terapêutico
Neoplasias/tratamento farmacológico
Polissacarídeos/imunologia
[Mh] Termos MeSH secundário: Animais
Anticorpos Monoclonais/imunologia
Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos
Seres Humanos
Imunomodulação/efeitos dos fármacos
Engenharia de Proteínas/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Antibodies, Monoclonal); 0 (Polysaccharides)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180306
[Lr] Data última revisão:
180306
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170504
[St] Status:MEDLINE
[do] DOI:10.1007/s40259-017-0223-8


  3 / 15265 MEDLINE  
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[PMID]:27778166
[Au] Autor:Fan R; Yuan Y; Zhang Q; Zhou XR; Jia L; Liu Z; Yu C; Luo SZ; Chen L
[Ad] Endereço:Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, People's Republic of China.
[Ti] Título:Isoleucine/leucine residues at "a" and "d" positions of a heptad repeat sequence are crucial for the cytolytic activity of a short anticancer lytic peptide.
[So] Source:Amino Acids;49(1):193-202, 2017 01.
[Is] ISSN:1438-2199
[Cp] País de publicação:Austria
[La] Idioma:eng
[Ab] Resumo:Many lytic peptides contain a heptad sequence with leucine or isoleucine residues at "a" and "d" positions. However, their roles in the peptide-induced cytolytic process remain unclear. We have recently reported an anticancer lytic peptide ZXR-2 (FKIGGFIKKLWRSLLA), which contains a shortened zipper-like sequence with Ile/Leu at "a" and "d" positions. To understand the roles of these Ile/Leu residues, a series of analogs were constructed by sequentially replacing the Ile or Leu residue with alanine (Ala). Significant reduction of the cytolytic activity was observed when the Ile (3rd and 7th) and Leu (10th and 14th) residues at the "a" and "d" positions were substituted, while the replacement of the separate Leu (15th) residue had less effect. Based on the quenching of the intrinsic fluorescence of the peptides and their induced surface pressure changes of lipid monolayer, it was conjectured that the peptide ZXR-2 might insert into cell membranes from the C-terminal and to a depth of the W position. Accordingly, I , I , and L residues which mainly exposed in aqueous solution were more responsible for the peptide self-association on cell membranes, while L , together with L , might help peptide insert and anchor to cell membranes. These results are significant to elucidate the crucial roles of such Ile/Leu residues at "a" and "d" positions in peptide-peptide and peptide-membrane interactions to exert the membrane disruption activity of lytic peptides. With further understanding about the structure-activity relationship of lytic peptides, it would be helpful for designing novel anticancer lytic peptides.
[Mh] Termos MeSH primário: Antineoplásicos/farmacologia
Isoleucina/química
Leucina/química
Peptídeos/farmacologia
[Mh] Termos MeSH secundário: 1,2-Dipalmitoilfosfatidilcolina/análogos & derivados
1,2-Dipalmitoilfosfatidilcolina/química
Alanina/química
Sequência de Aminoácidos
Substituição de Aminoácidos
Antineoplásicos/síntese química
Linhagem Celular Tumoral
Membrana Celular/efeitos dos fármacos
Sobrevivência Celular/efeitos dos fármacos
Colesterol/química
Células HEK293
Células HeLa
Seres Humanos
L-Lactato Desidrogenase/secreção
Lipossomos/química
Peptídeos/síntese química
Fosfatidilserinas/química
Engenharia de Proteínas
Estrutura Secundária de Proteína
Eletricidade Estática
Relação Estrutura-Atividade
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Antineoplastic Agents); 0 (Liposomes); 0 (Peptides); 0 (Phosphatidylserines); 04Y7590D77 (Isoleucine); 2644-64-6 (1,2-Dipalmitoylphosphatidylcholine); 3036-82-6 (dipalmitoylphosphatidylserine); 319X2NFW0A (colfosceril palmitate); 97C5T2UQ7J (Cholesterol); EC 1.1.1.27 (L-Lactate Dehydrogenase); GMW67QNF9C (Leucine); OF5P57N2ZX (Alanine)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180305
[Lr] Data última revisão:
180305
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161026
[St] Status:MEDLINE
[do] DOI:10.1007/s00726-016-2350-9


