Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.138
Filtrar
1.
Int J Mol Sci ; 20(15)2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31387209

RESUMO

Galectin-1 (Gal-1) is a 14 kDa protein that has been well characterized for promoting cancer metastasis and tumor immune evasion. By localizing to the cancer cell surface, Gal-1 induces T cell apoptosis through binding T cell surface receptors. The transmembrane protein, Sushi Domain Containing 2 (SUSD2), has been previously shown to be required for Gal-1 surface presentation in breast cancer cells. Western immunoblot analysis revealed that SUSD2 is cleaved into two fragments. However, the significance of this cleavage for Gal-1 surface localization has not been investigated. To define the location of cleavage, a mutagenesis analysis of SUSD2 was performed. Our studies demonstrated that SUSD2 is cleaved at its glycine-aspartic acid-proline-histidine (GDPH) amino acid sequence. Generation of a noncleavable SUSD2 mutant (GDPH∆-SUSD2) showed that SUSD2 cleavage was required for SUSD2 and Gal-1 plasma membrane localization. Noncleavable cysteine mutants were also unable to present Gal-1 at the cell surface, further demonstrating that SUSD2 cleavage is required for Gal-1 surface presentation. Treatment with the serine protease inhibitor, Pefabloc SC, inhibited SUSD2 cleavage in a dose dependent manner, suggesting that SUSD2 is cleaved by a serine protease. Therefore, identification and inhibition of this protease may provide a new therapeutic tool for inhibiting SUSD2 and Gal-1's combined tumorigenic function in breast cancer.


Assuntos
Motivos de Aminoácidos , Apresentação do Antígeno/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Galectina 1/imunologia , Glicoproteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Neoplasias da Mama/genética , Membrana Celular/imunologia , Membrana Celular/metabolismo , Dissulfetos , Retículo Endoplasmático/metabolismo , Feminino , Galectina 1/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteólise
2.
Food Chem ; 297: 124867, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253333

RESUMO

Broad molecular weight (MW) distribution and variability of mannan to protein ratio of purified mannoproteins (MP), isolated from yeast cell walls upon the enzymatic treatment, revealed their multiplicity. The main fraction of high-MW Agrimos®-MP1 and YCW-b-MP1' contained mannoproteins with a mannan to protein ratio of 3.5 and 6.9, respectively. Low-MW YCW-b-MP2' was mainly comprised of mannan, with a ratio of 181, whereas low-MW Agrimos®-MP2 was characterized by a ratio of 12.2. The solubility of MP1/MP2 was higher than that of MP1'/MP2'. Mannoproteins showed similar or lower solubility than mannan, and they exhibited a Newtonian behaviour. Sonication was the appropriate method for the formation of mannoproteins-based emulsions. Contrary to MP1/MP1'-based emulsions, MP2/MP2'-based ones showed higher affinity towards soybean oil than glyceryl-trioleate. pH affected the emulsifying ability of MP1/MP1'. MP1/MP1' showed similar or slightly inferior emulsifying properties than lecithin. This study is expected to broaden the applications of mannoproteins as value-added ingredients.


Assuntos
Mananas/química , Glicoproteínas de Membrana/química , Saccharomyces cerevisiae/metabolismo , Parede Celular/metabolismo , Concentração de Íons de Hidrogênio , Mananas/metabolismo , Glicoproteínas de Membrana/metabolismo , Peso Molecular , Solubilidade , Temperatura Ambiente , Viscosidade
3.
Molecules ; 24(11)2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163608

RESUMO

Interactions between grape seed tannin and either a mannoprotein or an arabinogalactan in model wine solutions of different ethanol concentrations were characterized with nanoparticle tracking analysis (NTA), UV-visible spectroscopy and dynamic light scattering (DLS). NTA results reflected a shift in particle size distribution due to aggregation. Furthermore, the light scattering intensity of each tracked particle measured by NTA demonstrated the presence of aggregates, even when a shift in particle size was not apparent. Mannoprotein and arabinogalactan behaved differently when combined with seed tannin. Mannoprotein formed large, highly light-scattering aggregates, while arabinogalactan exhibited only weak interactions with seed tannin. A 3% difference in alcohol concentration of the model solution (12 vs. 15% v/v) was sufficient to affect the interactions between mannoprotein and tannin when the tannin concentration was high. In summary, this study showed that NTA is a promising tool for measuring polydisperse samples of grape and wine macromolecules, and their aggregates under wine-like conditions. The implications for wine colloidal properties are discussed based on these results.


