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Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living ...

University of Florence

Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine − CIRMMP

In-cell NMR allows characterizing the folding state of a protein as well as posttranslational events at molecular level, in the cellular context. Here, the initial maturation steps of human copper, zinc superoxide dismutase 1 are characterized in the E. coli cytoplasm by in-cell NMR: from the apo protein, which is partially unfolded, to the zinc binding which causes its final quaternary structure. The protein selectively binds only one zinc ion, whereas in vitro also the copper site binds a non-physiological zinc ion. However, no intramolecular disulfide bridge formation occurs, nor copper uptake, suggesting the need of a specific chaperone for those purposes.

In-cell NMR in E. coli to monitor maturation steps of hSOD1.

We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.

Atomic-resolution monitoring of protein maturation in live human cells by NMR.

Most mitochondrial proteins are encoded by nuclear DNA, synthesized in the cytoplasm, and imported into mitochondria. Several proteins of the intermembrane space (IMS) are imported and localized through an oxidative process, being folded through the formation of structural disulfide bonds catalyzed by Mia40, and trapped in the IMS. To be imported, these proteins need to be reduced and unfolded; however, no structural information in situ exists on these proteins in the cytoplasm. In humans, Mia40 undergoes the same mechanism, although its folding state in the cytoplasm is unknown. We provide atomic-level details on the Mia40 folding state in the human cell cytoplasm through in-cell nuclear magnetic resonance. Overexpressed cytoplasmic Mia40 is folded, and its folding state depends on the glutaredoxin 1 (Grx1) and thioredoxin 1 (Trx1) systems. Specifically, increased Grx1 levels keep most Mia40 unfolded, while Trx1 is less effective. 

Visualization of redox-controlled protein fold in living cells.

Many eukaryotic proteins exert their physiological function in specific cellular compartments. Proteins of the inter-membrane space (IMS) of mitochondria, for example, are synthesized in the cytoplasm and translocate to the IMS, where they are further processed to their mature form. In-cell Nuclear Magnetic Resonance (NMR) has proven to be an ideal approach to investigate eukaryotic proteins at the atomic level, inside the cytoplasm. Here we show that proteins inside intact mitochondria isolated from human cells can be structurally characterized by NMR (in-mitochondria NMR). By this approach, we characterized the folding and maturation state of two human proteins in the IMS, SOD1 and Mia40. Both observed proteins were in the folded state. Mia40 was in the oxidized, functional state, while SOD1 disulfide bond formation was promoted by increasing the level of the SOD1 chaperone, CCS, in the IMS. 

Structural insights of proteins in sub-cellular compartments: In-mitochondria NMR.

Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone. 

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants.

In-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present, while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins. 

Protein interaction patterns in different cellular environments are revealed by in-cell NMR.

The redox properties of cellular environments are critical to many functional processes, and are strictly controlled in all living organisms. The glutathione-glutathione disulfide (GSH-GSSG) couple is the most abundant intracellular redox couple. A GSH redox potential can be calculated for each cellular compartment, which reflects the redox properties of that environment. This redox potential is often used to predict the redox state of a disulfide-containing protein, based on thermodynamic considerations. However, thiol-disulfide exchange reactions are often catalyzed by specific partners, and the distribution of the redox states of a protein may not correspond to the thermodynamic equilibrium with the GSH pool. Ideally, the protein redox state should be measured directly, bypassing the need to extrapolate from the GSH. Here, by in-cell NMR, we directly observe the redox state of three human proteins, Cox17, Mia40 and SOD1, in the cytoplasm of human and bacterial cells. We compare the observed distributions of redox states with those predicted by the GSH redox potential, and our results partially agree with the predictions. Discrepancies likely arise from the fact that the redox state of SOD1 is controlled by a specific partner, its copper chaperone (CCS), in a pathway which is not linked to the GSH redox potential. In principle, in-cell NMR allows determining whether redox proteins are at the equilibrium with GSH, or they are kinetically regulated. Such approach does not need assumptions on the redox potential of the environment, and provides a way to characterize each redox-regulating pathway separately. 

Direct structural evidence of protein redox regulation obtained by in-cell NMR.

In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

Algal autolysate medium to label proteins for NMR in mammalian cells.

In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection.

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells.

