Analysis of Ras/ERK Compartmentalization by Subcellular Fractionation.

A vast number of stimuli use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their cognate receptors, in order to regulate multiple cellular functions, including key processes such as proliferation, cell cycle progression, differentiation, and survival. The duration, intensity and specificity of the responses are, in part, controlled by the compartmentalization/subcellular localization of the signaling intermediaries. Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate a multitude of biological responses. The Ras effector pathway leading to ERKs activation is also subject to space-related regulatory processes. About half of ERK1/2 substrates are found in the nucleus and function mainly as transcription factors. The other half resides in the cytosol and other cellular organelles. Such subcellular distribution enhances the complexity of the Ras/ERK cascade and constitutes an essential mechanism to endow variability to its signals, which enables their participation in the regulation of a broad variety of functions. Thus, analyzing the subcellular compartmentalization of the members of the Ras/ERK cascade constitutes an important factor to be taken into account when studying specific biological responses evoked by Ras/ERK signals. Herein, we describe methods for such purpose.

Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes.

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

The grab-and-drop protocol: a novel strategy for membrane protein isolation and reconstitution from single cells.

We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.

A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease.

An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5 fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer.

GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples.

Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 µg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/µg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.

Highly sensitive and selective colorimetric genotyping of single-nucleotide polymorphisms based on enzyme-amplified ligation on magnetic beads.

Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.

In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases.

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P3]. PLCepsilon is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCepsilon variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation.

Genetic analyses of the role of RCE1 in RAS membrane association and transformation.

Proteins terminating with a CAAX motif, such as the nuclear lamins and the RAS family of proteins, undergo post-translational modification of a carboxyl-terminal cysteine with an isoprenyl lipid--a process called protein prenylation. After prenylation, the last three residues of CAAX proteins are clipped off by an endoprotease of the endoplasmic reticulum. RCE1 is responsible for the endoproteolytic processing of the RAS proteins and is likely responsible for endoproteolytic processing of the vast majority of CAAX proteins. Prenylation has been shown to be essential for the proper intracellular targeting and function of several CAAX proteins, but the physiologic importance of the endoprotease step has remained less certain. Here, we will review methods that have been used to define the physiologic importance of the endoproteolytic processing step of CAAX protein processing.

Semisynthesis of H-Ras with a glutamic acid methylester at position 61.

The mechanism of the guanosine triphosphate (GTP) hydrolysis reaction of small G-proteins such as Ras is generally understood; however, some important molecular details are still missing. One example concerns the role of Gln61 in the catalysis of the GTP hydrolysis reaction. This amino acid is frequently mutated in oncogenic Ras leading to constitutively active variants of the protein. To elucidate the role of Gln61, subtle structural changes were introduced at this position by exchanging the natural occurring glutamine against a glutamic acid methyl ester (GluOme). Thereby the H-bond donor properties of this residue are changed and analysis of the GTP hydrolysis reaction can provide information on the function of the native carboxamide moiety. Using a semisynthetic approach, Ras(1-166)Gln61GluOMe was synthesized by sequential native chemical ligation of three unprotected peptide segments. Peptides Ras(1-50) and Ras(51-79)Gln61GluOMe were synthesized using Boc chemistry. The C-terminal peptide Ras(80-166) was expressed in E. coli. Initial tests of this semisynthetic strategy were performed by synthesis of the N- and C-terminally truncated protein variant Ras(39-101)Gln61GluOMe. The identified optimal reaction conditions were then applied to the synthesis of Ras(1-166)Gln61GluOMe. Refolding of the semisynthetic product in the presence of GTP was successful and revealed intrinsic GTPase activity of Ras(1-166)Gln61GluOMe.

A phosphatase holoenzyme comprised of Shoc2/Sur8 and the catalytic subunit of PP1 functions as an M-Ras effector to modulate Raf activity.