Assuntos
Nanopartículas/química , Polissacarídeos/química , Taninos/química , Vinho/análise , Goma Arábica/química , Glicoproteínas de Membrana/química , Peso Molecular , Tamanho da Partícula , Espalhamento de Radiação , Sementes/química
4.
Arch Virol ; 164(9): 2309-2314, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31172288

RESUMO

The surface (SU) and transmembrane (TM) glycoproteins of many retroviruses are linked by disulphide bonds, and the interaction of SU with a cellular receptor results in disulphide bond isomerisation triggered by the CXXC motif in SU. This reaction leads to the fusion of viral and host cell membranes. In this work, we show that the cysteine at amino acid position 212 in the CAIC motif of the SU glycoprotein of bovine leukaemia virus has a free thiol group. A C-to-A mutation at position 212, either individually or in combination with a C-to-A mutation at position 215, was found to inhibit the maturation process, suggesting its involvement in the formation of the covalent bond with TM.


Assuntos
Cisteína/metabolismo , Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , Cisteína/genética , Vírus da Leucemia Bovina/química , Vírus da Leucemia Bovina/isolamento & purificação , Vírus da Leucemia Bovina/fisiologia , Glicoproteínas de Membrana/genética , Mutação
5.
J Agric Food Chem ; 67(26): 7428-7434, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31187991

RESUMO

Protein-polyphenol interactions play a very important role in wine stability assessment, especially in red varieties. Different polysaccharides can influence these interactions by protecting or disrupting charges and are even used as additives to stabilize colloidal solutions. The most common examples are mannoproteins and carboxymethyl cellulose (CMC). In some cases, the mechanisms that are involved in these reactions are not thoroughly understood and can lead to unexpected problems and delayed haze formation after CMC addition to red wines. Small-scale bench trials were conducted in model systems under different pH conditions to monitor the formation of turbidity and protection mechanisms during the interaction of proteins, polyphenols, and polysaccharides. Egg-white protein was chosen as a protein model due to its complex composition, a commercial grape tannin extract was used as polyphenol source, and pectin, glucomannan, mannoprotein, alginate, and CMC were applied as polysaccharides to model various wine conditions. Reactions were monitored in duplicate on a 50 mL scale by spectrophotometry at 860 nm over at least 30 days. Some of the polysaccharides interacted directly with proteins or polyphenols causing precipitation. Other polysaccharides delayed the reaction between proteins and other macromolecules depending on their concentration. The results of these experiments provide important insights into reaction dynamics between macromolecules that are involved in the physical stability of wine.


Assuntos
Carboximetilcelulose Sódica/química , Proteínas do Ovo/química , Extratos Vegetais/química , Polifenóis/química , Polissacarídeos/química , Vinho/análise , Animais , Galinhas , Glicoproteínas de Membrana/química , Taninos/química , Vitis/química
6.
Int J Mol Sci ; 20(12)2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31212749

RESUMO

Hac1p is a key transcription factor regulating the unfolded protein response (UPR) induced by abnormal accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. The accumulation of unfolded/misfolded proteins is sensed by protein Ire1p, which then undergoes trans-autophosphorylation and oligomerization into discrete foci on the ER membrane. HAC1 pre-mRNA, which is exported to the cytoplasm but is blocked from translation by its intron sequence looping back to its 5'UTR to form base-pair interaction, is transported to the Ire1p foci to be spliced, guided by a cis-acting bipartite element at its 3'UTR (3'BE). Spliced HAC1 mRNA can be efficiently translated. The resulting Hac1p enters the nucleus and activates, together with coactivators, a large number of genes encoding proteins such as protein chaperones to restore and maintain ER homeostasis and secretary protein quality control. This review details the translation regulation of Hac1p production, mediated by the nonconventional splicing, in the broad context of translation control and summarizes the evolution and diversification of the UPR signaling pathway among fungal, metazoan and plant lineages.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação Fúngica da Expressão Gênica , Processamento de RNA , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Íntrons , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Ligação Proteica , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Resposta a Proteínas não Dobradas
7.
Nat Commun ; 10(1): 2731, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227690

