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1.
BMC Bioinformatics ; 24(1): 137, 2023 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-37029385

RESUMO

Vesicle transport proteins not only play an important role in the transmembrane transport of molecules, but also have a place in the field of biomedicine, so the identification of vesicle transport proteins is particularly important. We propose a method based on ensemble learning and evolutionary information to identify vesicle transport proteins. Firstly, we preprocess the imbalanced dataset by random undersampling. Secondly, we extract position-specific scoring matrix (PSSM) from protein sequences, and then further extract AADP-PSSM and RPSSM features from PSSM, and use the Max-Relevance-Max-Distance (MRMD) algorithm to select the optimal feature subset. Finally, the optimal feature subset is fed into the stacked classifier for vesicle transport proteins identification. The experimental results show that the of accuracy (ACC), sensitivity (SN) and specificity (SP) of our method on the independent testing set are 82.53%, 0.774 and 0.836, respectively. The SN, SP and ACC of our proposed method are 0.013, 0.007 and 0.76% higher than the current state-of-the-art methods.


Assuntos
Algoritmos , Matrizes de Pontuação de Posição Específica , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Proteínas de Transporte , Máquina de Vetores de Suporte , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/isolamento & purificação
2.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33446503

RESUMO

Almost 150 papers about brain lymphatics have been published in the last 150 years. Recently, the information in these papers has been synthesized into a picture of central nervous system (CNS) "glymphatics," but the fine structure of lymphatic elements in the human brain based on imaging specific markers of lymphatic endothelium has not been described. We used LYVE1 and PDPN antibodies to visualize lymphatic marker-positive cells (LMPCs) in postmortem human brain samples, meninges, cavernous sinus (cavum trigeminale), and cranial nerves and bolstered our findings with a VEGFR3 antibody. LMPCs were present in the perivascular space, the walls of small and large arteries and veins, the media of large vessels along smooth muscle cell membranes, and the vascular adventitia. Lymphatic marker staining was detected in the pia mater, in the arachnoid, in venous sinuses, and among the layers of the dura mater. There were many LMPCs in the perineurium and endoneurium of cranial nerves. Soluble waste may move from the brain parenchyma via perivascular and paravascular routes to the closest subarachnoid space and then travel along the dura mater and/or cranial nerves. Particulate waste products travel along the laminae of the dura mater toward the jugular fossa, lamina cribrosa, and perineurium of the cranial nerves to enter the cervical lymphatics. CD3-positive T cells appear to be in close proximity to LMPCs in perivascular/perineural spaces throughout the brain. Both immunostaining and qPCR confirmed the presence of adhesion molecules in the CNS known to be involved in T cell migration.


Assuntos
Encéfalo/metabolismo , Sistema Linfático/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas de Transporte Vesicular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Autopsia , Encéfalo/diagnóstico por imagem , Movimento Celular/genética , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Dura-Máter/diagnóstico por imagem , Dura-Máter/metabolismo , Endotélio Linfático/diagnóstico por imagem , Endotélio Linfático/metabolismo , Feminino , Sistema Glinfático/metabolismo , Humanos , Imuno-Histoquímica/métodos , Sistema Linfático/diagnóstico por imagem , Vasos Linfáticos/diagnóstico por imagem , Vasos Linfáticos/metabolismo , Masculino , Glicoproteínas de Membrana/isolamento & purificação , Espaço Subaracnóideo/diagnóstico por imagem , Espaço Subaracnóideo/metabolismo , Linfócitos T/imunologia , Proteínas de Transporte Vesicular/isolamento & purificação
3.
Methods Mol Biol ; 2159: 17-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32529360

RESUMO

The dynamin-related proteins (DRPs) are self-assembling membrane remodeling machines that are indispensable for fundamental cellular trafficking and homeostatic processes. We describe in this chapter protocols developed in our laboratory for purification of full-length and minimal constructs of Chaetomium thermophilum Vps1, the model fungal DRP, using mammalian and Escherichia coli expression systems.


