High-throughput discovery of novel small-molecule inhibitors of acid Ceramidase.

Ceramide has a key role in the regulation of cellular senescence and apoptosis. As Ceramide levels are lowered by the action of acid ceramidase (AC), abnormally expressed in various cancers, the identification of AC inhibitors has attracted increasing interest. However, this finding has been mainly hampered by the lack of formats suitable for the screening of large libraries. We have overcome this drawback by adapting a fluorogenic assay to a 384-well plate format. The performance of this optimised platform has been proven by the screening a library of 4100 compounds. Our results show that the miniaturised platform is well suited for screening purposes and it led to the identification of several hits, that belong to different chemical classes and display potency ranges of 2-25 µM. The inhibitors also show selectivity over neutral ceramidase and retain activity in cells and can therefore serve as a basis for further chemical optimisation.

Dinuclear silver and gold bisNHC complexes as drug candidates for cancer therapy.

We report four dinuclear silver(I) and gold(I) complexes containing two different bidentate N-heterocyclic carbene ligands (bisNHC). One of these complexes 4, shows strong and selective anticancer activity against the human ovarian cancer cell line A2780. Mechanistically, 4 enhances the oxidative stress by stimulating reactive oxygen species production and inhibiting the scavenging activity of thioredoxin reductase. Our findings provide evidence that tuning ligand and electronic properties of metal-NHC complexes can modulate their reactivity and selectivity and it may result in potential novel anticancer drugs.

Activity-Based Imaging of Acid Ceramidase in Living Cells.

Acid ceramidase (AC) hydrolyzes ceramides into sphingoid bases and fatty acids. The enzyme is overexpressed in several types of cancer and Alzheimer's disease, and its genetic defect causes different incurable disorders. The availability of a method for the specific visualization of catalytically active AC in intracellular compartments is crucial for diagnosis and follow-up of therapeutic strategies in diseases linked to altered AC activity. This work was undertaken to develop activity-based probes for the detection of AC. Several analogues of the AC inhibitor SABRAC were synthesized and found to act as very potent (two-digit nM range) irreversible AC inhibitors by reaction with the active site Cys143. Detection of active AC in cell-free systems was achieved either by using fluorescent SABRAC analogues or by click chemistry with an azide-substituted analogue. The compound affording the best features allowed the unprecedented labeling of active AC in living cells.

Small molecule inhibition of the KRAS-PDEδ interaction impairs oncogenic KRAS signalling.

The KRAS oncogene product is considered a major target in anticancer drug discovery. However, direct interference with KRAS signalling has not yet led to clinically useful drugs. Correct localization and signalling by farnesylated KRAS is regulated by the prenyl-binding protein PDEδ, which sustains the spatial organization of KRAS by facilitating its diffusion in the cytoplasm. Here we report that interfering with binding of mammalian PDEδ to KRAS by means of small molecules provides a novel opportunity to suppress oncogenic RAS signalling by altering its localization to endomembranes. Biochemical screening and subsequent structure-based hit optimization yielded inhibitors of the KRAS-PDEδ interaction that selectively bind to the prenyl-binding pocket of PDEδ with nanomolar affinity, inhibit oncogenic RAS signalling and suppress in vitro and in vivo proliferation of human pancreatic ductal adenocarcinoma cells that are dependent on oncogenic KRAS. Our findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic RAS.

High Affinity Immobilization of Proteins Using the CrAsH/TC Tag.

Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC) tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC) and an optimized motif (FLNCCPGCCMEP). While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters.

Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins.

Lipoprotein-binding chaperones mediate intracellular transport of lipidated proteins and determine their proper localisation and functioning. Understanding of the exact structural parameters that determine recognition and transport by different chaperones is of major interest. We have synthesised several lipid-modified peptides, representative of different lipoprotein classes, and have investigated their binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins-1 and -3. Our results demonstrate that PDEδ recognises S-isoprenylated C-terminal peptidic structures but not N-myristoylated peptides. In contrast, UNC119 proteins bind only mono-N-myristoylated, but do not recognise doubly lipidated and S-isoprenylated peptides at the C terminus. For galectins-1 and -3, neither binding to N-acylated, nor to C-terminally prenylated peptides could be determined. These results shed light on the specificity of the chaperone-mediated cellular lipoprotein transport systems.