RESUMO

Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C. crescentus, we show that 2D protein self-assembly is sufficient to build and maintain the S-layer in living cells by efficient protein crystal nucleation and growth. We propose a model supported by single-molecule tracking whereby randomly secreted SLP monomers diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated at the edges of growing 2D S-layer crystals. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Unsupervised assembly poses challenges for therapeutics targeting S-layers. However, protein crystallization as an evolutionary driver rationalizes S-layer diversity and raises the potential for biologically inspired self-assembling macromolecular nanomaterials.


Assuntos
Proteínas de Bactérias/química , Parede Celular/química , Glicoproteínas de Membrana/química , Caulobacter crescentus/química , Cristalização , Lipopolissacarídeos/química , Substâncias Macromoleculares/química , Nanoestruturas/química , Nanotecnologia/métodos
8.
J Exp Clin Cancer Res ; 38(1): 214, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118109

RESUMO

BACKGROUND: Gastric cancer is one of the deadliest malignant tumours, with a high incidence in China, and is regulated by aberrantly overexpressed oncogenes. However, existing therapies are insufficient to meet patients' needs; thus, the identification of additional therapeutic targets and exploration of the underlying mechanism are urgently needed. GPAA1 is the subunit of the GPI transamidase that transfers the GPI anchor to proteins within the ER. The functional impacts of increased expression levels of GPAA1 in human cancers are not well understood. METHODS: Data mining was performed to determine the pattern of GPAA1 expression and the reason for its overexpression in tumour and adjacent normal tissues. In vitro and in vivo experiments evaluating proliferation and metastasis were performed using cells with stable deletion or overexpression of GPAA1. A tissue microarray established by the Ren Ji Hospital was utilized to analyse the expression profile of GPAA1 and its correlation with prognosis. Western blotting, an in situ proximity ligation assay, and co-immunoprecipitation (co-IP) were performed to reveal the mechanism of GPAA1 in gastric cancer. RESULTS: GPAA1 was a markedly upregulated oncogene in gastric cancer due to chromosomal amplification. GPAA1 overexpression was confirmed in specimens from the Ren Ji cohort and was associated with ERBB2 expression, predicting unsatisfactory patient outcomes. Aberrantly upregulated GPAA1 dramatically contributed to cancer growth and metastasis in in vitro and in vivo studies. Mechanistically, GPAA1 enhanced the levels of metastasis-associated GPI-anchored proteins to increase tumour metastasis and intensified lipid raft formation, which consequently promoted the interaction between EGFR and ERBB2 as well as downstream pro-proliferative signalling. CONCLUSIONS: GPAA1 facilitates the expression of cancer-related GPI-anchored proteins and supplies a more robust platform-the lipid raft-to promote EGFR-ERBB2 dimerization, which further contributes to tumour growth and metastasis and to cancer progression. GPAA1 could be a promising diagnostic biomarker and therapeutic target for gastric cancer.


Assuntos
Proteínas Ligadas por GPI/genética , Glicoproteínas de Membrana/genética , Receptor ErbB-2/genética , Neoplasias Gástricas/genética , Aciltransferases/genética , Idoso , Animais , Proliferação de Células/genética , Progressão da Doença , Intervalo Livre de Doença , Receptores ErbB/química , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Masculino , Glicoproteínas de Membrana/química , Microdomínios da Membrana/genética , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Multimerização Proteica/genética , Receptor ErbB-2/química , Transdução de Sinais/genética , Neoplasias Gástricas/patologia , Análise Serial de Tecidos
9.
Cancer Sci ; 110(7): 2237-2246, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31127873

