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Copper homeostasis is critical to the functioning of the brain, and its breakdown is linked with many brain diseases. Copper is also known to interact with the negatively charged lipid, phosphatidylserine (PS), as well as α-synuclein, an aggregation-prone protein enriched in the synapse, which plays a role in synaptic vesicle docking and fusion. However, the interplay between copper, PS lipid, and α-synuclein is not known. Herein, we report a detailed and predominantly kinetic study of the interactions among these three components pertinent to copper homeostasis and neurotransmission. We found that synaptic vesicle-mimicking small unilamellar vesicles (SUVs) can sequester any excess free Cu2+ within milliseconds, and bound Cu2+ on SUVs can be reduced to Cu+ by GSH at a nearly constant rate under physiological conditions. Moreover, we revealed that SUV-bound Cu2+ does not affect the binding between wild-type α-synuclein and SUVs but affect that between N-terminal acetylated α-synuclein and SUVs. In contrast, Cu2+ can effectively displace both types of α-synuclein from the vesicles. Our results suggest that synaptic vesicles may mediate copper transfer in the brain, while copper could participate in synaptic vesicle docking to the plasma membrane via its regulation of the interaction between α-synuclein and synaptic vesicle.
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Cobre , Homeostase , Fosfatidilserinas , Vesículas Sinápticas , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Fosfatidilserinas/metabolismo , Vesículas Sinápticas/metabolismo , Cobre/metabolismo , Homeostase/fisiologia , Humanos , Transmissão Sináptica/fisiologia , AnimaisRESUMO
Expression levels of microRNAs (miRNAs) in single cells are low and conventional miRNA detection methods require amplification that can be complex, time-consuming, costly and may bias results. Single cell microfluidic platforms have been developed; however, current approaches are unable to absolutely quantify single miRNA molecules expressed in single cells. Herein, we present an amplification-free sandwich hybridisation assay to detect single miRNA molecules in single cells using a microfluidic platform that optically traps and lyses individual cells. Absolute quantification of miR-21 and miR-34a molecules was achieved at a single cell level in human cell lines and validated using real-time qPCR. The sensitivity of the assay was demonstrated by quantifying single miRNA molecules in nasal epithelial cells and CD3+ T-cells, as well as nasal fluid collected non-invasively from healthy individuals. This platform requires ~50 cells or ~30 µL biofluid and can be extended for other miRNA targets therefore it could monitor miRNA levels in disease progression or clinical studies.
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Líquidos Corporais , MicroRNAs , Humanos , MicroRNAs/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Líquidos Corporais/metabolismoRESUMO
The multi-subunit chaperonin containing TCP-1 (CCT) is an essential molecular chaperone that functions in the folding of key cellular proteins. This paper reviews the interactome of the eukaryotic chaperonin CCT and its primary clients, the ubiquitous cytoskeletal proteins, actin and tubulin. CCT interacts with other nascent proteins, especially the WD40 propeller proteins, and also assists in the assembly of several protein complexes. A new proteomic dataset is presented for CCT purified from the human malarial parasite, P. falciparum (PfCCT). The CCT8 subunit gene was C-terminally FLAG-tagged using Selection Linked Integration (SLI) and CCT complexes were extracted from infected human erythrocyte cultures synchronized for maximum expression levels of CCT at the trophozoite stage of the parasite's asexual life cycle. We analyze the new PfCCT proteome and incorporate it into our existing model of the CCT system, supported by accumulated data from biochemical and cell biological experiments in many eukaryotic species. Together with measurements of CCT mRNA, CCT protein subunit copy number and the post-translational and chemical modifications of the CCT subunits themselves, a cumulative picture is emerging of an essential molecular chaperone system sitting at the heart of eukaryotic cell growth control and cell cycle regulation.
