RESUMO
Intracellular aggregation of the human amyloid protein α-synuclein is causally linked to Parkinson's disease. While the isolated protein is intrinsically disordered, its native structure in mammalian cells is not known. Here we use nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to derive atomic-resolution insights into the structure and dynamics of α-synuclein in different mammalian cell types. We show that the disordered nature of monomeric α-synuclein is stably preserved in non-neuronal and neuronal cells. Under physiological cell conditions, α-synuclein is amino-terminally acetylated and adopts conformations that are more compact than when in buffer, with residues of the aggregation-prone non-amyloid-ß component (NAC) region shielded from exposure to the cytoplasm, which presumably counteracts spontaneous aggregation. These results establish that different types of crowded intracellular environments do not inherently promote α-synuclein oligomerization and, more generally, that intrinsic structural disorder is sustainable in mammalian cells.
Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Acetilação , Linhagem Celular , Citoplasma/química , Citoplasma/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Células HeLa , Humanos , Neurônios/citologia , Neurônios/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
We propose a method to dynamically monitor the progress of an enzymatic reaction using NMR of hyperpolarized 129 Xe in a host-guest system. It is based on a displacement assay originally designed for fluorescence experiments that exploits the competitive binding of the enzymatic product on the one hand and a reporter dye on the other hand to a supramolecular host. Recently, this assay has been successfully transferred to NMR, using xenon as a reporter, cucurbit[6]uril as supramolecular host, and chemical exchange saturation transfer with hyperpolarized Xe (Hyper-CEST) as detection technique. Its advantage is that the enzyme acts on the unmodified substrate and that only the product is detected through immediate inclusion into the host. We here apply a method that drastically accelerates the acquisition of Hyper-CEST spectra in vitro using magnetic field gradients. This allows monitoring the dynamic progress of the conversion of lysine to cadaverine with a temporal resolution of ~30 s. Moreover, the method only requires to sample the very early onset of the reaction (<0.5% of substrate conversion where the host itself is required only at µM concentrations) at comparatively low reaction rates, thus saving enzyme material and reducing NMR acquisition time. The obtained value for the specific activity agrees well with previously published results from fluorescence assays. We furthermore outline how the Hyper-CEST results correlate with xenon T2 measurements performed during the enzymatic reaction. This suggests that ultrafast Hyper-CEST spectroscopy can be used for dynamically monitoring enzymatic activity with NMR.
Assuntos
Carboxiliases/química , Bacillus/enzimologia , Hidrocarbonetos Aromáticos com Pontes/química , Cadaverina/química , Catálise , Imidazóis/química , Cinética , Lisina/química , Espectroscopia de Ressonância Magnética/métodos , Xenônio/químicaRESUMO
Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Células K562 , Fosfotransferases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Fatores de TempoRESUMO
The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper-CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper-CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live-cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell-surface glycans at nanomolar concentrations.
Assuntos
Técnicas Biossensoriais , Imageamento por Ressonância Magnética , Polissacarídeos/metabolismo , Xenônio/química , Sobrevivência Celular , Meios de Contraste/química , Imagem Molecular , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Propriedades de SuperfícieRESUMO
We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
Assuntos
Butadienos/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Biocatálise/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Células HCT116 , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Reprodutibilidade dos Testes , Especificidade por Substrato , Fatores de TempoRESUMO
Methylation and acetylation of protein lysine residues constitute abundant post-translational modifications (PTMs) that regulate a plethora of biological processes. In eukaryotic proteins, lysines are often mono-, di-, or trimethylated, which may signal different biological outcomes. Deconvoluting these different PTM types and PTM states is not easily accomplished with existing analytical tools. Here, we demonstrate the unique ability of NMR spectroscopy to discriminate between lysine acetylation and mono-, di-, or trimethylation in a site-specific and quantitative manner. This enables mapping and monitoring of lysine acetylation and methylation reactions in a nondisruptive and continuous fashion. Time-resolved NMR measurements of different methylation events in complex environments including cell extracts contribute to our understanding of how these PTMs are established in vitro and in vivo.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Acetilação , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Lisina/análise , Espectroscopia de Ressonância Magnética , Metilação , Processamento de Proteína Pós-TraducionalRESUMO
Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.