Revealing conformational substates of lipidated N-Ras protein by pressure modulation.

Regulation of protein function is often linked to a conformational switch triggered by chemical or physical signals. To evaluate such conformational changes and to elucidate the underlying molecular mechanisms of subsequent protein function, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We apply pressure modulation in combination with FTIR spectroscopy to reveal equilibria between spectroscopically resolved substates of the lipidated signaling protein N-Ras. Pressure has the advantage that its thermodynamic conjugate is volume, a parameter that is directly related to structure. The conformational dynamics of N-Ras in its different nucleotide binding states in the absence and presence of a model biomembrane was probed by pressure perturbation. We show that not only nucleotide binding but also the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is accompanied by structural reorientations of the G domain, as also indicated by complementary ATR-FTIR and IRRAS measurements. These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner, which has consequences for the G-domain orientation of membrane-associated N-Ras, which in turn is known to be critical for its effector and modulator interactions. Finally, these results provide insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea where pressures reach the kbar range.

Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance--PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol--using 2H solid-state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Quantitative analysis of protein lipidation and acyl-CoAs reveals substrate preferences of the S-acylation machinery.

Protein palmitoylation or S-acylation has emerged as a key regulator of cellular processes. Increasing evidence shows that this modification is not restricted to palmitate but it can include additional fatty acids, raising the possibility that differential S-acylation contributes to the fine-tuning of protein activity. However, methods to profile the acyl moieties attached to proteins are scarce. Herein, we report a method for the identification and quantification of lipids bound to proteins that relies on hydroxylamine treatment and mass spectrometry analysis of fatty acid hydroxamates. This method has enabled unprecedented and extensive profiling of the S-acylome in different cell lines and tissues and has shed light on the substrate specificity of some S-acylating enzymes. Moreover, we could extend it to quantify also the acyl-CoAs, which are thioesters formed between a fatty acid and a coenzyme A, overcoming many of the previously described challenges for the detection of such species. Importantly, the simultaneous analysis of the lipid fraction and the proteome allowed us to establish, for the first time, a direct correlation between the endogenous levels of acyl-CoAs and the S-acylation profile of its proteome.

Semisynthetic lipidated LC3 protein mediates membrane fusion.

All together: Lipidated LC3 has been synthesized by expressed protein ligation. A TEV-cleavable MBP tag was employed to facilitate ligation under folding conditions and to solubilize the lipidated protein. The synthetic LC3-phosphatidylethanolamine (PE) mediates membrane tethering and fusion at the physiological concentration of PE, and could be a useful tool for autophagy studies.

Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo.

Lipidated Rho and Rab GTP-binding proteins are transported between membranes in complex with solubilizing factors called 'guanine nucleotide dissociation inhibitors' (GDIs). Unloading from GDIs using GDI displacement factors (GDFs) has been proposed but remains mechanistically elusive. PDEδ is a putative solubilizing factor for several prenylated Ras-subfamily proteins. Here we report the structure of fully modified farnesylated Rheb-GDP in complex with PDEδ. The structure explains the nucleotide-independent binding of Rheb to PDEδ and the relaxed specificity of PDEδ. We demonstrate that the G proteins Arl2 and Arl3 act in a GTP-dependent manner as allosteric release factors for farnesylated cargo. We thus describe a new transport system for farnesylated G proteins involving a GDI-like molecule and an unequivocal GDF. Considering the importance of PDEδ for proper Ras and Rheb signaling, this study is instrumental in developing a new target for anticancer therapy.

Rotational and translational dynamics of ras proteins upon binding to model membrane systems.

