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.

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.

Zinc controls operator affinity of human transcription factor YY1 by mediating dimerization via its N-terminal region.

Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.

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.

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.

The FDA-Approved Antiviral Raltegravir Inhibits Fascin1-Dependent Invasion of Colorectal Tumor Cells In Vitro and In Vivo.

BACKGROUND: Fascin1 is the key actin-bundling protein involved in cancer invasion and metastasis whose expression is associated with bad prognosis in tumor from different origins. METHODS: In the present study, virtual screening (VS) was performed for the search of Fascin1 inhibitors and RAL, an FDA-approved inhibitor of human immunodeficiency virus-1 (HIV-1) integrase, was identified as a potential Fascin1 inhibitor. Biophysical techniques including nuclear magnetic resonance (NMR) and differential scanning fluorimetry (DSF) were carried out in order to confirm RAL as a Fascin1 blocker. The effect of RAL on actin-bundling activity Fascin1 was assessed by transmission electron microscopy (TEM), immunofluorescence, migration, and invasion assays on two human colorectal adenocarcinoma cell lines: HCT-116 and DLD-1. In addition, the anti-metastatic potential of RAL was in vivo evaluated by using the zebrafish animal model. RESULTS: NMR and DSF confirmed in silico predictions and TEM demonstrated the RAL-induced disorganization of the actin structure compared to control conditions. The protrusion of lamellipodia in cancer cell line overexpressing Fascin1 (HCT-116) was abolished in the presence of this drug. By following the addition of RAL, migration of HCT-116 and DLD-1 cell lines was significantly inhibited. Finally, using endogenous and exogenous models of Fascin1 expression, the invasive capacity of colorectal tumor cells was notably impaired in the presence of RAL in vivo assays; without undesirable cytotoxic effects. CONCLUSION: The current data show the in vitro and in vivo efficacy of the antiretroviral drug RAL in inhibiting human colorectal cancer cells invasion and metastasis in a Fascin1-dependent manner.

Methylglyoxal interaction with superoxide dismutase 1.

Methylglyoxal (MG) is a highly reactive aldehyde spontaneously formed in human cells mainly as a by-product of glycolysis. Such endogenous metabolite reacts with proteins, nucleotides and lipids forming advanced glycation end-products (AGEs). MG binds to arginine, lysine and cysteine residues of proteins causing the formation of stable adducts that can interfere with protein function. Among the proteins affected by glycation, MG has been found to react with superoxide dismutase 1 (SOD1), a fundamental anti-oxidant enzyme that is abundantly expressed in neurons. Considering the high neuronal susceptibility to MG-induced oxidative stress, we sought to investigate by mass spectrometry and NMR spectroscopy which are the structural modifications induced on SOD1 by the reaction with MG. We show that MG reacts preferentially with the disulfide-reduced, demetallated form of SOD1, gradually causing its unfolding, and to a lesser extent, with the intermediate state of maturation - the reduced, zinc-bound homodimer - causing its gradual monomerization. These results suggest that MG could impair the correct maturation of SOD1 in vivo, thus both increasing cellular oxidative stress and promoting the cytotoxic misfolding and aggregation process of SOD1.

Conformational characterization of full-length X-chromosome-linked inhibitor of apoptosis protein (XIAP) through an integrated approach.

The X-chromosome-linked inhibitor of apoptosis protein (XIAP) is a multidomain protein whose main function is to block apoptosis by caspase inhibition. XIAP is also involved in other signalling pathways, including NF-κB activation and copper homeostasis. XIAP is overexpressed in tumours, potentiating cell survival and resistance to chemotherapeutics, and has therefore become an important target for the treatment of malignancy. Despite the fact that the structure of each single domain is known, the conformation of the full-length protein has never been determined. Here, the first structural model of the full-length XIAP dimer, determined by an integrated approach using nuclear magnetic resonance, small-angle X-ray scattering and electron paramagnetic resonance data, is presented. It is shown that XIAP adopts a compact and relatively rigid conformation, implying that the spatial arrangement of its domains must be taken into account when studying the interactions with its physiological partners and in developing effective inhibitors.