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Aggregation kinetics of proteins and peptides have been studied extensively due to their significance in many human diseases, including neurodegenerative disorders, and the roles they play in some key physiological processes. However, most of these studies have been performed as bulk measurements using Thioflavin T or other fluorescence turn-on reagents as indicators of fibrillization. Such techniques are highly successful in making inferences about the nucleation and growth mechanism of fibrils, yet cannot directly measure assembly reactions at low protein concentrations which is the case for amyloid-ß (Aß) peptide under physiological conditions. In particular, the evolution from monomer to low-order oligomer in early stages of aggregation cannot be detected. Single-molecule methods allow direct access to such fundamental information. We developed a high-throughput protocol for single-molecule photobleaching experiments using an automated fluorescence microscope. Stepwise photobleaching analysis of the time profiles of individual foci allowed us to determine stoichiometry of protein oligomers and probe protein aggregation kinetics. Furthermore, we investigated the potential application of supervised machine learning with support vector machines (SVMs) as well as multilayer perceptron (MLP) artificial neural networks to classify bleaching traces into stoichiometric categories based on an ensemble of measurable quantities derivable from individual traces. Both SVM and MLP models achieved a comparable accuracy of more than 80% against simulated traces up to 19-mer, although MLP offered considerable speed advantages, thus making it suitable for application to high-throughput experimental data. We used our high-throughput method to study the aggregation of Aß40 in the presence of metal ions and the aggregation of α-synuclein in the presence of gold nanoparticles.
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The front cover artwork is provided by the group of Liming Ying at Imperial College London. The image shows that N-terminal acetylation of α-synuclein shifts the binding from the N-terminus to His50 and significantly slows down the binding reaction. Read the full text of the Article at 10.1002/cphc.202100651.
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Cobre/metabolismo , alfa-Sinucleína/metabolismo , Acetilação , Sítios de Ligação , Cobre/química , Humanos , Cinética , Mutação , alfa-Sinucleína/genéticaRESUMO
The interaction between α-synuclein (αSyn) and Cu2+ has been suggested to be closely linked to brain copper homeostasis. Disruption of copper levels could induce misfolding and aggregation of αSyn, and thus contribute to the progression of Parkinson's disease (PD). Understanding the molecular mechanism of αSyn-Cu2+ interaction is important and controversies in Cu2+ coordination geometry with αSyn still exists. Herein, we find that the pathological H50Q mutation has no impact on the kinetics of Cu2+ binding to the high-affinity site of wild type αSyn (WT-αSyn), indicating the non-involvement of His50 in high-affinity Cu2+ binding to WT-αSyn. In contrast, the physiological N-terminally acetylated αSyn (NAc-αSyn) displays several orders of magnitude weaker Cu2+ binding affinity than WT-αSyn. Cu2+ coordination mode to NAc-αSyn has also been proposed based on EPR spectrum. In addition, we find that Cu2+ coordinated WT-αSyn is reduction-active in the presence of GSH, but essentially inactive towards ascorbate. Our work provides new insights into αSyn-Cu2+ interaction, which may help understand the multifaceted normal functions of αSyn as well as pathological consequences of αSyn aggregation.
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Cobre/metabolismo , alfa-Sinucleína/metabolismo , Acetilação , Sítios de Ligação , Cobre/química , Humanos , Cinética , Mutação , alfa-Sinucleína/genéticaRESUMO
The cellular thermal shift assay (CETSA) has been used extensively since its introduction to study drug-target engagement within both live cells and cellular lysate. This has proven to be a useful tool in early stage drug discovery and is used to study a wide range of protein classes. We describe the application of a single-cell CETSA workflow within a microfluidic affinity capture (MAC) chip. This has enabled us to quantitatively determine the active FOXO1 single-molecule count and observe FOXO1 stabilization and destabilization in the presence of three small molecule inhibitors, including demonstrating the determination of EC50. The successful use of the MAC chip for single-cell CETSA paves the way for the study of precious clinical samples owing to the low number of cells needed by the chip. It also provides a useful tool for studying any underlying population heterogeneity that exists within a cellular system, a feature that is usually masked when conducting ensemble measurements.