RESUMO

Glycoprotein NMB (GPNMB) is highly expressed in many types of malignant tumors and thought to be a poor prognostic factor in those cancers, including breast cancer. Glycoprotein NMB is a type IA transmembrane protein that has a long extracellular domain (ECD) and a short intracellular domain (ICD). In general, the ECD of a protein is involved in protein-protein or protein-carbohydrate interactions, whereas the ICD is important for intracellular signaling. We previously reported that GPNMB contributes to the initiation and malignant progression of breast cancer through the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) in its ICD. Furthermore, we showed that the tyrosine residue in hemITAM is involved in induction of the stem-like properties of breast cancer cells. However, the contribution of the ECD to its tumorigenic function has yet to be fully elucidated. In this study, we focused on the region, the so-called kringle-like domain (KLD), that is conserved among species, and made a deletion mutant, GPNMB(ΔKLD). Enhanced expression of WT GPNMB induced sphere and tumor formation in breast epithelial cells; in contrast, GPNMB(ΔKLD) lacked these activities without affecting its molecular properties, such as subcellular localization, Src-induced tyrosine phosphorylation at least in overexpression experiments, and homo-oligomerization. Additionally, GPNMB(ΔKLD) lost its cell migration promoting activity, even though it reduced E-cadherin expression. Although the interaction partner binding to KLD has not yet been identified, we found that the KLD of GPNMB plays an important role in its tumorigenic potential.


Assuntos
Neoplasias da Mama/patologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Mutação , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Sequência Conservada , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Kringles , Glicoproteínas de Membrana/genética , Camundongos , Transplante de Neoplasias
10.
PLoS One ; 14(5): e0216863, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31075115

RESUMO

S-layers commonly cover archaeal cell envelopes and are composed of proteins that self-assemble into a paracrystalline surface structure. Despite their detection in almost all archaea, there are few reports investigating the structural properties of these proteins, with no reports exploring this topic for halophilic S-layers. The objective of the present study was to investigate the secondary and tertiary organization of the Haloferax volcanii S-layer protein. Such investigations were performed using circular dichroism, fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. The protein secondary structure is centered on ß-sheets and is affected by environmental pH, with higher disorder in more alkaline conditions. The pH can also affect the protein's tertiary structure, with higher tryptophan side-chain exposure to the medium under the same conditions. The concentrations of Na, Mg and Ca ions in the environment also affect the protein structures, with small changes in α-helix and ß-sheet content, as well as changes in tryptophan side chain exposure. These changes in turn influence the protein's functional properties, with cell envelope preparations revealing striking differences when in different salt conditions. Thermal denaturation assays revealed that the protein is stable. It has been reported that the S-layer protein N-glycosylation process is affected by external factors and the present study indicates for the first time changes in the protein structure.


Assuntos
Haloferax volcanii/química , Temperatura Alta , Glicoproteínas de Membrana/química , Metais/química , Salinidade , Haloferax volcanii/metabolismo , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismo , Metais/metabolismo , Desnaturação Proteica , Estrutura Secundária de Proteína
11.
Int J Mol Sci ; 20(10)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108847

RESUMO

Of the three interleukin-22 binding protein (IL-22BP) isoforms produced by the human IL22RA2 gene, IL-22BPi2 and IL-22BPi3 are capable of neutralizing IL-22. The longest isoform, IL-22BPi1, does not bind IL-22, is poorly secreted, and its retention within the endoplasmic reticulum (ER) is associated with induction of an unfolded protein response (UPR). Therapeutic modulation of IL-22BPi2 and IL-22BPi3 production may be beneficial in IL-22-dependent disorders. Recently, we identified the ER chaperones GRP94 and cyclophilin B in the interactomes of both IL-22BPi1 and IL-22BPi2. In this study, we investigated whether secretion of the IL-22BP isoforms could be modulated by pharmacological targeting of GRP94 and cyclophilin B, either by means of geldanamycin, that binds to the ADP/ATP pocket shared by HSP90 paralogs, or by cyclosporin A, which causes depletion of ER cyclophilin B levels through secretion. We found that geldanamycin and its analogs did not influence secretion of IL-22BPi2 or IL-22BPi3, but significantly enhanced intracellular and secreted levels of IL-22BPi1. The secreted protein was heterogeneously glycosylated, with both high-mannose and complex-type glycoforms present. In addition, cyclosporine A augmented the secretion of IL-22BPi1 and reduced that of IL-22BPi2 and IL-22BPi3. Our data indicate that the ATPase activity of GRP94 and cyclophilin B are instrumental in ER sequestration and degradation of IL-22BPi1, and that blocking these factors mobilizes IL-22BPi1 toward the secretory route.