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

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.

Cadmium effects on superoxide dismutase 1 in human cells revealed by NMR.

Cadmium is a toxic pollutant that in recent decades has become more widespread in the environment due to anthropogenic activities, significantly increasing the risk of exposure. Concurrently, a continually growing body of research has begun to enumerate the harmful effects that this heavy metal has on human health. Consequently, additional research is required to better understand the mechanism and effects of cadmium at the molecular level. The main mechanism of cadmium toxicity is based on the indirect induction of severe oxidative stress, through several processes that unbalance the anti-oxidant cellular defence system, including the displacement of metals such as zinc from its native binding sites. Such mechanism was thought to alter the in vivo enzymatic activity of SOD1, one of the main antioxidant proteins of many tissues, including the central nervous system. SOD1 misfolding and aggregation is correlated with cytotoxicity in neurodegenerative diseases such as amyotrophic lateral sclerosis. We assessed the effect of cadmium on SOD1 folding and maturation pathway directly in human cells through in-cell NMR. Cadmium does not directly bind intracellular SOD1, instead causes the formation of its intramolecular disulfide bond in the zinc-bound form. Metallothionein overexpression is strongly induced by cadmium, reaching NMR-detectable levels. The intracellular availability of zinc modulates both SOD1 oxidation and metallothionein overexpression, strengthening the notion that zinc-loaded metallothioneins help maintaining the redox balance under cadmium-induced acute stress.

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.

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

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.

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.

Identification of a novel nucleophosmin-interaction motif in the tumor suppressor p14arf.

The tumor suppressor p14arf interacts, in response to oncogenic signals, with the p53 E3-ubiquitin ligase HDM2, thereby resulting in p53 stabilization and activation. In addition, it also exerts tumor-suppressive functions in p53-independent contexts. The activities of p14arf are regulated by the nucleolar chaperone nucleophosmin (NPM1), which controls its levels and cellular localization. In acute myeloid leukemia with mutations in the NPM1 gene, mutated NPM1 aberrantly translocates in the cytosol carrying with itself p14arf that is subsequently degraded, thus impairing the p14arf-HDM2-p53 axis. In this work we investigated the complex between these two proteins by means of NMR and other techniques. We identified a novel NPM1-interacting motif in the C-terminal region of p14arf, which corresponds to its predicted nucleolar localization signal. This motif recognizes a specific region of the NPM1 N-terminal domain and, upon binding, the two proteins form soluble high molecular weight complexes. By NMR, we identified critical residues on both proteins involved in the interaction. Collectively, our data provide a structural framework to rationalize the overall assembly of the p14arf-NPM1 supramolecular complexes. A number of p14arf cancer-associated mutations cluster in this motif and their effect on the interaction with NPM1 was also analyzed.

Solution structure and interaction with copper in vitro and in living cells of the first BIR domain of XIAP.

The X-chromosome linked inhibitor of apoptosis (XIAP) is a multidomain metalloprotein involved in caspase inhibition and in copper homeostasis. It contains three zinc-binding baculoviral IAP repeats (BIR) domains, which are responsible for caspase interaction. Recently, it has been suggested that the BIR domains can bind copper, however high resolution data on such interaction is missing. Here we characterize by NMR the structural properties of BIR1 in solution, and the effects of its interaction with copper both in vitro and in physiological environments. BIR1 is dimeric in solution, consistent with the X-ray structure. Cysteine 12, located in the unfolded N-terminal region, has a remarkably low redox potential, and is prone to oxidation even in reducing physiological environments. Interaction of BIR1 with copper(II) results in the oxidation of cysteine 12, with the formation of either an intermolecular disulfide bond between two BIR1 molecules or a mixed disulfide bond with glutathione, whereas the zinc binding site is not affected by the interaction.

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

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.

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.

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

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.