Assuntos
Chaetomium/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Expressão Gênica , Proteínas Recombinantes de Fusão , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/isolamento & purificação , Animais , Linhagem Celular , Chaetomium/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Vetores Genéticos/genética , Humanos , Transfecção , Proteínas de Transporte Vesicular/metabolismo
4.
Nat Commun ; 10(1): 5708, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31836717

RESUMO

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48-UN complex and its assembly.


Assuntos
Proteínas de Transporte Nucleocitoplasmático/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Ubiquitina/ultraestrutura , Proteínas de Transporte Vesicular/ultraestrutura , Cristalografia por Raios X , Proteínas de Transporte Nucleocitoplasmático/isolamento & purificação , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteína com Valosina/metabolismo , Proteínas de Transporte Vesicular/isolamento & purificação , Proteínas de Transporte Vesicular/metabolismo
5.
Biochim Biophys Acta Mol Cell Res ; 1866(12): 118551, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31487505

RESUMO

In spite of its basic and applied interest, the regulation of ER exit by filamentous fungi is insufficiently understood. In previous work we isolated a panel of conditional mutations in sarA encoding the master GTPase SarASAR1 in A. nidulans and demonstrated its key role in exocytosis and hyphal morphogenesis. However, the SAR1 guanine nucleotide exchange factor (GEF), Sec12, has not been characterized in any filamentous fungus, largely due to the fact that SEC12 homologues share little amino acid sequence identity beyond a GGGGxxxxGϕxN motif involved in guanine nucleotide exchange. Here we demonstrate that AN11127 encodes A. nidulans Sec12, which is an essential protein that localizes to the ER and that, when overexpressed, rescues the growth defect resulting from a hypomorphic sarA6ts mutation at 37 °C. Using purified, bacterially expressed proteins we demonstrate that the product of AN11127 accelerates nucleotide exchange on SarASAR1, but not on its closely related GTPase ArfAARF1, as expected for a bona fide GEF. The unequivocal characterization of A. nidulans Sec12 paves the way for the tailored modification of ER exit in a model organism that is closely related to industrial species of filamentous fungi.


Assuntos
Aspergillus nidulans/metabolismo , Fatores de Troca do Nucleotídeo Guanina/análise , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Transporte Vesicular/isolamento & purificação
6.
Methods Mol Biol ; 1998: 227-238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250306

RESUMO

Most endosomal sorting complex required for transport (ESCRT)-III proteins are not fully functional when expressed as fusion of fluorescent or epitope tags, frequently making the use of specific antibodies the only available method for their detection. Heterologous expression of ESCRT-III proteins in bacteria often results in the formation of insoluble aggregates or inclusion bodies that interfere with their purification. However, inclusion bodies are usually pure protein aggregates with high antigenicity. In addition, since proteins within inclusion bodies are presented in a range of folding states, immunization with inclusion bodies can potentially result in antibodies with specificity for different folding states of the protein under study. We describe here a protocol to isolate bacterial inclusion bodies of plant ESCRT-III proteins for production of polyclonal antibodies. We also describe a nitrocellulose-based immunoaffinity purification method that allows the immobilization of ESCRT-III proteins and the subsequent isolation of specific antibodies from a crude serum.


Assuntos
Anticorpos/isolamento & purificação , Proteínas de Arabidopsis/isolamento & purificação , Complexos Endossomais de Distribuição Requeridos para Transporte/isolamento & purificação , Corpos de Inclusão/metabolismo , Proteínas de Transporte Vesicular/isolamento & purificação , Animais , Anticorpos/imunologia , Proteínas de Arabidopsis/administração & dosagem , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/administração & dosagem , Complexos Endossomais de Distribuição Requeridos para Transporte/imunologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Escherichia coli/genética , Vetores Genéticos/genética , Imunização/métodos , Plasmídeos/genética , Dobramento de Proteína , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Purificação por Afinidade em Tandem/métodos , Transformação Bacteriana , Proteínas de Transporte Vesicular/administração & dosagem , Proteínas de Transporte Vesicular/imunologia , Proteínas de Transporte Vesicular/metabolismo
7.
Methods Mol Biol ; 1860: 211-220, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317507