Plasma-membrane-associated Ras proteins typically control signal transduction processes. As nanoclustering and membrane viscosity sensing provide plausible signaling mechanisms, determination of the rotational and translational dynamics of membrane-bound Ras isoforms can help to link their dynamic mobility to their function. Herein, by using time-resolved fluorescence anisotropy and correlation spectroscopic measurements, we obtain the rotational-correlation time and the translational diffusion coefficient of lipidated boron-dipyrromethene-labeled Ras, both in bulk Ras and upon membrane binding. The results show that the second lipidation motif of N-Ras triggers dimer formation in bulk solution, whereas K-Ras4B is monomeric. Upon membrane binding, an essentially free rotation of the G-domain is observed, along with a high lateral mobility; the latter is essentially limited by the viscosity of the membrane and by lipid-mediated electrostatic interactions. This high diffusional mobility warrants rapid recognition-binding sequences in the membrane-bound state, thereby facilitating efficient interactions between the Ras proteins and scaffolding or effector proteins. The lipid-like rapid lateral diffusion observed here complies with in vivo data.

N-Ras forms dimers at POPC membranes.

Ras is a central regulator of cellular signaling pathways. It is mutated in 20-30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between ß2 and ß3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants.

Dissociation of the K-Ras4B/PDEδ complex upon contact with lipid membranes: membrane delivery instead of extraction.

K-Ras4B is a small GTPase whose selective membrane localization and clustering into microdomains are mediated by its polybasic farnesylated C-terminus. The importance of the subcellular distribution for the signaling activity of K-Ras4B became apparent from recent in vivo studies, showing that the delta subunit of cGMP phosphodiesterase (PDEδ), which possesses a hydrophobic prenyl-binding pocket, is able to function as a potential binding partner for farnesylated proteins, thereby leading to a modulation of the spatiotemporal organization of K-Ras. Even though PDEδ has been suggested to serve as a cytosolic carrier for Ras, the functional transport mechanism still remains largely elusive. In this study, the effect of PDEδ on the interaction of GDP- and GTP-loaded K-Ras4B with neutral and anionic model biomembranes has been investigated by a combination of different spectroscopic and imaging techniques. The results show that PDEδ is not able to extract K-Ras4B from membranes. Rather, the K-Ras4B/PDEδ complex formed in bulk solution turned out to be unstable in the presence of heterogeneous membranes, resulting in a release of farnesylated K-Ras4B upon membrane contact. With the additional observation of enhanced membrane affinity for the K-Ras4B/PDEδ complex, a molecular mechanism for the PDEδ-K-Ras4B-membrane interaction could be proposed. This includes an effective delivery of PDEδ-solubilized K-Ras4B to the plasma membrane, probably through cytoplasmic diffusion, the dissociation of the K-Ras4B/PDEδ complex upon plasma membrane contact, and finally the membrane binding of released farnesylated K-Ras4B that leads to K-Ras4B-enriched microdomain formation.

Chemical-biological exploration of the limits of the Ras de- and repalmitoylating machinery.

A dynamic de-/repalmitoylation cycle determines localization and activity of H- and N-Ras. This combined cellular de- and repalmitoylation machinery has been shown to be substrate tolerant--it accepts variation of amino acid sequence, structure and configuration. Here, semisynthetic Ras-proteins in which the C-terminal amino acids are replaced by peptoid residues are used to reveal the first limitations of substrate recognition by the de- and repalmitoylating machinery.

Bioorthogonal chemistry for site-specific labeling and surface immobilization of proteins.

Understanding protein structure and function is essential for uncovering the secrets of biology, but it remains extremely challenging because of the high complexity of protein networks and their wiring. The daunting task of elucidating these interconnections requires the concerted application of methods emerging from different disciplines. Chemical biology integrates chemistry, biology, and pharmacology and has provided novel techniques and approaches to the investigation of biological processes. Among these, site-specific protein labeling with functional groups such as fluorophors, spin probes, and affinity tags has greatly facilitated both in vitro and in vivo studies of protein structure and function. Bioorthogonal chemical reactions, which enable chemo- and regioselective attachment of small-molecule probes to proteins, are particularly attractive and relevant for site-specific protein labeling. The introduction of powerful labeling techniques also has inspired the development of novel strategies for surface immobilization of proteins to create protein biochips for in vitro characterization of biochemical activities or interactions between proteins. Because this process requires the efficient immobilization of proteins on surfaces while maintaining structure and activity, tailored methods for protein immobilization based on bioorthogonal chemical reactions are in high demand. In this Account, we summarize recent developments and applications of site-specific protein labeling and surface immobilization of proteins, with a special focus on our contributions to these fields. We begin with the Staudinger ligation, which involves the formation of a stable amide bond after the reaction of a preinstalled azide with a triaryl phosphine reagent. We then examine the Diels-Alder reaction, which requires the protein of interest to be functionalized with a diene, enabling conjugation to a variety of dienophiles under physiological conditions. In the oxime ligation, an oxyamine is condensed with either an aldehyde or a ketone to form an oxime; we successfully pursued the inverse of the standard technique by attaching the oxyamine, rather than the aldehyde, to the protein. The click sulfonamide reaction, which involves the Cu(I)-catalyzed reaction of sulfonylazides with terminal alkynes, is then discussed. Finally, we consider in detail the photochemical thiol-ene reaction, in which a thiol adds to an ene group after free radical initiation. Each of these methods has been successfully developed as a bioorthogonal transformation for oriented protein immobilization on chips and for site-specific protein labeling under physiological conditions. Despite the tremendous progress in developing such transformations over the past decade, however, the demand for new bioorthogonal methods with improved kinetics and selectivities remains high.