Assuntos
Benzoquinonas/farmacologia , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Lactamas Macrocíclicas/farmacologia , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ciclofilinas/química , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Monócitos/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteólise , Receptores de Interleucina/química , Receptores de Interleucina/genética
12.
Adv Exp Med Biol ; 1135: 139-160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31098815

RESUMO

In recent years, a growing number of studies have implicated the coordinated action of NPC1 and NPC2 in intralysosomal transport and efflux of cholesterol. Our current understanding of this process developed with just over two decades of research. Since the cloning of the genes encoding the NPC1 and NPC2 proteins, studies of the biochemical defects observed when either gene is mutated along with computational and structural studies have unraveled key steps in the underlying mechanism. Here, we summarize the major contributions to our understanding of the proposed cholesterol transport controlled by NPC1 and NPC2, and briefly discuss recent findings of cholesterol binding and transport proteins beyond NPC1 and NPC2. We conclude with key questions and major challenges for future research on cholesterol transport by the NPC1 and NPC2 proteins.


Assuntos
Proteínas de Transporte/química , Colesterol/química , Glicoproteínas/química , Glicoproteínas de Membrana/química , Transporte Biológico , Humanos
13.
Soft Matter ; 15(22): 4525-4540, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31099376

RESUMO

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.


Assuntos
HIV-1/química , Manose/química , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/química , Proteínas do Envelope Viral/química , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Manose/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Nanopartículas Metálicas/química , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
14.
EMBO J ; 38(12)2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31109937

RESUMO

γ-Secretase complexes (GSECs) are multimeric membrane proteases involved in a variety of physiological processes and linked to Alzheimer's disease (AD). Presenilin (PSEN, catalytic subunit), Nicastrin (NCT), Presenilin Enhancer 2 (PEN-2), and Anterior Pharynx Defective 1 (APH1) are the essential subunits of GSECs. Mutations in PSEN and the Amyloid Precursor Protein (APP) cause early-onset AD GSECs successively cut APP to generate amyloid-ß (Aß) peptides of various lengths. AD-causing mutations destabilize GSEC-APP/Aßn interactions and thus enhance the production of longer Aßs, which elicit neurotoxic events underlying pathogenesis. Here, we investigated the molecular strategies that anchor GSEC and APP/Aßn during the sequential proteolysis. Our studies reveal that a direct interaction between NCT ectodomain and APPC99 influences the stability of GSEC-Aßn assemblies and thereby modulates Aß length. The data suggest a potential link between single-nucleotide variants in NCSTN and AD risk. Furthermore, our work indicates that an extracellular interface between the protease (NCT, PSEN) and the substrate (APP) represents the target for compounds (GSMs) modulating Aß length. Our findings may guide future rationale-based drug discovery efforts.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Secretases da Proteína Precursora do Amiloide/química , Precursor de Proteína beta-Amiloide/química , Animais , Células Cultivadas , Ativação Enzimática , Espaço Extracelular , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteólise , Relação Estrutura-Atividade
15.
PLoS One ; 14(4): e0215237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30973916