RESUMO

Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means of screening for specific protein-lipid interactions. Liposomes are prepared by mixing phospholipid combinations of interest before drying and rehydration. Sonication of the lipid mixture produces small unilamellar vesicles (SUVs) which are incubated with a protein of interest to allow for any binding to occur. Liposomes and liposome-protein complexes are floated on a sucrose gradient by centrifugation to separate them from unbound protein. Bound protein levels are easily determined by SDS-PAGE and Western blotting. This approach provides a reliable means of assaying novel protein-lipid interactions in vitro. Here we use liposome floatation to show the binding of the SNARE-activating protein Sec18 (mammalian NSF) to phosphatidic acid.


Assuntos
Adenosina Trifosfatases/metabolismo , Lipossomos/metabolismo , Fosfolipídeos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Lipossomos/química , Fusão de Membrana , Fosfolipídeos/química , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/isolamento & purificação
8.
New Phytol ; 222(2): 907-922, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30570158

RESUMO

Abscisic acid-insensitive 5 (ABI5) is an essential and conserved plant basic leucine zipper transcription factor whose level controls seed germination and postgerminative development. It has been demonstrated that activity of ABI5 is transcriptionally and post-translationally regulated. However, transcriptional regulation of ABI5 is not fully understood. Here, we identified SAB1 (Sensitive to ABA 1) as a novel negative regulator of ABI5 that simultaneously regulates its stability, promoter binding activity and histone methylation-mediated gene silencing of ABI5. SAB1 encodes a Regulator of Chromatin Condensation 1 (RCC1) family protein and is expressed in an opposite pattern to that of ABI5 during early seedling growth in response to abscisic acid (ABA). SAB1 mutation results in enhanced ABA sensitivity and acts upstream of ABI5. SAB1 physically interacts with ABI5 at phosphoamino acid Ser-145, and reduces the phosphorylation of ABI5 and the protein stability. SAB1 reduces ABI5 binding activity to its own promoter, leading to reduced transcriptional level of ABI5. SAB1 inactivates ABI5 transcription by increasing the level of histone H3K27me2 in the ABI5 promoter. Our findings have identified SAB1 as a crucial new component of ABA signaling which modulates early development of plant by precisely controlling ABI5 activity through multiple mechanisms.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/isolamento & purificação , Germinação , Proteínas de Transporte Vesicular/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Cromatina/metabolismo , Germinação/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Fosfoaminoácidos/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/crescimento & desenvolvimento , Transcrição Gênica/efeitos dos fármacos , Proteínas de Transporte Vesicular/isolamento & purificação
9.
Protein Expr Purif ; 142: 68-74, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28918196

RESUMO

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis.


Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/genética , Transportador Equilibrativo 2 de Nucleosídeo/genética , Plasmídeos/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Detergentes/química , Transportador Equilibrativo 1 de Nucleosídeo/biossíntese , Transportador Equilibrativo 1 de Nucleosídeo/isolamento & purificação , Transportador Equilibrativo 2 de Nucleosídeo/biossíntese , Transportador Equilibrativo 2 de Nucleosídeo/isolamento & purificação , Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/isolamento & purificação
10.
PLoS One ; 12(6): e0177920, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28591131

RESUMO

Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.


Assuntos
Células Dendríticas/ultraestrutura , Interações Hospedeiro-Patógeno , Vírus da Influenza A/ultraestrutura , Vírion/ultraestrutura , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Endossomos/virologia , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Antígenos HLA-DR/isolamento & purificação , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Proteínas de Membrana Lisossomal/isolamento & purificação , Microscopia , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Transporte Vesicular/isolamento & purificação , Proteínas do Core Viral/genética , Proteínas do Core Viral/isolamento & purificação , Vírion/genética , Vírion/patogenicidade , Replicação Viral/genética
11.
Protein Expr Purif ; 120: 7-15, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26690372

RESUMO

Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.