Ras family GTPases (RFGs) are known to share many regulatory and effector proteins. How signaling and biological specificity is achieved is poorly understood. Using a proteomics approach, we have identified a complex comprised of Shoc2/Sur-8 and the catalytic subunit of protein phosphatase 1 (PP1c) as a highly specific M-Ras effector. M-Ras targets Shoc2-PP1c to stimulate Raf activity by dephosphorylating the S259 inhibitory site of Raf proteins bound to other molecules of M-Ras or Ras. Therefore, distinct RFGs, through independent effectors, can regulate different steps in the activation of Raf kinases. Shoc2 function is essential for activation of the MAPK pathway by growth factors. Furthermore, in tumor cells with Ras gene mutations, inhibition of Shoc2 expression inhibits MAPK, but not PI3K activity. We propose that the Shoc2-PP1c holoenzyme provides an attractive therapeutic target for inhibition of the MAPK pathway in cancer.

Purification of ARAP3 and characterization of GAP activities.

ARAP3 is a dual Arf and Rho GTPase activating protein (GAP) that was identified from pig leukocyte cytosol using a phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns[3,4,5]P3) affinity matrix in a targeted proteomics study. ARAP3's domain structure includes five PH domains, an Arf GAP domain, three ankyrin repeats, a Rho GAP domain, and a Ras association domain. ARAP3 is a PtdIns(3,4,5)P3-dependent GAP for Arf6 both in vitro and in vivo. It acts as a Rap-GTP-activated RhoA GAP in vitro, and this activation depends on a direct interaction between ARAP3 and Rap-GTP; in vivo PtdIns(3,4,5)P3 seems to be required to allow ARAP3's activation as a RhoA GAP by Rap-GTP. Overexpression of ARAP3 in pig aortic endothelial (PAE) cells causes the PI3K-dependent loss of adhesion to the substratum and interferes with lamellipodium formation. This overexpression phenotype depends on ARAP3's intact abilities to bind PtdIns(3,4,5)P3, to interact with Rap-GTP, and to be a catalytically active RhoA and Arf6 GAP.

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.

Expression, purification, crystallization and preliminary crystallographic analysis of human Rad GTPase.

Human Rad is a new member of the Ras GTPase superfamily and is overexpressed in human skeletal muscle of individuals with type II diabetes. The GTPase core domain was overexpressed in Escherichia coli and purified for crystallization. Crystals were obtained at 293 K by vapour diffusion using a crystallization robot. The crystals were found to belong to space group P2(1), with unit-cell parameters a = 52.2, b = 58.6, c = 53.4 A, beta = 97.9 degrees , and contained two Rad molecules in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 1.8 A using synchrotron radiation at SPring-8.

Characterization of Saccharomyces cerevisiae Ras1p and chimaeric constructs of Ras proteins reveals the hypervariable region and farnesylation as critical elements in the adenylyl cyclase signaling pathway.

Ras1p and Ras2p, from Saccharomyces cerevisiae, are GTP-binding proteins that are essential elements in the signaling cascade leading to the activation of adenylyl cyclase. To overcome proteolytic activities that have hampered biochemical studies of Ras1p so far, its gene was genetically modified after which full-length Ras1p could be obtained. The interaction of farnesylated and unprenylated Ras1p with guanine nucleotides, guanine nucleotide exchange factors, GTPase activating proteins, and adenylyl cyclase was compared to Ras2p and human Ha-Ras interactions. Farnesylation of Ras proteins was demonstrated to be a prerequisite for membrane-bound guanine nucleotide exchange factor dependent formation of Ras-GTP complexes, and for efficient Ras-mediated adenylyl cyclase activation. To relate observed functional deviations with sequence differences between Ras1p and Ras2p, which reside almost exclusively within the hypervariable region, truncated versions and chimaeras of the Ras proteins were made. The characteristics of these constructs point to the presence of the hypervariable region of yeast Ras proteins for an efficient activation of adenylyl cyclase. The importance of the latter was confirmed as inhibition of the activation of adenylyl cyclase by an isolated farnesylated hypervariable region of Ras2p could be shown. This strongly suggests that the hypervariable region of Ras proteins can interact directly with adenylyl cyclase.

ATPase and GTPase activities copurifying with GTP-binding proteins in E. coli.