RESUMO

We investigated the significance of MUC21 in EGFR-mutated lung adenocarcinoma (LADC). Two-hundred forty-one surgically resected LADCs (116 EGFR-mutated and 125 wild-type tumors) were examined for immunohistochemical expression of MUC21 protein. A polyclonal antibody and two monoclonal antibodies (heM21C and heM21D) that bind differentially glycosylated MUC21 epitopes were used, and MUC21 proteins detected by these antibodies were named MUC21P, MUC21C, and MUC21D, respectively. MUC21 mRNA levels were semi-quantified and classified into "high" and "low". Among the immunohistochemical expression detected by three different antibodies, high expressors tended to be related to EGFR mutations. The three varieties of the immunohistochemical expressions were related to different histological elements in the EGFR-mutated LADCs. Either MUC21P or MUC21C high expressors had a higher proportion of lepidic elements with low papillary structure and micropapillary elements. MUC21D high expressors had a significantly higher proportion of micropapillary elements (Mann-Whitney test P ≤0.0001). Furthermore, MUC21D high expressors showed high incidence of lymphatic canal invasion and lymph node metastasis (Pearson x2 test, P = 0.0021, P = 0.0125), and a significantly higher recurrence rate (5-year recurrence-free survival 50.7% vs. 73.8%, log-rank test P = 0.0495). MUC21 proteins with a specific glycosylation status may be involved in the progression of EGFR-mutated LADCs, particularly at the stage where tumors are transforming from pure lepidic to micropapillary through low papillary lepidic lesions.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Adenocarcinoma de Pulmão/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Receptores ErbB/genética , Feminino , Glicosilação , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Mucinas/química , Mucinas/genética , Mutação , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Cell Mol Life Sci ; 76(19): 3899-3914, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30993352

RESUMO

The P3H1/CRTAP/PPIB complex is essential for prolyl 3-hydroxylation and folding of procollagens in the endoplasmic reticulum (ER). Deficiency in any component of this ternary complex is associated with the misfolding of collagen and the onset of osteogenesis imperfecta. However, little structure information is available about how this ternary complex is assembled and retained in the ER. Here, we assessed the role of the KDEL sequence of P3H1 and probed the spatial interactions of PPIB in the complex. We show that the KDEL sequence is essential for retaining the P3H1 complex in the ER. Its removal resulted in co-secretion of P3H1 and CRTAP out of the cell, which was mediated by the binding of P3H1 N-terminal domain with CRTAP. The secreted P3H1/CRTAP can readily bind PPIB with their C-termini close to PPIB in the ternary complex. Cysteine modification, crosslinking, and mass spectrometry experiments identified PPIB surface residues involved in the complex formation, and showed that the surface of PPIB is extensively covered by the binding of P3H1 and CRTAP. Most importantly, we demonstrated that one disease-associated pathological PPIB mutation on the binding interface did not affect the PPIB prolyl-isomerase activity, but disrupted the formation of P3H1/CRTAP/PPIB ternary complex. This suggests that defects in the integrity of the P3H1 ternary complex are associated with pathological collagen misfolding. Taken together, these results provide novel structural information on how PPIB interacts with other components of the P3H1 complex and indicate that the integrity of P3H1 complex is required for proper collagen formation.


Assuntos
Ciclofilinas/química , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas da Matriz Extracelular/genética , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Mutação , Prolil Hidroxilases , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoglicanas/química , Proteoglicanas/genética , Deleção de Sequência
17.
J Phys Chem Lett ; 10(9): 2235-2243, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30995409

RESUMO

Rhodopsins are photoreceptive proteins using light to drive a plethora of biological functions such as vision, proton and ion pumping, cation and anion channeling, and gene and enzyme regulation. Here we combine organic synthesis, NMR structural studies, and photochemical characterization to show that it is possible to prepare a fully synthetic mimic of rhodopsin photoreceptors. More specifically, we conjugate a bile acid binding protein with a synthetic mimic of the rhodopsin protonated Schiff base chromophore to achieve a covalent complex featuring an unnatural protein host, photoswitch, and photoswitch-protein linkage with a reverse orientation. We show that, in spite of its molecular-level diversity, light irradiation of the prepared mimic fuels a photochromic cycle driven by sequential photochemical and thermal Z/E isomerizations reminiscent of the photocycles of microbial rhodopsins.


