The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.     

Analysis of Ras-induced oncogenic transformation of NIH-3T3 cells using differential-display 2-DE proteomics

Ras proteins control at least three crucial signalling networks responsible for several cellular processes including anchorage independence, survival, and proliferation. Point mutations in one of the three ras genes are frequent in human tumours. In these tumours, Ras oncoproteins contribute significantly to the malignant phenotype, including deregulation of tumour-cell growth, apoptosis and invasiveness, and the ability to induce angiogenesis. Although significant strides have been made in understanding Ras biology, the collaborative actions of Ras effectors are still poorly understood. Here, we describe a proteomics approach to study global changes in protein expression in Ras-transformed NIH3T3 cells. We exploited 2-D difference gel electrophoresis (DIGE) for pre-separation fluorescent protein labelling with three separate dyes to reduce gel-to-gel variability, to increase sensitivity and dynamic range of protein detection, and to enhance quantification of dysregulated proteins. Proteins dysregulated (> 1.5-fold) by oncogenic Ras transformation reported to be implicated in Ras-regulated pathways include S-methyl-5-thioadenosine phosphorylase, stress-induced-phosphoprotein 1, galectin-1, annexin A7 (synexin), 60S acidic ribosomal protein P0, serine/threonine protein phosphatase type 1 (PP1alpha) and prohibitin. Significantly, we report for the first time the expression of the newly discovered cytokine IL-25 (or IL-17E) in mouse embryonic fibroblast cells and its down-regulation (2.1-fold) upon Ras-induced oncogenic transformation.     

Synthesis of functional Ras lipoproteins and fluorescent derivatives.

For the study of biological signal transduction, access to correctly lipidated proteins is of utmost importance. Furthermore, access to bioconjugates that embody the correct structure of the protein but that may additionally carry different lipid groups or labels (i.e., fluorescent tags) by which the protein can be traced in biological systems, could provide invaluable reagents. We report here of the development of techniques for the synthesis of a series of modified Ras proteins. These modified Ras proteins carry a number of different, natural and non-natural lipid residues, and the process was extended to also provide access to a number of fluorescently labeled derivatives. The maleimide group provided the key to link chemically synthesized lipopeptide molecules in a specific and efficient manner to a truncated form of the H-Ras protein. Furthermore, a preliminary study on the biological activity of the natural Ras protein derivative (containing the normal farnesyl and palmitoyl lipid residues) has shown full biological activity. This result highlights the usefulness of these compounds as invaluable tools for the study of Ras signal transduction processes and the plasma membrane localization of the Ras proteins.     

A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitors

Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 microm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties.     

Synthesis and biological activity of photoactivatable N-ras peptides and proteins

A modular strategy for the assembly of farnesylated N-Ras heptapeptides carrying a photoactivatable benzophenone (BP) group within the lipid residue is described. This strategy is based on the fragment condensation of a N-terminal hexapeptide synthesized on the solid support with a cysteine methyl ester which is modified with different farnesyl analogues, incorporating the photophor. At the N-terminus of the peptides different functional groups can be attached, e.g., biotin for product enrichment and detection after photoactivation or a maleimido (MIC) linker, allowing for the coupling to proteins carrying a C-terminal free cysteine. Using this strategy, 24 peptides were synthesized, incorporating farnesyl analogues with four different chain lengths. Two of these photoactivatable conjugates were ligated to oncogenic human N-RasG12V Delta 181. A cellular transformation assay revealed that the semisynthetic proteins retain their biological activity despite the photolabel. The first photolabeling experiments with a geranyl-BP-labeled N-Ras construct and the farnesyl-sensitive guanine nucleotide exchange factor hSos1 indicate that this photoaffinity labeling system can be particularly useful for studying protein-protein interactions, e.g., the participation of the farnesyl group in Ras signaling, which is still discussed with controversy.     

Synthesis of Ras proteins and their application in biofunctional studies

We summarized the developed strategies including chemical total synthesis, biosynthesis and semi-synthesis for producing Ras proteins with modification and their application in biological studies.     

The Ras dimer structure

Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein–protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target.

By combining the incorporation of unnatural amino acids, click chemistry, FRET and EPR distance measurements, protein modeling and biomolecular simulations, we obtained an unambiguous Ras dimer structural model and disrupt the dimer by mutagenesis.     

Translational Dynamics of Lipidated Ras Proteins in the Presence of Crowding Agents and Compatible Osmolytes

Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N‐Ras and K‐Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N‐Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K‐Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20 wt % Ficoll N‐Ras forms trimers and K‐Ras4B dimers. Concentrations of sucrose up to 10 wt % foster formation of N‐Ras trimers and K‐Ras dimers as well. The results can be rationalized in terms of the excluded‐volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited‐hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state—monomeric or clustered—influences their membrane‐partitioning behavior and their interplay with cytosolic interaction partners.     

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.     

脂蛋白合成新进展

生物体内的信号传导蛋白在膜上的定位与其生物功能的发挥依赖于特定脂肪链的修饰,然而传统的基因表达法合成脂蛋白,得到的纯品产率很低.在近10年中,逐渐发展起来一种新的合成方法,即将化学合成脂修饰的多肽与基因表达培养蛋白相结合,可以合成出具有多条脂肪链修饰的蛋白缀合物,并且整个合成过程在非常温和的环境中进行,产品能保持较高的纯度和活性.采用该方法合成的脂蛋白用于体外的实验中,其结果与生物体内的现象非常接近.脂蛋白合成方法的发展对研究细胞中的信号传导过程具有重要的意义,并在药物合成和提高药效方面都有很多应用,这对于研究恶性肿瘤等疾病的发病机理起到了重要的推动作用.同时该脂蛋白合成的成功是采用化学法合成生物大分子解释生物体内的现象一个重大的突破,是化学生物学发展重要的一步.     

Proteomic analysis of macrophages stimulated by lipopolysaccharide: Lipopolysaccharide inhibits the cleavage of nucleophosmin

Lipopolysaccharide (LPS) is a complex glycolipid composed of a hydrophilic polysaccharide and a hydrophobic domain that is responsible for the biological activity of LPS. There are many reports about LPS stimulation, and many activated proteins have been detected after LPS stimulation in various cell types. Furthermore, most of the LPS signaling pathways are clear. However, we were interested in examining the changes of LPS-induced total cytosolic proteins expression and the LPS signaling pathway by the proteomics technique during LPS-induced macrophage activation. Our study employed two-dimensional gel electrophoresis and mass spectrometry to analyze the proteins involved in LPS-induced activation in RAW 264.7 cells. We found 11 protein spots whose expression was different between untreated cells and LPS-treated cells. Ten protein spots were identified, seven of which, tubulin beta-4 chain (49.6 kDa, pI 4.78), nucleophosmin (32.6 kDa, pI 4.62, two spots), 40S ribosomal protein SA (P40) (32.7 kDa, pI 4.74), transforming protein RhoA (21.8 kDa, pI 5.83), nucleolin (76.6 kDa, pI 4.69), and T-complex protein 1 zeta subunit (58 kDa, pI 6.63) were down-regulated, and three of which, nucleophosmin (32.6 kDa, pI 4.62, two spots) and proteosome subunit alpha type-1 (29.5 kDa, pI 6.00), were up-regulated. The suppression of the proteolytic degradation of nucleophosmin was associated with LPS-induced RAW 264.7 cell activation. Cleaved caspase-3 decreased, thus it might be involved in proteolysis of nucleophosmin in LPS-induced macrophage activation. Our study also demonstrated that there was no change of the expression of nucleophosmin at the mRNA level.     

Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors

Lipid-modified proteins play decisive roles in important biological processes such as signal transduction, organisation of the cytoskeleton and vesicular transport. Lipidation of these proteins is essential for correct biological function. Among the modifications with lipids, prenylation and myristoylation are well understood. However, the machinery of palmitoylation is still under investigation. Recently, an enzyme, acyl protein thioesterase 1 (APT1), that may play a regulatory role in the palmitoylation cycle of H-Ras and G-protein alpha subunits, was purified. Motivated by this work, several inhibitors of APT1 were designed, synthesized and biologically evaluated leading to highly active compounds.     

A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.

A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer-aided 2-D gel electrophoresis. The protein numbers in this database differ from those reported in an earlier version due to changes in the scanning hardware (Celis et al., Electrophoresis 1990, 11, 242-254). Annotation categories reported include: "protein name" (listing 207 known proteins in alphabetical order), "basal cell markers", "differentiation markers", "proteins highly up-regulated in psoriatic skin", "microsequenced proteins" and "human autoantigens". For reference, we have also included 2-D gel (isoelectric focusing) patterns of cultured normal and psoriatic keratinocytes, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis.     

Engineering unnatural nucleotide specificity to probe G protein signaling

G proteins comprise approximately 0.5% of proteins encoded by mammalian genomes. To date, there exists a lack of small-molecule modulators that could contribute to their functional study. In this report, we present the use of H-Ras to develop a system that answers this need. Small molecules that allow for the highly specific inhibition or activation of the engineered G protein were developed. The rational design preserved binding of the natural substrates to the G protein, and the mutations were functionally innocuous in a cellular context. This tool can be used for isolating specific G protein effectors, as we demonstrate with the identification of Nol1 as a putative effector of H-Ras. Finally, the generalization of this system was confirmed by applying it to Rap1B, suggesting that this method will be applicable to other G proteins.     

Curcumin Inhibits Lysophosphatidic Acid Mediated MCP-1 Expression via Blocking ROCK Signalling

Curcumin is a natural compound that has been widely used as a food additive and medicine in Asian countries. Over several decades, diverse biological effects of curcumin have been elucidated, such as anti-inflammatory and anti-oxidative activities. Monocyte chemoattractant protein-1 (MCP-1) is a key inflammatory marker during the development of atherosclerosis, and curcumin blocks MCP-1 expression stimulated by various ligands. Hence, we studied the action of curcumin on lysophosphatidic acid (LPA) mediated MCP-1 expression and explored the specific underlying mechanisms. In human vascular smooth muscle cells, LPA induces Rho-associated protein kinase (ROCK) dependent transforming growth factor receptor (TGFBR1) transactivation, leading to glycosaminoglycan chain elongation. We found that LPA also signals via the TGFBR1 transactivation pathway to regulate MCP-1 expression. Curcumin blocks LPA mediated TGFBR1 transactivation and subsequent MCP-1 expression by blocking the ROCK signalling. In the vasculature, ROCK signalling regulates smooth muscle cell contraction, inflammatory cell recruitment, endothelial dysfunction and vascular remodelling. Therefore, curcumin as a ROCK signalling inhibitor has the potential to prevent atherogenesis via multiple ways.     

Transgenic peas expressing an α‐amylase inhibitor gene from beans show altered expression and modification of endogenous proteins

Differential in‐gel electrophoresis showed contrasting effects of the transgenic expression of an α‐amylase inhibitor from beans on the proteomes of two pea cultivars. One cultivar showed minor changes relative to its non‐transgenic parent with only one protein changing by more than about twofold. Changes in the abundance of certain endogenous proteins in the other cultivar were of greater number and magnitude with some endogenous proteins undetected while some new protein spots appeared in the transgenic proteome. The sets of proteins with altered expression were generally different between the two cultivars. Some of the proteins that were differentially expressed were identified by MS. Most were seed storage globulins, which are sited together with the transgenic product. Some of the changes may be due to alterations in expression levels but there were also changes due to post‐translational processing.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.     

Identification of differentially expressed, tumor-associated proteins in oral squamous cell carcinoma by proteomic analysis

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis and therefore the discovery of early markers to discriminate malignant from normal cells would be of critical importance in clinical diagnosis. Subcellular fractions from oral squamous cell carcinoma (OSCC) and control samples, enriched in mitochondrial and cytosolic proteins, were analyzed by 2-DE, followed by MALDI-TOF-MS. Twenty proteins showed altered expression levels in OSCC; 14 were up- and 6 were down-regulated in comparison with the control samples. For 11 proteins, cofilin, C-reactive protein precursor, creatine kinase m-chain, fatty acid-binding protein, keratin type II, myosin light chain 2 and 3, nucleoside diphosphate kinase A, phosphoglycerate mutase 1, plakoglobulin, and retinoic acid-binding protein II, it is shown for the first time that they are differentially expressed in OSCC. Proteins with highly up-regulated levels may be of interest as potential diagnostic markers and consequently of clinical interest.     

Inhibition of extracellular signal-regulated kinase 1/2 activity of the breast cancer cell line MDA-MB-231 leads to major alterations in the pattern of protein expression

Biochemical and genetic strategies have implied that aberrant signaling in the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathway contributes significantly to transformed phenotypes. Using PD98059, an inhibitor of the ERK-kinase MEK1, we have here assessed the effects of ERK inhibition on the pattern of protein expression in the metastatic human breast cancer cell line MDA-MB-231. At a concentration of inhibitor which did not significantly affect cell growth, PD98059 induced large changes in the expression of MDA-MB-231 polypeptides. The majority of these changes were due to decreased expression of low-abundance proteins. Decreases of more abundant proteins such as glutathione-S-transferase pi, hsp80 and hsp100 were also recorded. The levels of a few proteins increased, among them cytokeratin 8. We conclude that PD98059 treatment of MDA-MB-231 cells induces large changes in protein expression.