Intrinsic GTPase activity of GTP-binding proteins plays the vital role in regulating the downstream activation pathway. We examined the GTP and ATP hydrolyzing (NTPase) abilities of various bacterial and human GTP-binding proteins under different metabolic conditions. Two metabolic components, acetate and 3-phosphoglyceric acid (3-PG), have shown significant stimulatory action on NTPase activity of G-protein preparations. Acetyl phosphate and 2,3-bisphosphoglyceric acid (2,3-BPG) blocked these stimulations. From gel filtration analyses, we have determined two fractions containing metabolite-inducible NTPase activities which are independent of GTP-binding protein enzymatic actions. Therefore, one should be cautious when NTPase activity is examined in a buffer containing acetate often used for NTPase assay.

Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.

Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae.

Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal CaaX motif (C is cysteine, a is any aliphatic residue, X is the carboxy terminal residue). To better understand the relationship between posttranslational processing and the subcellular localization of Ras, a yeast genetic screen was undertaken based on the loss of function of a palmitoylation-dependent RAS2 allele. Mutations were identified in an uncharacterized open reading frame (YLR246w) that we have designated ERF2 and a previously described suppressor of hyperactive Ras, SHR5. ERF2 encodes a 41-kDa protein with four predicted transmembrane (TM) segments and a motif consisting of the amino acids Asp-His-His-Cys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD. Mutations within the DHHC-CRD abolish Erf2 function. Subcellular fractionation and immunolocalization experiments reveal that Erf2 tagged with a triply iterated hemagglutinin epitope is an integral membrane protein that colocalizes with the yeast endoplasmic reticulum marker Kar2. Strains lacking ERF2 are viable, but they have a synthetic growth defect in the absence of RAS2 and partially suppress the heat shock sensitivity resulting from expression of the hyperactive RAS2(V19) allele. Ras2 proteins expressed in an erf2Delta strain have a reduced level of palmitoylation and are partially mislocalized to the vacuole. Based on these observations, we propose that Erf2 is a component of a previously uncharacterized Ras subcellular localization pathway. Putative members of an Erf2 family of proteins have been uncovered in yeast, plant, worm, insect, and mammalian genome databases, suggesting that Erf2 plays a role in Ras localization in all eucaryotes.

Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.

We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

Identification of peptide superagonists for a self-K-ras-reactive CD4+ T cell clone using combinatorial peptide libraries and mass spectrometry.

The proliferative responses of a human CD4+ T cell clone 29.15.2, reactive with a self-K-ras-derived peptide (3EYKLVVVGAGGVGKSALT20), were tested using a set of X9 combinatorial peptide libraries containing the flanking residues (EYKLVXXXXXXXXXSALT, where X indicates random amino acids). Certain peptide libraries, such as EYKLVXXXXXXM XXSALT and EYKLVXXXXXXXH XSALT, stimulated a marked proliferation of 29.15.2. However, no combinations of substitutions tested, such as EYKLVXXXXXXMH XSALT, exhibited additive effects. We subsequently synthesized peptides with degenerate sequences (a mixture of 480 species), where each position is composed of the wild-type (wt) residue or of amino acids that induced the proliferation of 29.15.2, in positional scanning. Interestingly, one fraction of degenerate peptides, separated by reverse-phase HPLC, stimulated much higher proliferation than did the wt; in addition, the retention time of this fraction was distinct from that of the wt. Mass spectrometry analysis of this fraction and flanking fractions identified five peptide species that exhibit strong signals in a manner that parallels the antigenic activity. Finally, 17 candidate peptide sequences were deduced from mass spectrometry and hydrophobicity scoring results, of which two peptides (EYKLVVVGAGGML KSALT and EYKLVVVGAGGMI KSALT) did induce 52- and 61-fold stronger proliferation, respectively, compared with the wt. These findings indicate that: 1) synthetic peptides that carry "the best" residue substitution at each position of combinatorial peptide libraries do not always exhibit superagonism, and 2) such a drawback can be overcome with the use of mass spectrometry. This approach provides new perspectives for the accurate and efficient identification of peptide superagonists.