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Descoberta de Drogas , Microfluídica , ProteínasRESUMO
Aggregates of amyloid-ß (Aß) are characteristic of Alzheimer's disease, but there is no consensus as to either the nature of the toxic molecular complex or the mechanism by which toxic aggregates are produced. We report on a novel feature of amyloid-lipid interactions where discontinuities in the lipid continuum can serve as catalytic centers for a previously unseen microscale aggregation phenomenon. We show that specific lipid membrane conditions rapidly produce long contours of lipid-bound peptide, even at sub-physiological concentrations of Aß. Using single molecule fluorescence, time-lapse TIRF microscopy and AFM imaging we characterize this phenomenon and identify some exceptional properties of the aggregation pathway which make it a likely contributor to early oligomer and fibril formation, and thus a potential critical mechanism in the etiology of AD. We infer that these amyloidogenic events occur only at areas of high membrane curvature, which suggests a range of possible mechanisms by which accumulated physiological changes may lead to their inception. The speed of the formation is in hours to days, even at 1 nM peptide concentrations. Lipid features of this type may act like an assembly line for monomeric and small oligomeric subunits of Aß to increase their aggregation states. We conclude that under lipid environmental conditions, where catalytic centers of the observed type are common, key pathological features of AD may arise on a very short timescale under physiological concentration.
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Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Humanos , Lipídeos de Membrana/metabolismoRESUMO
[This corrects the article DOI: 10.1039/C9SC05710B.].
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Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional 'snapshot' of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10-20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.
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Nanotecnologia , Pinças Ópticas , Análise de Célula Única , Animais , Axônios/metabolismo , Biópsia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA/química , Eletricidade , Eletrodos , Fluorescência , Humanos , Camundongos , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SoluçõesRESUMO
Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores, we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed the unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, filament-growth, and drug-binding at the single-molecule level demonstrating the promise of nanopore sensing for in-depth understanding of protein folding landscapes and for drug discovery.
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The cytosolic chaperonin CCT (chaperonin containing TCP-1) is an ATP-dependent double-ring protein machine mediating the folding of members of the eukaryotic cytoskeletal protein families. The actins and tubulins are obligate substrates of CCT because they are completely dependent on CCT activity to reach their native states. Genetic and proteomic analysis of the CCT interactome in the yeast Saccharomyces cerevisiae revealed a CCT network of approximately 300 genes and proteins involved in many fundamental biological processes. We classified network members into sets such as substrates, CCT cofactors and CCT-mediated assembly processes. Many members of the 7-bladed propeller family of proteins are commonly found tightly bound to CCT isolated from human and plant cells and yeasts. The anaphase promoting complex (APC/C) cofactor propellers, Cdh1p and Cdc20p, are also obligate substrates since they both require CCT for folding and functional activation. In vitro translation analysis in prokaryotic and eukaryotic cell extracts of a set of yeast propellers demonstrates their highly differential interactions with CCT and GroEL (another chaperonin). Individual propeller proteins have idiosyncratic interaction modes with CCT because they emerged independently with neo-functions many times throughout eukaryotic evolution. We present a toy model in which cytoskeletal protein biogenesis and folding flux through CCT couples cell growth and size control to time dependent cell cycle mechanisms.This article is part of a discussion meeting issue 'Allostery and molecular machines'.
