Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR.

A detailed knowledge of the complex processes that make cells and organisms alive is fundamental in order to understand diseases and to develop novel drugs and therapeutic treatments. To this aim, biological macromolecules should ideally be characterized at atomic resolution directly within the cellular environment. Among the existing structural techniques, solution NMR stands out as the only one able to investigate at high resolution the structure and dynamic behavior of macromolecules directly in living cells. With the advent of more sensitive NMR hardware and new biotechnological tools, modern in-cell NMR approaches have been established since the early 2000s. At the coming of age of in-cell NMR, we provide a detailed overview of its developments and applications in the 20 years that followed its inception. We review the existing approaches for cell sample preparation and isotopic labeling, the application of in-cell NMR to important biological questions, and the development of NMR bioreactor devices, which greatly increase the lifetime of the cells allowing real-time monitoring of intracellular metabolites and proteins. Finally, we share our thoughts on the future perspectives of the in-cell NMR methodology.

Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy.

In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into the folding, maturation, interactions, and ligand binding of important pharmacological targets directly in human cells. However, its widespread application is hampered by the fact that soluble globular proteins often interact with large cellular components, causing severe line broadening in conventional heteronuclear NMR experiments. 19F NMR can overcome this issue, as fluorine atoms incorporated in proteins can be detected by simple background-free 1D NMR spectra. Here, we show that fluorinated amino acids can be easily incorporated in proteins expressed in human cells by employing a medium switch strategy. This straightforward approach allows the incorporation of different fluorinated amino acids in the protein of interest, reaching fluorination efficiencies up to 60%, as confirmed by mass spectrometry and X-ray crystallography. The versatility of the approach is shown by performing 19F in-cell NMR on several proteins, including those that would otherwise be invisible by 1H-15N in-cell NMR. We apply the approach to observe the interaction between an intracellular target, carbonic anhydrase 2, and its inhibitors, and to investigate how the formation of a complex between superoxide dismutase 1 and its chaperone CCS modulates the interaction of the chaperone subunit with the cellular environment.

Protein in-cell NMR spectroscopy at 1.2 GHz.

In-cell NMR spectroscopy provides precious structural and functional information on biological macromolecules in their native cellular environment at atomic resolution. However, the intrinsic low sensitivity of NMR imposes a big limitation in the applicability of the methodology. In this respect, the recently developed commercial 1.2 GHz NMR spectrometer is expected to introduce significant benefits. However, cell samples may suffer from detrimental effects at ultrahigh fields, that must be carefully evaluated. Here we show the first in-cell NMR spectra recorded at 1.2 GHz on human cells, and we compare resolution and sensitivity against those obtained at 900 and 950 MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular α-synuclein and carbonic anhydrase. Major improvements are observed at 1.2 GHz when analyzing unfolded proteins, such as α-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins.

Biophysical characterization of the interaction between the full-length XIAP and Smac/DIABLO.

XIAP is multi-functional protein which regulates apoptosis acting as a direct caspase inhibitor. It is overexpressed in cancer cells, where it antagonizes the pro-apoptotic action of chemotherapeutics, and therefore it has become an important target for the treatment of cancer. In cells undergoing programmed cell death, the pro-apoptotic protein Smac is released by the mitochondria and binds to XIAP, thereby blocking caspase inhibition. Thus, Smac is considered a master regulator of apoptosis in mammals. In this regard, several Smac mimetic compounds have been developed to inhibit XIAP activity in cancer tissues. These compounds have shown low efficacy, partly due to the lack of structural knowledge of the XIAP-Smac interaction. In this work, through SEC-MALS and circular dichroism, we provide the first biophysical characterization of the interaction between the full-length form of XIAP and Smac, determining the stoichiometry of the complex and providing important information to develop more effective XIAP inhibitors.

Real-Time Quantitative In-Cell NMR: Ligand Binding and Protein Oxidation Monitored in Human Cells Using Multivariate Curve Resolution.

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.

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency.