Assuntos
Doença de Parkinson/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Motivos de Aminoácidos , Humanos , Espectroscopia de Ressonância Magnética , Metionina/metabolismo , Oxirredução , Estresse Oxidativo , Fosforilação , Serina/metabolismoRESUMO
A nanoparticulate carrier system is used to efficiently deliver a contrast agent for highly sensitive xenon Hyper-CEST MRI. The carrier system not only improves the biocompatibility and solubility of the contrast agent, it also allows selective cell targeting as demonstrated by the discrimination of human brain capillary and aortic endothelial cells.
Assuntos
Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/metabolismo , Células Endoteliais/diagnóstico por imagem , Células Endoteliais/metabolismo , Angiografia por Ressonância Magnética , Peptídeos/farmacologia , Células Cultivadas , Humanos , Lipossomos , Peptídeos/química , RadiografiaRESUMO
We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the ¹5N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D ¹H-¹5N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.
Assuntos
Extratos Celulares/análise , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/metabolismo , Serina/metabolismo , Treonina/metabolismo , Animais , Humanos , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas/análise , Serina/análise , Treonina/análise , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Delivery of isotope-labeled IDPs into mammalian cells for the purpose of generating suitable in-cell NMR samples can also be facilitated by action of pore-forming bacterial toxins. In the course of this procedure, mammalian cell membranes are permeated for short periods of time in order to enable the influx of exogenous proteins via a concentration gradient between the outside and the inside of the targeted "host" cells. In contrast to CPP-mediated IDP uptake, toxins offer the advantage that cellular protein transduction does not rely on active biological processes like endocytosis, but on simple passive diffusion. Therefore, proteins that are to be delivered into mammalian cells are not required to contain additional "targeting" sequences, and can be employed in their native contexts. The protocol outlined here employs isotope-labeled human α-synuclein, adherent human HeLa cells, and the Streptococcus pyogenes endotoxin Streptolysin O (SLO).
Assuntos
Ressonância Magnética Nuclear Biomolecular , alfa-Sinucleína/química , Proteínas de Bactérias/farmacologia , Soluções Tampão , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células HeLa , Humanos , Conformação Proteica , Proteínas Recombinantes/química , Estreptolisinas/farmacologiaRESUMO
Many mammalian IDPs exert important biological functions in key cellular processes and often in highly specialized subsets of cells. For these reasons, tools to characterize the structural and functional characteristics of IDPs inside mammalian cells are of particular interest. Moving from bacterial and amphibian in-cell NMR experiments to mammalian systems offers the unique opportunity to advance our knowledge about general IDP properties in native cellular environments. This is never more relevant than for IDPs that exhibit pathological structural rearrangements under certain cellular conditions, as is the case for human α-synuclein in dopaminergic neurons of the substantia nigra in the course of Parkinson's disease, for example. To efficiently deliver isotope-labeled IDPs into mammalian cells is one of the first challenges when preparing a mammalian in-cell NMR sample. The method presented here provides a detailed protocol for the transduction of isotope-labeled α-synuclein, as a model IDP, into cultured human HeLa cells. Cellular IDP delivery is afforded by action of a cell-penetrating peptide (CPP) tag. In the protocol outlined below, the CPP tag is "linked" to the IDP cargo moiety via an oxidative, disulfide-coupling reaction.
Assuntos
Ressonância Magnética Nuclear Biomolecular , alfa-Sinucleína/química , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/isolamento & purificação , Células HeLa , Humanos , Acoplamento Oxidativo , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Irrespective of how isotope-labeled proteins are delivered into mammalian cells, laboratory routines are needed to assess the quality of the resulting in-cell NMR samples. These include methods to evaluate overall cell viability, protein transduction efficiency, intracellular protein concentration, localization, and stability. In addition, quality control experiments to assess protein leakage from manipulated cells are of particular importance for in-cell NMR experiments. The purpose of this chapter is to outline qualitative and quantitative methods to determine general biological properties of in-cell NMR samples in order to ensure the highest possible standards for in-cell NMR studies.