DJ-1 is a conserved, ubiquitous protein associated to a large number of intracellular processes. Human DJ-1 has been linked to several pathologies, including hereditary forms of Parkinson's disease, cancer, and amyotrophic lateral sclerosis. Several cytoprotective functions of DJ-1 have been reported, however, its actual mechanisms of action remain elusive. In vitro, DJ-1 has been shown to bind zinc and copper(II) at its active site, which contains a conserved cysteine (C106), and copper(I) at a different binding site. C106 is essential to DJ-1 function, and is easily oxidized upon oxidative stress. Here, we investigated the metal-binding- and redox properties of DJ-1 in living human cells by in-cell NMR. Intracellular DJ-1 is surprisingly free from interactions with any other cellular components and as such is clearly detectable by NMR. Metal-bound forms of DJ-1 were not observed upon treating the cells with excess zinc or copper. No copper binding was observed when co-expressing DJ-1 with the copper chaperone for superoxide dismutase 1 (SOD1). Co-expression of DJ-1 with SOD1 itself did not promote copper binding to SOD1, excluding a previously suggested function of DJ-1 as a copper chaperone. Overall, our data do not support the role of DJ-1 as a metalloprotein. Conversely, oxidative treatment to the cells caused the complete and selective oxidation of C106 to sulfinic acid, consistent with the reported role of DJ-1 as a redox sensor.

Intracellular metal binding and redox behavior of human DJ-1.

Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.

A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants.

Superoxide dismutase-1 (SOD1) mutants, including those with unaltered enzymatic activity, are known to cause amyotrophic lateral sclerosis (ALS). Several destabilizing factors contribute to pathogenicity including a reduced ability to complete the normal maturation process which comprises folding, metal cofactor acquisition, intra-subunit disulphide bond formation and dimerization. Immature SOD1 forms toxic oligomers and characteristic large insoluble aggregates within motor system cells. Here we report that the cysteine-reactive molecule ebselen efficiently confers the SOD1 intra-subunit disulphide and directs correct SOD1 folding, depopulating the globally unfolded precursor associated with aggregation and toxicity. Assisted formation of the unusual SOD1 cytosolic disulphide bond could have potential therapeutic applications. In less reducing environments, ebselen forms a selenylsulphide with Cys111 and restores the monomer-dimer equilibrium of A4V SOD1 to wild-type. Ebselen is therefore a potent bifunctional pharmacological chaperone for SOD1 that combines properties of the SOD1 chaperone hCCS and the recently licenced antioxidant drug, edaravone.

The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation.

FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies.

Real-Time Insights into Biological Events: In-Cell Processes and Protein-Ligand Interactions.

DJ-1 is a highly conserved soluble protein that is associated to several cellular pathways. In humans, DJ-1 has been implicated in several pathologies such as cancer, Parkinson's disease and amyotrophic lateral sclerosis. Several roles have been attributed to DJ-1, including defense against oxidative stress, chaperone activity and proteasome regulation. The recent finding that DJ-1 acts as a protein and DNA deglycase further confirms the protective function of DJ-1 and suggests a common mechanism of action in the various pathways in which DJ-1 is involved. Cysteine 106, located in the putative active site of DJ-1, is critical for the biological activity of DJ-1 and is easily oxidized to cysteine-sulfinate. While such oxidation modulates DJ-1 activity, the underlying molecular mechanism has not yet been elucidated. Cysteine oxidation does not perturb the protein structure, therefore changes in protein dynamics in solution could modulate its function. Here, we report a revised and completed (98%) backbone assignment of reduced DJ-1, together with the backbone assignment of oxidized DJ-1. Chemical shift perturbation is observed in several regions across the sequence, while no changes in secondary structure are observed. These data will provide the starting point for further characterization of the changes in the backbone dynamics of DJ-1 upon oxidation in solution at physiological temperature.

Backbone resonance assignment of human DJ-1 in the reduced state and in the cysteine sulfinic acid state.

In-cell NMR can investigate protein conformational changes at atomic resolution, such as those changes induced by drug binding or chemical modifications, directly in living human cells, and therefore has great potential in the context of drug development as it can provide an early assessment of drug potency. NMR bioreactors can greatly improve the cell sample stability over time and, more importantly, allow for recording in-cell NMR data in real time to monitor the evolution of intracellular processes, thus providing unique insights into the kinetics of drug-target interactions. However, current implementations are limited by low cell viability at >24 h times, the reduced sensitivity compared to "static" experiments and the lack of protocols for automated and quantitative analysis of large amounts of data. Here, we report an improved bioreactor design which maintains human cells alive and metabolically active for up to 72 h, and a semiautomated workflow for quantitative analysis of real-time in-cell NMR data relying on Multivariate Curve Resolution. We apply this setup to monitor protein-ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing further analysis by fitting with kinetic models. This unique approach can therefore be applied to investigate complex kinetic behaviors of macromolecules in a cellular setting, and could be extended in principle to any real-time NMR application in live cells.

Letizia Barbieri

Magnetic Resonance Center − CERM

University of Florence

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

Letizia Barbieri

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Magnetic Resonance Center − CERM

University of Florence

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

Magnetic Resonance Center − CERM