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Chaperonina com TCP-1/química , Saccharomyces cerevisiae/química , Regulação Alostérica , Citosol/química , Células Eucarióticas/química , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
Actin filament turnover underpins several processes in the life cycle of the malaria parasite, Plasmodium falciparum. Polymerization and depolymerization are especially important for gliding motility, a substrate-dependent form of cell movement that underpins the protozoan parasite's ability to disseminate and invade host cells. To date, given difficulties in extraction of native actins directly from parasites, much of our biochemical understanding of malarial actin has instead relied on recombinant protein extracted and purified from heterologous protein expression systems. Here, using in vitro transcription-translation methodologies and quantitative protein-binding assays, we explored the folding state of heterologously expressed P. falciparum actin 1 (PfACTI) with the aim of assessing the reliability of current recombinant-protein-based data. We demonstrate that PfACTI, when expressed in non-native systems, is capable of binding to and release from bacterial, yeast, and mammalian chaperonin complexes but appears to be incompletely folded. Characterization of the native Plasmodium folding machinery in silico, the chaperonin containing t-complex protein-1 complex, highlights key divergences between the different chaperonin systems that likely underpins this incomplete folded state. These results highlight the importance of characterizing actin's folded state and raise concerns about the interpretation of actin polymerization kinetics based solely on protein derived from heterologous expression systems.
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Actinas/química , Chaperoninas/metabolismo , Plasmodium falciparum/metabolismo , Dobramento de Proteína , Proteínas de Protozoários/química , Actinas/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 µm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies.
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Fracionamento Celular/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Som , Proteína Supressora de Tumor p53/análise , Linhagem Celular Tumoral , Desenho de Equipamento , HumanosRESUMO
Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy.
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Peptídeos/química , Espectrofotometria Infravermelho , Tirosina/análogos & derivados , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Teoria Quântica , Tirosina/análiseRESUMO
Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.
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Análise Serial de Proteínas , Análise de Célula Única , Linhagem Celular Tumoral , Humanos , MicrofluídicaRESUMO
We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6 × 10(6) molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to calibrate data retrospectively to determine the absolute protein copy number at the single cell level in two human cancer cell lines.
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Técnicas Analíticas Microfluídicas/instrumentação , Análise Serial de Proteínas/instrumentação , Linhagem Celular Tumoral , Desenho de Equipamento , Proteínas de Fluorescência Verde/análise , Humanos , Neoplasias/química , Análise de Célula Única/instrumentação , Proteína Supressora de Tumor p53/análiseRESUMO
A central aspect of cellular systems biology is the study of cell-to-cell variability driven by network control of molecular noise. Proteins are produced in stochastic bursts and, although time averaging smoothes their accumulated levels, variation in their copy number is substantial in members of environmental sensing and signalling networks. We have developed a label-free, microfluidic antibody capture chip platform called the MAC chip, to quantify precisely the copy numbers of many proteins from a single cell in a multiplexed single assay format. We intend to investigate protein noise in circulating tumour cells (CTCs) isolated from biopsies of cancer patients through the identification of biomolecular signatures, such as p53 tumour suppressor protein, which correlate with biological properties and clinical outcomes during treatment.
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Microfluídica , Proteômica/métodos , Análise de Célula Única , Biologia de Sistemas/métodos , Humanos , Nanotecnologia , Neoplasias/patologia , Proteínas/análiseRESUMO
Measuring protein expression in single cells is the basis of single cell proteomics. The sensitivity and dynamic range of a single cell immunoassay should ideally be such that proteins that are expressed between 1-10(6) copies per cell can be detected and counted. We have investigated the effect of miniaturizing antibody microarrays by reducing capture spot sizes from 100 µm to 15 µm using dip-pen nanolithography. We demonstrate that protocols developed for printing and passivating antibody capture spots using conventional pin-based contact printing can be directly transferred to dip-pen lithography whilst retaining the capture activity per unit area. Using a simple kinetic model, we highlight how the limit of detection and dynamic range of a sandwich immunoassay, respectively, increase and decrease when spot size is reduced. However, we show that reducing spot size is more effective than increasing assay chamber volume when seeking to multiplex such a microfluidic immunoassay. Although we make particular reference to single cell microfluidic immunoassays, the topics discussed here are applicable to capture assays in general.
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Imunoensaio , Técnicas Analíticas Microfluídicas , Análise de Célula Única , Proteína Supressora de Tumor p53/análiseRESUMO
The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.