Assuntos
Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Western Blotting , Dicroísmo Circular , Clonagem Molecular , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Estágios do Ciclo de Vida , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Plasmodium falciparum/fisiologia , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/isolamento & purificação
12.
Nat Struct Mol Biol ; 23(1): 59-66, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26656853

RESUMO

The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst complexes from Saccharomyces cerevisiae and showed that they are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin induced-degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer and demonstrated that several known exocyst-binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex by using negative-stain electron microscopy; our results indicate that the exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests that the subunits are contiguous helical bundles packed together into a bundle of long rods.


Assuntos
Exocitose , Substâncias Macromoleculares/química , Substâncias Macromoleculares/isolamento & purificação , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/isolamento & purificação , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica de Transmissão , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas de Transporte Vesicular/ultraestrutura
13.
Methods Mol Biol ; 1298: 85-98, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25800834
14.
Proteomics Clin Appl ; 9(5-6): 586-96, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25644331

RESUMO

PURPOSE: Cancer of the upper digestive tract (uGI) is a major contributor to cancer-related death worldwide. Due to a rise in occurrence, together with poor survival rates and a lack of diagnostic or prognostic clinical assays, there is a clear need to establish molecular biomarkers. EXPERIMENTAL DESIGN: Initial assessment was performed on urine samples from 60 control and 60 uGI cancer patients using MS to establish a peak pattern or fingerprint model, which was validated by a further set of 59 samples. RESULTS: We detected 86 cluster peaks by MS above frequency and detection thresholds. Statistical testing and model building resulted in a peak profiling model of five relevant peaks with 88% overall sensitivity and 91% specificity, and overall correctness of 90%. High-resolution MS of 40 samples in the 2-10 kDa range resulted in 646 identified proteins, and pattern matching identified four of the five model peaks within significant parameters, namely programmed cell death 6 interacting protein (PDCD6IP/Alix/AIP1), Rabenosyn-5 (ZFYVE20), protein S100A8, and protein S100A9, of which the first two were validated by Western blotting. CONCLUSIONS AND CLINICAL RELEVANCE: We demonstrate that MS analysis of human urine can identify lead biomarker candidates in uGI cancers, which makes this technique potentially useful in defining and consolidating biomarker patterns for uGI cancer screening.


Assuntos
Biomarcadores Tumorais/urina , Proteínas de Ligação ao Cálcio/urina , Proteínas de Ciclo Celular/urina , Complexos Endossomais de Distribuição Requeridos para Transporte/urina , Neoplasias Esofágicas/urina , Neoplasias Gástricas/urina , Proteínas de Transporte Vesicular/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/isolamento & purificação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Estudos de Casos e Controles , Proteínas de Ciclo Celular/isolamento & purificação , Cromatografia de Afinidade , Complexos Endossomais de Distribuição Requeridos para Transporte/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte Vesicular/isolamento & purificação , Adulto Jovem
15.
Mol Biotechnol ; 55(3): 227-35, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23780701

RESUMO

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.


Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Proteínas de Bactérias , Proteínas de Transporte/química , Proteínas de Transporte/genética , DNA Polimerase Dirigida por DNA/biossíntese , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/isolamento & purificação , Escherichia coli/metabolismo , Vetores Genéticos , Proteínas Ligantes de Grupo Heme , Hemeproteínas/química , Histidina/química , Histidina/genética , Ratos , Proteínas Recombinantes de Fusão/química , Sefarose/análogos & derivados , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/isolamento & purificação
16.
Artigo em Inglês | MEDLINE | ID: mdl-22750874

RESUMO

Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6 Šand a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS-PAGE analysis of dissolved crystals.