Assuntos
Materiais Biomiméticos/química , Proteínas de Transporte/química , Corantes/química , Glicoproteínas de Membrana/química , Fotorreceptores Microbianos/química , Rodopsina/química , Isomerismo , Luz , Processos Fotoquímicos , Conformação Proteica , Prótons , Relação Estrutura-Atividade
18.
Gene ; 703: 102-111, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-30928364

RESUMO

Haemonchus contortus (HC) causes Haemonchosis in sheep and goats, with high mortality and morbidity due to lack of effective vaccine and increasing resistance to anthelmintic drugs. The present study was aimed at developing the 3D model of HCP24 protein and to identify the candidate epitopic peptides for effective humoral and cell-mediated immune-response. The HCP24 protein was homology modelled using the Swiss server and developed model was validated by ERRAT, VERIFY3D, PROQ, RAMPAGE and PROCHECK servers. Linear and prominent antigenic epitopes were predicted by SVMTrip and Immuno-medicine group tool. Conformational B-cell epitopes were predicted by Ellipro. MHC-I and MHC-II binding peptides were predicted by MHCPRED2, MHC2PRED and Propred I server. Proteosomal cleavage sites were predicted by Netchop server, to assess the stability of peptides. Reverse and three frame translation was done by EMBOSS tool. Bepipred and IEDB analysis also confirmed that both the predicted peptides (pep-1 and pep-2) were important antigenic region but pep-1 should have better hydrophobicity and stability. The degree of confidence achieved on scientific validation of the generated 3D model of the protein allows us to prescribe its use for research purpose. We could determine the peptide Pep-1(EDCKCTNCVCSRDEAL) should be a conformational B cell epitope with high antigenic potential and should demonstrate good binding affinity with host MHC-II and MHC-I alleles as well as stability inside host. Thus, it could be an ideal vaccine candidate for developing sub-unit vaccine against the parasite and should be assessed for protective immune response by in vitro and in-vivo studies.


Assuntos
Epitopos/química , Haemonchus/metabolismo , Glicoproteínas de Membrana/química , Animais , Sítios de Ligação , Simulação por Computador , Epitopos/imunologia , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Glicoproteínas de Membrana/imunologia , Modelos Moleculares , Conformação Proteica , Homologia Estrutural de Proteína
19.
J Agric Food Chem ; 67(14): 4031-4042, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30892885

RESUMO

The increase of temperature can cause a decoupling of sugar and anthocyanin accumulation in grapes, leading to wines poor in color. Different enological techniques are nowadays under study to overcome this problem. Among them, the present study has evaluated the color and pigment composition modifications caused by the addition during Syrah winemaking of either Pedro Ximénez seeds (intended to increase chemical stability) or/and two different mannoproteins (color-protective and astringency-modulator) to increase colloidal stability. Color and pigment composition modifications were assessed from CIELAB color parameters and from HPLC-DAD-MS n results before and after cold-treatment (used to force colloidal instability). The addition of both seeds and mannoproteins increased color stability against cold and, additionally, against SO2-bleaching in the case of mannoproteins. However, the initial pigment composition and color of the samples were differently affected by these additions, being clearly affected (Δ E* ab > 3) in the cases of seeds and with the astringency-modulator mannoprotein and hardly modified with the other one.


Assuntos
Aditivos Alimentares/química , Manipulação de Alimentos/métodos , Glicoproteínas de Membrana/química , Pigmentos Biológicos/química , Sementes/química , Vitis/química , Vinho/análise , Cor , Fermentação , Aditivos Alimentares/metabolismo , Glicoproteínas de Membrana/metabolismo , Pigmentos Biológicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Vitis/metabolismo , Vitis/microbiologia , Vinho/microbiologia
20.
Methods Mol Biol ; 1952: 219-232, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825178

RESUMO

Matrigel is extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma in C57BL/6 mice, a tumor rich in extracellular matrix (ECM) proteins. It consists mainly of laminin (approximately 60%), collagen IV (approximately 30%), and nidogen-1/entactin (approximately 8%), while it also contains heparan sulfate proteoglycans, such as perlecan, other ECM proteins, as well as growth factors bound to the ECM. Matrigel mimics the physiological cell matrix and is the most commonly used matrix substrate to study in vitro and in vivo angiogenesis. Here, we describe the in vivo Matrigel plug assay and how it can be used for both qualitative and quantitative assessment of angiogenesis.


Assuntos
Colágeno/química , Células Endoteliais/citologia , Laminina/química , Glicoproteínas de Membrana/química , Neovascularização Fisiológica , Proteoglicanas/química , Animais , Separação Celular/métodos , Combinação de Medicamentos , Citometria de Fluxo/métodos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Microtomia/métodos , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA