Surface change of Ras enabling effector binding monitored in real time at atomic resolution

Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP-to-GTP exchange. This modifies the Ras surface (Ras(on)GTP) and enables effector binding, which then activates signal-transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to "off" (Ras(off)GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras(off)-to-Ras(on) transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time-resolved FTIR (trFTIR) spectroscopy. In the transition from Ras(off) to Ras(on) a GTP-bound intermediate is now identified, in which effector binding is still prevented (Ras(off)GTP). The loop movement from Ras(off)GTP to Ras(on)GTP was directly monitored by the C=O vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s(-1) at 260 K. A small molecule that shifted the equilibrium from the Ras(on)GTP state towards the Ras(off)GTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules.     

Identification and Characterization of a Peptidic Ligand for Ras

The development of new ligands for the oncoprotein Ras can provide tools for the study of this important signaling component or potentially serve as therapeutic agents for the treatment of Ras‐associated diseases. Herein, we report a peptidic Ras ligand identified through naïve phage display. Panning a phage library with a diversity of 10⁹ transormants successfully identified a peptide dodecamer that contains two internal consensus motifs and binds Ras in both the active GTP‐ and inactive GDP‐bound conformations with low micromolar dissociation constants. The dodecamer does not alter the intrinsic GTPase activity of Ras, does not compete for Ras binding to the Ras binding domain of Raf, and does not alter cell viability. This novel Ras ligand has the potential to serve in the development of higher‐affinity ligands and chemical tools targeting Ras.     

Site-specific functionalization of proteins by organopalladium reactions

A new carbon-carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki-Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild-type Ras protein. Finally, the iF-Ras proteins containing a 4-iodo-L-phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS.     

Sequence-Selective Covalent CaaX-Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3 K Signaling

Oncogenic Ras proteins are implicated in the most common life-threatening cancers. Despite intense research over the past two decades, the progress towards small-molecule inhibitors has been limited. One reason for this failure is that Ras proteins interact with their effectors only via protein-protein interactions, which are notoriously difficult to address with small organic molecules. Herein we describe an alternative strategy, which prevents farnesylation and subsequent membrane insertion, a prerequisite for the activation of Ras proteins. Our approach is based on sequence-selective supramolecular receptors which bind to the C-terminal farnesyl transferase recognition unit of Ras and Rheb proteins and covalently modify the essential cysteine in the so-called CaaX-box.     

Millisecond Allosteric Dynamics of Activated Ras Reproduced with a Slowly Hydrolyzable GTP Analogue

The millisecond timescale dynamics of activated Ras transiently sample a low-populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small-molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non-hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP. It was found that GTPγS, in addition to its better mimicking of the exchange kinetics than the commonly used non-hydrolyzable analogues GppNHp and GppCH₂p, can rigorously reproduce the natural dynamics network in active Ras, thus indicating its fitness for use in the development of allosteric inhibitors.     

Synthesis and application of fluorescent ras proteins for live-cell imaging

Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.     

Synthesis of GTP-derived Ras ligands

A practical and convenient method for the synthesis of acid- and base-sensitive GTP analogues carrying a further substituent at the terminal phosphate has been developed. Key to the successful synthesis of these potential ligands of the Ras protein is the use of Pd0-sensitive allyl protecting groups in a one-pot synthesis that avoids evaporation steps. Initial biochemical analysis of a representative compound revealed that such GTP analogues can bind to Ras and might open up the possibility of developing small molecules that can act as deactivators of oncogenic Ras.     

Boron‐Based Inhibitors of Acyl Protein Thioesterases 1 and 2

Ras proteins are of importance in cell proliferation, and hence their mutated forms play causative roles in many kinds of cancer in different tissues. Inhibition of the Ras‐depalmitoylating enzyme acyl protein thioesterases APT1 and ‐2 is a new approach to modulating the Ras cycle. Here we present boronic and borinic acid derivatives as a new class of potent and nontoxic APT inhibitors. These compounds were detected by extensive library screening using chemical arrays and turned out to inhibit human APT1 and ‐2 in a competitive mode. Furthermore, one of the molecules was demonstrated to inhibit Erk1/2 phosphorylation significantly.     

A method for computational combinatorial peptide design of inhibitors of Ras protein

A computational combinatorial approach is proposed for the designof a peptide inhibitor of Ras protein. The procedure involvesthree steps. First, a `Multiple Copy Simultaneous Search' identifiesthe location of specific functional groups on the Ras surface.This search method allowed us to identify an important bindingsurface consisting of two ß strands (residues 5–8and 52–56), in addition to the well known Ras effectorloop and switch II region. The two ß strands had not previouslybeen reported to be involved in Ras–Raf interaction. Second,after constructing the peptide inhibitor chain based on thelocation of N-methylacetamide (NMA) minima, functional groupsare selected and connected to the main chain C atom. This stepgenerates a number of possible peptides with different sequenceson the Ras surface. Third, potential inhibitors are designedbased on a sequence alignment of the peptides generated in thesecond step. This computational approach reproduces the conservedpattern of hydrophobic, hydrophilic and charged amino acidsidentified from the Ras effectors. The advantages and limitationsof this approach are discussed.     

Photochemical modulation of Ras-mediated signal transduction using caged farnesyltransferase inhibitors: activation by one- and two-photon excitation

The creation of caged molecules involves the attachment of protecting groups to biologically active compounds such as ligands, substrates and drugs that can be removed under specific conditions. Photoremovable caging groups are the most common due to their ability to be removed with high spatial and temporal resolution. Here, the synthesis and photochemistry of a caged inhibitor of protein farnesyltransferase is described. The inhibitor, FTI, was caged by alkylation of a critical thiol group with a bromohydroxycoumarin (Bhc) moiety. While Bhc is well established as a protecting group for carboxylates and phosphates, it has not been extensively used to cage sulfhydryl groups. The resulting caged molecule, Bhc-FTI, can be photolyzed with UV light to release the inhibitor that prevents Ras farnesylation, Ras membrane localization and downstream signaling. Finally, it is shown that Bhc-FTI can be uncaged by two-photon excitation to produce FTI at levels sufficient to inhibit Ras localization and alter cell morphology. Given the widespread involvement of Ras proteins in signal transduction pathways, this caged inhibitor should be useful in a plethora of studies.     

Imaging activation of two Ras isoforms simultaneously in a single cell

Fluorescence resonance energy transfer (FRET) microscopy approaches have been used to study protein interactions in living cells. Up to now, due to the spectral requirements for FRET detection, this has been limited to the measurement of single protein interactions. Here we present a novel time-resolved fluorescence imaging method for simultaneously monitoring the activation state of two proteins in a single cell. A Ras sensor, consisting of fluorescently labelled Ras and a fluorescently labelled Ras binding domain (RBD) of Raf, which reads out Ras activation by its interaction with RBD as a FRET signal, has been adapted for this purpose. By using yellow (YFP) and cyan (CFP) versions of the green fluorescent protein from Aquorea victoria as donors and a tandem construct of Heteractis crispa Red (tHcRed) as acceptor for both donors, two independent FRET signals can be measured at the same time. Measuring the YFP and CFP donor lifetimes by fluorescence-lifetime imaging microscopy (FLIM) allows us to distinguish the two different FRET signals in a single cell. Using this approach, we show that different Ras isoforms and mutants that localize to the plasma membrane, to the Golgi or to both compartments display distinct activation profiles upon growth-factor stimulation; this indicates that there is a differential regulation in cellular compartments. The method presented here is especially useful when studying spatiotemporal aspects of protein regulation as part of larger cellular signalling networks.     

Linking the fields--the interplay of organic synthesis, biophysical chemistry, and cell biology in the chemical biology of protein lipidation

Research in the biological sciences has undergone a fundamental and dramatic change during the last decades. Whereas biology was more phenomenologically oriented for a long time, today many biological processes are investigated and understood in molecular detail. It has become evident that all biological phenomena have a chemical basis: Biology is based on chemical principles. In the past, this insight had led to the development of biochemistry, molecular biology, and modern pharmacology. Today it increasingly determines the manner in which various biological phenomena are studied. The tools provided by classical biological techniques often are not sufficient to address the prevailing issues in precise molecular detail. Instead, the strengths of both chemical and biological methodology have to be used. Several recent research projects have proven that combining the power of organic synthesis with cell biology may open up entirely new and alternative opportunities for the study of biological problems. In this review we summarize the successful interplay between three disciplines-organic synthesis, biophysics, and cell biology-in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in precise molecular detail. This interplay is highlighted by using the Ras protein as a representative example. The development of methods for the synthesis of Ras-derived peptides and fully functional Ras proteins, the determination of their biophysical properties, in particular the ability to bind to model membranes, and finally the use of synthetic Ras peptides and Ras proteins in cell biological experiments are addressed. The successful combination of these three disciplines has led to a better understanding of the factors governing the selective targeting of Ras and related lipid-modified proteins to the plasma membrane.     

Mapping the isoprenoid binding pocket of PDEdelta by a semisynthetic, photoactivatable N-Ras lipoprotein

Biologically functional Ras isoforms undergo post-translational modifications starting with farnesylation of the most C-terminal cysteine. Combined with further processing steps, this isoprenylation allows for the anchoring of these proteins in endomembranes, where signal transduction events take place. The specific localization is subject to dynamic regulation and assumed to modulate the activity of Ras proteins by governing their spatiotemporal distribution. The delta subunit of phosphodiesterase (PDEdelta) has attracted attention as a solubilization factor of isoprenylated Ras. In this study, we demonstrate that critical residues in the putative isoprenoid pocket of PDEdelta can be mapped by coupling with a semisynthetic N-Ras lipoprotein in which the native farnesyl group of the processed protein was replaced by a photoactivatable geranyl benzophenone moiety. The crosslinked product included parts of beta-sheet 9 of PDEdelta, which contains the highly conserved amino acids V145 and L147. Modeling of the PDEdelta-geranyl benzophenone (GerBP) complex supports the conclusion that the photolabeled sequence is embedded in the putative isoprenoid pocket of PDEdelta.     

Ras-mediated cleavage of a GTP analogue by a novel mechanism

The small guanosine triphosphate (GTP) binding protein Ras is involved in many cellular signal transduction processes leading to cell growth, differentiation and apoptosis. Mutations in ras genes are found in a large number of human tumours. GTP hydrolysis, the process that normally leads to the transition of the Ras protein from the active (GTP-bound) form to the inactive (GDP-bound) form is impaired due to these oncogenic mutations. In contrast, the GTP analogue 3,4-diaminobenzophenone(DABP)-phosphoramidate-GTP, a substrate for GTP-binding proteins, enables switching to the inactive GDP form in both wild-type and oncogenic Ras. Here we show by HPLC, mass spectrometry and NMR spectroscopy that the mechanism of this DABP-GTPase reaction is different from the physiological GTPase reaction. The gamma-phosphate group is not attacked by a nucleophilic water molecule, but rather by the aromatic amino group of the analogue, which leads to the generation of a stable cyclic diamidate product. These findings have potential implications for the development of anti-Ras drugs.     

Enhanced antiproliferative and apoptotic response of HT-29 adenocarcinoma cells to combination of photoactivated hypericin and farnesyltransferase inhibitor manumycin A

Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells.     

Modular Assembly of Allosteric MEK Inhibitor Structural Elements Unravels Potency and Feedback‐Modulation Handles

Having recently identified a so‐far unexplored area adjacent to the known binding site of allosteric mitogen‐activated protein kinase kinase (MEK) inhibitors, we now report an extension of these studies by combining our new side chains with different MEK inhibitor cores in a modular manner. Replacement of the amide headgroup with inverse sulfonamides resulted in the identification of new MEK inhibitors with at least 10‐fold higher cellular potency against K‐Ras‐mutated tumor cells. A selected inhibitor from this new series retained the favorable pharmacokinetic profile of its predecessor in rodent and non‐rodent species and displayed significant in vivo efficacy at once‐daily oral doses of 0.25–1 mg kg^?1 in a K‐Ras‐mutated xenograft model. The brain penetration potential of this analogue was significantly attenuated relative to PD325901. In a second series, the central fluorophenyl core was replaced by a pyridine moiety which gave rise to a similar boost in cellular potency. Most notably, analogues from this second series do not show MEK feedback phosphorylation in K‐Ras‐mutated A549 cells. Our results complement recent reports on the structural intricacies of MEK–Raf feedback interactions.     