Assuntos
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Clonagem Molecular , Cristalização , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/isolamento & purificação
17.
Cereb Cortex ; 22(5): 1203-14, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21810784

RESUMO

ATP is known to be coreleased with glutamate at certain central synapses. However, the nature of its release is controversial. Here, we demonstrate that ATP release from cultured rat hippocampal neurons is sensitive to RNAi-mediated knockdown of the recently identified vesicular nucleotide transporter (VNUT or SLC17A9). In the intact brain, light microscopy showed particularly strong VNUT immunoreactivity in the cerebellar cortex, the olfactory bulb, and the hippocampus. Using immunoelectron microscopy, we found VNUT immunoreactivity colocalized with synaptic vesicles in excitatory and inhibitory terminals in the hippocampal formation. Moreover, VNUT immunolabeling, unlike that of the vesicular glutamate transporter VGLUT1, was enriched in preterminal axons and present in postsynaptic dendritic spines. Immunoisolation of synaptic vesicles indicated presence of VNUT in a subset of VGLUT1-containing vesicles. Thus, we conclude that VNUT mediates transport of ATP into synaptic vesicles of hippocampal neurons, thereby conferring a purinergic phenotype to these cells.


Assuntos
Trifosfato de Adenosina/metabolismo , Neurônios/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Western Blotting , Células Cultivadas , Imunofluorescência , Hipocampo/metabolismo , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Neurônios/ultraestrutura , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Proteínas de Transporte Vesicular/isolamento & purificação
18.
Nat Struct Mol Biol ; 17(11): 1298-304, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20972447

RESUMO

Multisubunit tethering complexes participate in the process of vesicle tethering--the initial interaction between transport vesicles and their acceptor compartments. TRAPPII (named for transport protein particle II) is a highly conserved tethering complex that functions in the late Golgi apparatus and consists of all of the subunits of TRAPPI and three additional, specific subunits. We have purified native yeast TRAPPII and characterized its structure and subunit organization by single-particle EM. Our data show that the nine TRAPPII components form a core complex that dimerizes into a three-layered, diamond-shaped structure. The TRAPPI subunits assemble into TRAPPI complexes that form the outer layers. The three TRAPPII-specific subunits cap the ends of TRAPPI and form the middle layer, which is responsible for dimerization. TRAPPII binds the Ypt1 GTPase and probably uses the TRAPPI catalytic core to promote guanine nucleotide exchange. We discuss the implications of the structure of TRAPPII for coat interaction and TRAPPII-associated human pathologies.


Assuntos
Proteínas de Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Dimerização , Complexo de Golgi/metabolismo , Imageamento Tridimensional , Modelos Moleculares , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/isolamento & purificação , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo
19.
Proc Natl Acad Sci U S A ; 105(15): 5683-6, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18375752

RESUMO

ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Nucleotídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Difosfato de Adenosina , Animais , Exocitose , Guanosina Trifosfato , Humanos , Camundongos , Proteínas de Transporte de Nucleotídeos/isolamento & purificação , Células PC12 , Ratos , Transfecção , Proteínas de Transporte Vesicular/isolamento & purificação
20.
Biochem Biophys Res Commun ; 371(3): 366-70, 2008 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-18439908

RESUMO

Synip and Stx4 complex plays a key role in GLUT4 vesicle trafficking and fusion with plasma membrane. The interaction of Synip with Stx4 prevents interaction of VAMP2 located in GLUT4 vesicle with Stx4 in basal state. Insulin induces the dissociation of the Synip and Stx4 complex, and then triggers VAMP2 to interact with Stx4 to form the SNARE complex, thus promoting the vesicle fusion. In this report, we adopt a novel system for co-expression of the Synip and Stx4 by using two common vectors pGEX6p-1 and pET28a(+) to investigate their expression, purification, and interaction. Through this co-expression system, we successfully co-expressed the Synip and Stx4 complex with high yield, and co-purified at an approximate 1:1 molar ratio with high purity (95%). We also demonstrate that the 1-28 residues of Stx4 are dispensable for interaction with Synip using this co-expression system.


Assuntos
Proteínas Qa-SNARE/biossíntese , Proteínas Qa-SNARE/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/isolamento & purificação , Animais , Linhagem Celular , Cromatografia em Gel , Vetores Genéticos/genética , Métodos , Camundongos , Estrutura Terciária de Proteína , Proteínas Qa-SNARE/genética , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas de Transporte Vesicular/genética
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