The role of G-domain orientation and nucleotide state on the Ras isoform-specific membrane interaction.

Ras proteins are proto-oncogenes that function as molecular switches linking extracellular stimuli with an overlapping but distinctive range of biological outcomes. Although modulatable interactions between the membrane and the Ras C-terminal hypervariable region (HVR) harbouring the membrane anchor motifs enable signalling specificity to be determined by their location, it is becoming clear that the spatial orientation of different Ras proteins is also crucial for their functions. To reveal the orientation of the G-domain at membranes, we conducted an extensive study on different Ras isoforms anchored to model raft membranes. The results show that the G-domain mediates the Ras-membrane interaction by inducing different sets of preferred orientations in the active and inactive states with largely parallel orientation relative to the membrane of most of the helices. The distinct locations of the different isoforms, exposing them to different effectors and regulators, coupled with different G-domain-membrane orientation, suggests synergy between this type of recognition motif and the specificity conferred by the HVR, thereby validating the concept of isoform specificity in Ras.

Visualizing association of lipidated signaling proteins in heterogeneous membranes--partitioning into subdomains, lipid sorting, interfacial adsorption, and protein association.

In a combined chemical biological and biophysical approach, we studied the partitioning of differently fluorescent-labeled palmitoyl and/or farnesyl lipidated peptides, which represent membrane recognition model systems, as well as the full lipidated N-Ras protein into various model membrane systems including canonical model raft mixtures. To this end, two-photon fluorescence microscopy on giant unilamellar vesicles, complemented by tapping-mode atomic force microscopy (AFM) measurements, was carried out. The measurements were performed over a wide temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (solid-ordered (gel), fluid/gel, liquid-ordered/liquid-disordered, all-fluid). The results provide direct evidence that partitioning of the lipidated peptides and N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is liquid-disordered>liquid-ordered>>solid-ordered. Intriguingly, we detect - using the better spatial resolution of AFM - also a large proportion of the lipidated protein located at the liquid-disordered/liquid-ordered phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of neighboring domains. In an all-liquid-ordered, cholesterol-rich phase, phase separation can be induced by an effective lipid sorting mechanism owing to the high affinity of the lipidated peptides and proteins to a fluid-like lipid environment. At low temperatures, where the overall acyl chain order parameter of the lipid bilayer has markedly increased, such an efficient lipid sorting mechanism is energetically too costly and self-association of the peptide into small clusters takes place. These data reveal the interesting ability of the lipidated peptides and proteins to induce formation of fluid microdomains at physiologically relevant high cholesterol concentrations. Furthermore, our results reveal self-association of the N-Ras protein at the domain boundaries which may serve as an important vehicle for association processes and nanoclustering, which has also been observed in in vivo studies.

Membrane-mediated induction and sorting of K-Ras microdomain signaling platforms.

The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (l(d)) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the l(d) phase and concentrate at the l(d)/l(o) phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.

Identification of acyl protein thioesterases 1 and 2 as the cellular targets of the Ras-signaling modulators palmostatin B and M.

Finding the target: activity-based proteomic profiling probes based on the depalmitoylation inhibitors palmostatin B and M have been synthesized and were found to target acyl protein thioesterase 1 (APT1) and 2 (APT2) in cells.