  4 / 15265 MEDLINE  
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[PMID]:28458031
[Au] Autor:Yin L; Yuvienco C; Montclare JK
[Ad] Endereço:Department of Chemical and Biomolecular Engineering, NYU Tandon School of Engineering, Brooklyn, NY 11201, United States.
[Ti] Título:Protein based therapeutic delivery agents: Contemporary developments and challenges.
[So] Source:Biomaterials;134:91-116, 2017 Jul.
[Is] ISSN:1878-5905
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:As unique biopolymers, proteins can be employed for therapeutic delivery. They bear important features such as bioavailability, biocompatibility, and biodegradability with low toxicity serving as a platform for delivery of various small molecule therapeutics, gene therapies, protein biologics and cells. Depending on size and characteristic of the therapeutic, a variety of natural and engineered proteins or peptides have been developed. This, coupled to recent advances in synthetic and chemical biology, has led to the creation of tailor-made protein materials for delivery. This review highlights strategies employing proteins to facilitate the delivery of therapeutic matter, addressing the challenges for small molecule, gene, protein and cell transport.
[Mh] Termos MeSH primário: Materiais Biocompatíveis/química
Biopolímeros/química
Proteínas/química
[Mh] Termos MeSH secundário: Sistemas de Liberação de Medicamentos/métodos
Terapia Genética/métodos
Nanotecnologia/métodos
Engenharia de Proteínas/métodos
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Biocompatible Materials); 0 (Biopolymers); 0 (Proteins)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180228
[Lr] Data última revisão:
180228
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170502
[St] Status:MEDLINE


  5 / 15265 MEDLINE  
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[PMID]:29251421
[Au] Autor:Muñoz-López Á; Summerer D
[Ad] Endereço:Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 4a, 44227, Dortmund.
[Ti] Título:Recognition of Oxidized 5-Methylcytosine Derivatives in DNA by Natural and Engineered Protein Scaffolds.
[So] Source:Chem Rec;18(1):105-116, 2018 Jan.
[Is] ISSN:1528-0691
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Methylation of genomic cytosine to 5-methylcytosine is a central regulatory element of mammalian gene expression with important roles in development and disease. 5-methylcytosine can be actively reversed to cytosine via oxidation to 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine by ten-eleven-translocation dioxygenases and subsequent base excision repair or replication-dependent dilution. Moreover, the oxidized 5-methylcytosine derivatives are potential epigenetic marks with unique biological roles. Key to a better understanding of these roles are insights into the interactions of the nucleobases with DNA-binding protein scaffolds: Natural scaffolds involved in transcription, 5-methylcytosine-reading and -editing as well as general chromatin organization can be selectively recruited or repulsed by oxidized 5-methylcytosines, forming the basis of their biological functions. Moreover, designer protein scaffolds engineered for the selective recognition of oxidized 5-methylcytosines are valuable tools to analyze their genomic levels and distribution. Here, we review recent structural and functional insights into the molecular recognition of oxidized 5-methylcytosine derivatives in DNA by selected protein scaffolds.
[Mh] Termos MeSH primário: 5-Metilcitosina/química
DNA/química
Proteínas/química
Proteínas/síntese química
[Mh] Termos MeSH secundário: 5-Metilcitosina/metabolismo
DNA/metabolismo
Oxirredução
Engenharia de Proteínas
Proteínas/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Proteins); 6R795CQT4H (5-Methylcytosine); 9007-49-2 (DNA)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171219
[St] Status:MEDLINE
[do] DOI:10.1002/tcr.201700088


  6 / 15265 MEDLINE  
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[PMID]:29377907
[Au] Autor:Pezeshgi Modarres H; Mofrad MR; Sanati-Nezhad A
[Ad] Endereço:BioMEMS and Bioinspired Microfluidic Laboratory, Department of Mechanical and Manufacturing Engineering, University of Calgary, Calgary, Alberta, Canada.
[Ti] Título:ProtDataTherm: A database for thermostability analysis and engineering of proteins.
[So] Source:PLoS One;13(1):e0191222, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Protein thermostability engineering is a powerful tool to improve resistance of proteins against high temperatures and thereafter broaden their applications. For efficient protein thermostability engineering, different thermostability-classified data sources including sequences and 3D structures are needed for different protein families. However, no data source is available providing such data easily. It is the first release of ProtDataTherm database for analysis and engineering of protein thermostability which contains more than 14 million protein sequences categorized based on their thermal stability and protein family. This database contains data needed for better understanding protein thermostability and stability engineering. Providing categorized protein sequences and structures as psychrophilic, mesophilic and thermophilic makes this database useful for the development of new tools in protein stability prediction. This database is available at http://profiles.bs.ipm.ir/softwares/protdatatherm. As a proof of concept, the thermostability that improves mutations were suggested for one sample protein belonging to one of protein families with more than 20 mesophilic and thermophilic sequences and with known experimentally measured ΔT of mutations available within ProTherm database.
[Mh] Termos MeSH primário: Bases de Dados de Proteínas
Engenharia de Proteínas
Estabilidade Proteica
[Mh] Termos MeSH secundário: Algoritmos
Sequência de Aminoácidos
Proteínas de Bactérias/química
Proteínas de Bactérias/genética
Temperatura Alta
Modelos Moleculares
Mutação
Termodinâmica
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180130
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0191222


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[PMID]:29373591
[Au] Autor:Xu K; Zhao Q; Wen X; Wu R; Wen Y; Huang X; Huang Y; Yan Q; Han X; Ma X; Chang YF; Cao S
[Ad] Endereço:Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
[Ti] Título:A trivalent Apx-fusion protein delivered by E. coli outer membrane vesicles induce protection against Actinobacillus pleuropneumoniae of serotype 1 and 7 challenge in a murine model.
[So] Source:PLoS One;13(1):e0191286, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Actinobacillus pleuropneumoniae (APP) causes serious economic losses in the swine industry, and is the etiologic agent of porcine pleuropneumonia. In this study we have engineered a trivalent Apx fusion protein enclosed in outer membrane vesicles (Apxr-OMV) and studied its immunoprotective efficacy against APP serotypes 1 and 7 challenge in mice. The results showed that the IgG levels in the Apxr-OMVs immune group were significantly higher than those of the negative control (P < 0.05). Up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokines were detected in splenocytes of Apxr-OMVs immune group. The survival rates 87.5% and 62.5% were observed against APP strain 1516 of serotype 7 and APP strain 2701 of serotype 1 in the groups of Apxr-OMVs immune group, respectively. Histopathological lesions of the pulmonary structure alveoli were found to be minimal in APX-OMV group challenged with APP serotypes 1 and 7. These results strongly indicated that engineered OMVs could effectively induce specific humoral or cellular immune responses. Moreover, Apxr-OMVs used as novel vaccine provides cross-protective immunity against different serotype 1 and 7 of APP infection in a mouse model. In contrast, the OMV-empty and PBS as negative controls or inactivated strain of APP-2701 and APP-1516 as positive controls for the animal study cannot provide protection or cross-protection.
[Mh] Termos MeSH primário: Actinobacillus pleuropneumoniae/fisiologia
Vacinas Bacterianas/imunologia
Membrana Celular/metabolismo
Escherichia coli/citologia
Escherichia coli/genética
Engenharia de Proteínas
Proteínas Recombinantes de Fusão/imunologia
[Mh] Termos MeSH secundário: Actinobacillus pleuropneumoniae/imunologia
Animais
Vacinas Bacterianas/genética
Proliferação Celular
Citocinas/metabolismo
Feminino
Imunidade Celular
Linfócitos/citologia
Camundongos
Camundongos Endogâmicos BALB C
Proteínas Recombinantes de Fusão/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Vaccines); 0 (Cytokines); 0 (Recombinant Fusion Proteins)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180127
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0191286


  8 / 15265 MEDLINE  
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[PMID]:29352320
[Au] Autor:Zhou X; Desai R; Zhang Y; Stec WJ; Miller KW; Jounaidi Y
[Ad] Endereço:Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
[Ti] Título:High-level production and purification in a functional state of an extrasynaptic gamma-aminobutyric acid type A receptor containing α4ß3δ subunits.
[So] Source:PLoS One;13(1):e0191583, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The inhibitory γ-aminobutyric acid type A receptors are implicated in numerous physiological processes, including cognition and inhibition of neurotransmission, rendering them important molecular targets for many classes of drugs. Functionally, the entire GABAAR family of receptors can be subdivided into phasic, fast acting synaptic receptors, composed of α-, ß- and γ-subunits, and tonic extrasynaptic receptors, many of which contain the δ-subunit in addition to α- and ß-subunits. Whereas the subunit arrangement of the former group is agreed upon, that of the αßδ GABAARs remains unresolved by electrophysiological and pharmacological research. To resolve such issues will require biophysical techniques that demand quantities of receptor that have been previously unavailable. Therefore, we have engineered a stable cell line with tetracycline inducible expression of human α4-, ß3- and N-terminally Flag-tagged δ-subunits. This cell line achieved a specific activity between 15 and 20 pmol [3H]muscimol sites/mg of membrane protein, making it possible to obtain 1 nmole of purified α4ß3δ GABAAR from sixty 15-cm culture dishes. When induced, these cells exhibited agonist-induced currents with characteristics comparable to those previously reported for this receptor and a pharmacology that included strong modulation by etomidate and the δ-subunit-specific ligand, DS2. Immunoaffinity purification and reconstitution in CHAPS/asolectin micelles resulted in the retention of equilibrium allosteric interactions between the separate agonist, anesthetic and DS2 sites. Moreover, all three subunits retained glycosylation. The establishment of this well-characterized cell line will allow molecular level studies of tonic receptors to be undertaken.
[Mh] Termos MeSH primário: Receptores de GABA-A/biossíntese
[Mh] Termos MeSH secundário: Fenômenos Eletrofisiológicos
Células HEK293
Seres Humanos
Cinética
Engenharia de Proteínas
Subunidades Proteicas
Ensaio Radioligante
Receptores de GABA-A/genética
Receptores de GABA-A/isolamento & purificação
Proteínas Recombinantes de Fusão/biossíntese
Proteínas Recombinantes de Fusão/genética
Proteínas Recombinantes de Fusão/isolamento & purificação
Transfecção
Ácido gama-Aminobutírico/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Protein Subunits); 0 (Receptors, GABA-A); 0 (Recombinant Fusion Proteins); 56-12-2 (gamma-Aminobutyric Acid)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180121
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0191583