Structure-based drug development is often hampered by the lack of in vivo activity of promising compounds screened in vitro, due to low membrane permeability or poor intracellular binding selectivity. Herein, we show that ligand screening can be performed in living human cells by "intracellular protein-observed" NMR spectroscopy, without requiring enzymatic activity measurements or other cellular assays. Quantitative binding information is obtained by fast, inexpensive 1 H NMR experiments, providing intracellular dose- and time-dependent ligand binding curves, from which kinetic and thermodynamic parameters linked to cell permeability and binding affinity and selectivity are obtained. The approach was applied to carbonic anhydrase and, in principle, can be extended to any NMR-observable intracellular target. The results obtained are directly related to the potency of candidate drugs, that is, the required dose. The application of this approach at an early stage of the drug design pipeline could greatly increase the low success rate of modern drug development.

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

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.

In-Cell NMR in Human Cells: Direct Protein Expression Allows Structural Studies of Protein Folding and Maturation.

Cellular structural biology methods are needed to characterize biological processes at atomic resolution in the physiological environment of the cell. Toward this goal, solution in-cell NMR is a powerful approach because it provides structural and dynamic data on macromolecules inside living cells. Several approaches have been developed for in-cell NMR in cultured human cells, which are needed to study processes related to human diseases that rely on the delivery of exogenous macromolecules to the cells. Such strategies, however, may not be applicable to proteins that are sensitive to the external environment or prone to aggregate and can introduce artifacts during protein purification or delivery. As a complementary approach, direct protein expression for in-cell NMR in human cells was developed. This strategy is especially useful when studying processes like protein folding, maturation, and post-translational modification, starting right after protein synthesis. Compared with the protein expression techniques in mammalian cells commonly used in cellular biology, the low sensitivity of NMR requires higher protein levels. Among the cell lines used for high-yield protein expression, the HEK293T cell line was chosen, as it can be efficiently transfected with a cost-effective reagent. A vector originally designed for secreted proteins allows high-level cytosolic protein expression. For isotopic labeling, commercially available or homemade labeled media are employed. Uniform or amino acid type-selective labeling strategies are possible. Protein expression can be targeted to specific organelles (e.g., mitochondria), allowing for in organello NMR applications. A variant of the approach was developed that allows the sequential expression of two or more proteins, with only one selectively labeled. Protein expression in HEK293T cells was applied to recapitulate the maturation steps of intracellular superoxide dismutase 1 (SOD1) and to study the effect of mutations linked to familial amyotrophic lateral sclerosis (fALS) by in-cell NMR. Intracellular wild-type SOD1 spontaneously binds zinc, while it needs the copper chaperone for superoxide dismutase (CCS) for copper delivery and disulfide bond formation. Some fALS-linked mutations impair zinc binding and cause SOD1 to irreversibly unfold, likely forming the precursor of cytotoxic aggregates. The SOD-like domain of CCS acts as a molecular chaperone toward mutant SOD1, stabilizing its folding and allowing zinc binding and correct maturation. Changes in protein redox state distributions can also be investigated by in-cell NMR. Mitochondrial proteins require the redox-regulating partners glutaredoxin 1 (Grx1) and thioredoxin (Trx) to remain in the reduced, import-competent state in the cytosol, whereas SOD1 requires CCS for disulfide bond formation. In both cases, the proteins do not equilibrate with the cytosolic redox pool. Cysteine oxidation in response to oxidative stress can also be monitored. In the near future, in-cell NMR in human cells will likely benefit from technological advancements in NMR hardware, the development of bioreactor systems for increased sample lifetime, the application of paramagnetic NMR to obtain structural restraints, and advanced tools for genome engineering and should be increasingly integrated with advanced cellular imaging techniques.

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

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.

A Unique Tool for Cellular Structural Biology: In-cell NMR.

Conventional structural and chemical biology approaches are applied to macromolecules extrapolated from their native context. When this is done, important structural and functional features of macromolecules, which depend on their native network of interactions within the cell, may be lost. In-cell nuclear magnetic resonance is a branch of biomolecular NMR spectroscopy that allows macromolecules to be analyzed in living cells, at the atomic level. In-cell NMR can be applied to several cellular systems to obtain biologically relevant structural and functional information. Here we summarize the existing approaches and focus on the applications to protein folding, interactions, and post-translational modifications.

Direct structural evidence of protein redox regulation obtained 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.

Sequential protein expression and selective labeling for in-cell NMR in human cells.