Regioselective carbon-carbon bond formation in proteins with palladium catalysis; new protein chemistry by organometallic chemistry

Palladium-catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki-Heck reaction for site-specific carbon-carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6-fused Ras protein containing 4-iodo-L-phenylalanine at position 32 (iF32-Ras-His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32-Ras-His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography-mass spectrometry (LC-MS). The regioselectivity of the Mizoroki-Heck reaction was furthermore confirmed by LC-MS/MS analysis. However, in addition to the biotinylated product (bF32-Ras-His), a dehalogenated product (F32-Ras-His) was detected by LC-MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki-Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras-binding domain as its downstream target, Raf-1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry.     

The Tyrosine Phosphatase hPTPRβ Controls the Early Signals and Dopaminergic Cells Viability via the P2X7 Receptor

ATP, one of the signaling molecules most commonly secreted in the nervous system and capable of stimulating multiple pathways, binds to the ionotropic purinergic receptors, in particular, the P2X₇ receptor (P2X₇R) and stimulates neuronal cell death. Given this effect of purinergic receptors on the viability of dopaminergic neurons model cells and that Ras GTPases control Erk1/2-regulated mitogen-activated cell proliferation and survival, we have investigated the role of the small GTPases of the Ras superfamily, together with their regulatory and effector molecules as the potential molecular intermediates in the P2X₇R-regulated cell death of SN4741 dopaminergic neurons model cells. Here, we demonstrate that the neuronal response to purinergic stimulation involves the Calmodulin/RasGRF1 activation of the small GTPase Ras and Erk1/2. We also demonstrate that tyrosine phosphatase PTPRβ and other tyrosine phosphatases regulate the small GTPase activation pathway and neuronal viability. Our work expands the knowledge on the intracellular responses of dopaminergic cells by identifying new participating molecules and signaling pathways. In this sense, the study of the molecular circuitry of these neurons is key to understanding the functional effects of ATP, as well as considering the importance of these cells in Parkinson’s Disease.     

CAST基因多态性与西门塔尔杂交牛胴体和肉质性状的相关性分析

目的分析钙蛋白酶抑制蛋白(Calpastatin,CAST)基因多态性与西门塔尔杂交牛胴体和肉质性状的相关性。方法选取95头西门塔尔杂交牛,采用限制性片段长度多态性聚合酶链反应(Restriction fragment length polymor-phism-polymerase chain reaction,RFLP-PCR)对CAST基因外显子1(Exon1)和外显子5(Exon5)区域的片段的多态性进行检测,并对突变位点与西门塔尔肉牛胴体和肉质性状的相关性进行分析。结果在所分析的片段中,仅引物4扩增的片段(Exon1)存在一个G→C碱基的置换突变(E1 256 g﹥c),为同义突变;该突变位点为RasⅠ酶的自然酶切位点,酶切后存在GG、GC和CC 3种基因型,CC基因型与西门塔尔牛的净肉率性状显著相关(P﹤0.05),而对其他胴体和肉质性状无显著影响。结论 CAST基因Exon1处存在的突变位点与西门塔尔肉牛的净肉率显著相关,因此该位点将有助于筛选与牛肉质相关的辅助选择标记,该基因将为鉴定与肉质相关的功能基因提供参考。     

AMPK Phosphorylation Is Controlled by Glucose Transport Rate in a PKA-Independent Manner

To achieve growth, microbial organisms must cope with stresses and adapt to the environment, exploiting the available nutrients with the highest efficiency. In Saccharomyces cerevisiae, Ras/PKA and Snf1/AMPK pathways regulate cellular metabolism according to the supply of glucose, alternatively supporting fermentation or mitochondrial respiration. Many reports have highlighted crosstalk between these two pathways, even without providing a comprehensive mechanism of regulation. Here, we show that glucose-dependent inactivation of Snf1/AMPK is independent from the Ras/PKA pathway. Decoupling glucose uptake rate from glucose concentration, we highlight a strong coordination between glycolytic metabolism and Snf1/AMPK, with an inverse correlation between Snf1/AMPK phosphorylation state and glucose uptake rate, regardless of glucose concentration in the medium. Despite fructose-1,6-bisphosphate (F1,6BP) being proposed as a glycolytic flux sensor, we demonstrate that glucose-6-phosphate (G6P), and not F1,6BP, is involved in the control of Snf1/AMPK phosphorylation state. Altogether, this study supports a model by which Snf1/AMPK senses glucose flux independently from PKA activity, and thanks to conversion of glucose into G6P.