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1.
Int J Mol Sci ; 24(8)2023 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-37108454

RESUMO

The post-synaptic density protein 95 (PSD95) is a crucial scaffolding protein participating in the organization and regulation of synapses. PSD95 interacts with numerous molecules, including neurotransmitter receptors and ion channels. The functional dysregulation of PSD95 as well as its abundance and localization has been implicated with several neurological disorders, making it an attractive target for developing strategies able to monitor PSD95 accurately for diagnostics and therapeutics. This study characterizes a novel camelid single-domain antibody (nanobody) that binds strongly and with high specificity to rat, mouse, and human PSD95. This nanobody allows for more precise detection and quantification of PSD95 in various biological samples. We expect that the flexibility and unique performance of this thoroughly characterized affinity tool will help to further understand the role of PSD95 in normal and diseased neuronal synapses.


Assuntos
Neurônios , Sinapses , Ratos , Camundongos , Humanos , Animais , Proteína 4 Homóloga a Disks-Large/metabolismo , Sinapses/metabolismo , Neurônios/metabolismo , Densidade Pós-Sináptica/metabolismo , Canais Iônicos/metabolismo , Fatores de Transcrição/metabolismo
2.
Biomolecules ; 11(2)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673130

RESUMO

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects.


Assuntos
Cromatografia de Afinidade/métodos , Temperatura Baixa , Proteínas/isolamento & purificação , Soluções Tampão , Epitopos/química , Imunoprecipitação , Anticorpos de Domínio Único/isolamento & purificação
3.
Nat Commun ; 10(1): 4403, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562305

RESUMO

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.


Assuntos
Epitopos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/metabolismo , Células 3T3 , Animais , Células COS , Chlorocebus aethiops , Epitopos/química , Epitopos/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética
4.
Sci Rep ; 7(1): 17629, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29247166

RESUMO

The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.


Assuntos
Antibacterianos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/metabolismo , Proteínas Periplásmicas de Ligação/antagonistas & inibidores , Triazinas/farmacologia , Vancomicina/farmacologia , 1-Naftilamina/análogos & derivados , 1-Naftilamina/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Proteínas Periplásmicas de Ligação/genética
5.
Mol Cell Proteomics ; 14(1): 216-26, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25403562

RESUMO

How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM.


Assuntos
Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Proteômica
6.
Nat Commun ; 5: 5396, 2014 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-25369922

RESUMO

Gram-negative bacteria use the type-V secretion pathway to expose proteins at their cell surface, many of which have virulence functions. Translocation of those proteins across the outer membrane occurs either by means of dedicated translocator proteins (two-partner secretion) or covalently fused translocator domains (autotransporters). Translocator proteins and translocator domains are ß-barrels requiring the ß-barrel assembly machinery (BAM) for membrane integration. However, the molecular details of their passage across the envelope and insertion into the outer membrane remain enigmatic, owing in part to the fact that in vitro systems are not available. Here we describe a versatile in vitro reconstitution system that faithfully reproduces both branches of the type-V secretion pathway and the assembly of ß-barrel outer membrane proteins. This system will allow an in-depth analysis of protein secretion across and integration into outer membranes.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Sistemas de Secreção Tipo V , Sistema Livre de Células , Proteolipídeos
7.
J Biol Chem ; 289(27): 19089-97, 2014 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-24855643

RESUMO

Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades Proteicas/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Escherichia coli/citologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Deleção de Genes , Chaperonas Moleculares/genética , Estresse Oxidativo , Periplasma/metabolismo , Transporte Proteico , Canais de Translocação SEC
8.
Biochim Biophys Acta ; 1838(7): 1862-70, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24726609

RESUMO

The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.


Assuntos
Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Glucosiltransferases/metabolismo , Acholeplasma laidlawii/enzimologia , Acholeplasma laidlawii/genética , Acholeplasma laidlawii/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Estrutura Secundária de Proteína
9.
Biochemistry ; 52(33): 5533-44, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23869703

RESUMO

Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by (31)P NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a "feed-forward" stimulation of lipid synthesis in E. coli. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.


Assuntos
Acholeplasma laidlawii/enzimologia , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Glucosiltransferases/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipídeos/biossíntese , Acetatos/metabolismo , Acholeplasma laidlawii/genética , Ânions/química , Ânions/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucosiltransferases/química , Glucosiltransferases/genética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/química , Modelos Moleculares , Análise Multivariada , Mutação , Fosfolipídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Transformação Genética
10.
J Proteome Res ; 10(4): 1848-59, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21210718

RESUMO

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.


Assuntos
Proteínas de Escherichia coli/análise , Escherichia coli/química , Proteínas de Membrana/análise , Chaperonas Moleculares/análise , Complexos Multiproteicos/química , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel Bidimensional/métodos , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/isolamento & purificação , Espectrometria de Massas/métodos , Proteínas de Membrana/classificação , Proteínas de Membrana/isolamento & purificação , Chaperonas Moleculares/classificação , Chaperonas Moleculares/isolamento & purificação , Peso Molecular , Complexos Multiproteicos/isolamento & purificação , Filogenia , Proteoma/análise , Proteômica/métodos
11.
J Bacteriol ; 192(17): 4317-26, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20581204

RESUMO

The molecular basis for the recognition of glucose as a germinant molecule by spores of Bacillus megaterium QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed gerWB) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70- to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.


Assuntos
Bacillus megaterium/fisiologia , Glucose/metabolismo , Ligantes , Proteínas de Membrana , Esporos Bacterianos/fisiologia , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Cromossomos Bacterianos/genética , Leucina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Análise de Sequência de DNA , Esporos Bacterianos/metabolismo
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