  9 / 15265 MEDLINE  
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[PMID]:29329339
[Au] Autor:Granger BL
[Ad] Endereço:Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, United States of America.
[Ti] Título:Accessibility and contribution to glucan masking of natural and genetically tagged versions of yeast wall protein 1 of Candida albicans.
[So] Source:PLoS One;13(1):e0191194, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Yeast wall protein 1 (Ywp1) is an abundant glycoprotein of the cell wall of the yeast form of Candida albicans, the most prevalent fungal pathogen of humans. Antibodies that bind to the polypeptide backbone of isolated Ywp1 show little binding to intact yeast cells, presumably because the Ywp1 epitopes are masked by the polysaccharides of the mannoproteins that form the outer layer of the cell wall. Rare cells do exhibit much greater anti-Ywp1 binding, however, and one of these was isolated and characterized. No differences were seen in its Ywp1, but it exhibited greater adhesiveness, sensitivity to wall perturbing agents, and exposure of its underlying ß-1,3-glucan layer to external antibodies. The molecular basis for this greater epitope accessibility has not been determined, but has facilitated exploration of how these properties change as a function of cell growth and morphology. In addition, previously engineered strains with reduced quantities of Ywp1 in their cell walls were also found to have greater ß-1,3-glucan exposure, indicating that Ywp1 itself contributes to the masking of wall epitopes, which may be important for understanding the anti-adhesive effect of Ywp1. Ectopic production of Ywp1 by hyphae, which reduces the adhesivity of these filamentous forms of C. albicans, was similarly found to reduce exposure of the ß-1,3-glucan in their walls. To monitor Ywp1 in the cell wall irrespective of its accessibility, green fluorescent protein (Gfp) was genetically inserted into wall-anchored Ywp1 using a bifunctional cassette that also allowed production from a single transfection of a soluble, anchor-free version. The wall-anchored Ywp1-Gfp-Ywp1 accumulated in the wall of the yeast forms but not hyphae, and appeared to have properties similar to native Ywp1, including its adhesion-inhibiting effect. Some pseudohyphal walls also detectably accumulated this probe. Strains of C. albicans with tandem hemagglutinin (HA) epitopes inserted into wall-anchored Ywp1 were previously created by others, and were further explored here. As above, rare cells with much greater accessibility of the HA epitopes were isolated, and also found to exhibit greater exposure of Ywp1 and ß-1,3-glucan. The placement of the HA cassette inhibited the normal N-glycosylation and propeptide cleavage of Ywp1, but the wall-anchored Ywp1-HA-Ywp1 still accumulated in the cell wall of yeast forms. Bifunctional transformation cassettes were used to additionally tag these molecules with Gfp, generating soluble Ywp1-HA-Gfp and wall-anchored Ywp1-HA-Gfp-Ywp1 molecules. The former revealed unexpected electrophoretic properties caused by the HA insertion, while the latter further highlighted differences between the presence of a tagged Ywp1 molecule (as revealed by Gfp fluorescence) and its accessibility in the cell wall to externally applied antibodies specific for HA, Gfp and Ywp1, with accessibility being greatest in the rapidly expanding walls of budding daughter cells. These strains and results increase our understanding of cell wall properties and how C. albicans masks itself from recognition by the human immune system.
[Mh] Termos MeSH primário: Candida albicans/genética
Candida albicans/metabolismo
Proteínas Fúngicas/genética
Proteínas Fúngicas/metabolismo
beta-Glucanas/metabolismo
[Mh] Termos MeSH secundário: Anticorpos Antifúngicos
Antígenos de Fungos/química
Antígenos de Fungos/genética
Antígenos de Fungos/metabolismo
Candida albicans/imunologia
Parede Celular/genética
Parede Celular/imunologia
Parede Celular/metabolismo
Epitopos/química
Epitopos/genética
Epitopos/metabolismo
Proteínas Fúngicas/imunologia
Glicosilação
Proteínas de Fluorescência Verde/genética
Proteínas de Fluorescência Verde/imunologia
Proteínas de Fluorescência Verde/metabolismo
Hemaglutininas/genética
Hemaglutininas/imunologia
Hemaglutininas/metabolismo
Seres Humanos
Glicoproteínas de Membrana/genética
Glicoproteínas de Membrana/imunologia
Glicoproteínas de Membrana/metabolismo
Engenharia de Proteínas
Proteínas Recombinantes de Fusão/genética
Proteínas Recombinantes de Fusão/imunologia
Proteínas Recombinantes de Fusão/metabolismo
beta-Glucanas/química
beta-Glucanas/imunologia
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antibodies, Fungal); 0 (Antigens, Fungal); 0 (Epitopes); 0 (Fungal Proteins); 0 (Hemagglutinins); 0 (Membrane Glycoproteins); 0 (Recombinant Fusion Proteins); 0 (beta-Glucans); 0 (mannoproteins); 147336-22-9 (Green Fluorescent Proteins); 9051-97-2 (beta-1,3-glucan)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180113
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0191194


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[PMID]:29228311
[Au] Autor:Hinrichsen M; Lenz M; Edwards JM; Miller OK; Mochrie SGJ; Swain PS; Schwarz-Linek U; Regan L
[Ad] Endereço:Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT06511, USA.
[Ti] Título:A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking.
[So] Source:Protein Eng Des Sel;30(12):771-780, 2017 12 01.
[Is] ISSN:1741-0134
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data.
[Mh] Termos MeSH primário: Rastreamento de Células/métodos
Corantes Fluorescentes/metabolismo
Engenharia de Proteínas/métodos
Processamento de Proteína Pós-Traducional
Análise de Célula Única/métodos
Espectrometria de Fluorescência/métodos
[Mh] Termos MeSH secundário: Corantes Fluorescentes/análise
Corantes Fluorescentes/química
Saccharomyces cerevisiae/genética
Saccharomyces cerevisiae/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Fluorescent Dyes)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180223
[Lr] Data última revisão:
180223
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171212
[St] Status:MEDLINE
[do] DOI:10.1093/protein/gzx059



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