BACKGROUND: In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein-protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells. METHODS: We established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR. RESULTS: SOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by (1)H-(15)N heteronuclear NMR. CONCLUSIONS AND GENERAL SIGNIFICANCE: We showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein-protein interactions by NMR in human cells.

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

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.

The Casein Kinase 2-Dependent Phosphorylation of NS5A Domain†3 from Hepatitis†C Virus Followed by Time-Resolved NMR Spectroscopy.

Hepatitis C virus (HCV) chronically affects millions of individuals worldwide. The HCV nonstructural protein 5A (NS5A) plays a critical role in the viral assembly pathway. Domain 3 (D3) of NS5A is an unstructured polypeptide responsible for the interaction with the core particle assembly structure. Casein kinase 2 (CK2) phosphorylates NS5A-D3 at multiple sites that have mostly been predicted and only observed indirectly. In order to identify the CK2-dependent phosphorylation sites, we monitored the reaction between NS5A-D3 and CK2 in vitro by time-resolved NMR. We unambiguously identified four serine residues as substrates of CK2. The apparent rate constant for each site was determined from the reaction curves. Ser408 was quickly phosphorylated, whereas the three other serine residues reacted more slowly. These results provide a starting point from which to elucidate the role of phosphorylation in the mechanisms of viral assembly-and in the modulation of the viral activity-at the molecular level.

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

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.

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

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.

Ligand-Based Competition Binding by Real-Time 19F NMR in Human Cells.

The development of more effective drugs requires knowledge of their bioavailability and binding efficacy directly in the native cellular environment. In-cell nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for investigating ligand-target interactions directly in living cells. However, the target molecule may be NMR-invisible due to interactions with cellular components, while observing the ligand by 1H NMR is impractical due to the cellular background. Such limitations can be overcome by observing fluorinated ligands by 19F in-cell NMR as they bind to the intracellular target. Here we report a novel approach based on real-time in-cell 19F NMR that allows measuring ligand binding affinities in human cells by competition binding, using a fluorinated compound as a reference. The binding of a set of compounds toward Hsp90α was investigated. In principle, this approach could be applied to other pharmacologically relevant targets, thus aiding the design of more effective compounds in the early stages of drug development.

Controlling the incorporation of fluorinated amino acids in human cells and its structural impact.

Fluorinated aromatic amino acids (FAAs) are promising tools when studying protein structure and dynamics by NMR spectroscopy. The incorporation FAAs in mammalian expression systems has been introduced only recently. Here, we investigate the effects of FAAs incorporation in proteins expressed in human cells, focusing on the probability of incorporation and its consequences on the 19 F NMR spectra. By combining 19 F NMR, direct MS and x-ray crystallography, we demonstrate that the probability of FAA incorporation is only a function of the FAA concentration in the expression medium and is a pure stochastic phenomenon. In contrast with the MS data, the x-ray structures of carbonic anhydrase II reveal that while the 3D structure is not affected, certain positions lack fluorine, suggesting that crystallization selectively excludes protein molecules featuring subtle conformational modifications. This study offers a predictive model of the FAA incorporation efficiency and provides a framework for controlling protein fluorination in mammalian expression systems.

VDAC1-interacting molecules promote cell death in cancer organoids through mitochondrial-dependent metabolic interference.

The voltage-dependent anion-selective channel isoform 1 (VDAC1) is a pivotal component in cellular metabolism and apoptosis with a prominent role in many cancer types, offering a unique therapeutic intervention point. Through an in-silico-to-in-vitro approach we identified a set of VA molecules (VDAC Antagonists) that selectively bind to VDAC1 and display specificity toward cancer cells. Biochemical characterization showed that VA molecules can directly interact with VDAC1 with micromolar affinity by competing with the endogenous ligand NADH for a partially shared binding site. NADH displacement results in mitochondrial distress and reduced cell proliferation, especially when compared to non-cancerous cells. Experiments performed on organoids derived from intrahepatic cholangiocarcinoma patients demonstrated a dose-dependent reduction in cell viability upon treatment with VA molecules with lower impact on healthy cells than conventional treatments like gemcitabine. VA molecules are chemical entities representing promising candidates for further optimization and development as cancer therapy strategies through precise metabolic interventions.

Monastrol suppresses invasion and metastasis in human colorectal cancer cells by targeting fascin independent of kinesin-Eg5 pathway.

Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.