Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

PubMed Central

Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

2013-01-01

The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination*

PubMed Central

Baker, Rachael; Wilkerson, Emily M.; Sumita, Kazutaka; Isom, Daniel G.; Sasaki, Atsuo T.; Dohlman, Henrik G.; Campbell, Sharon L.

2013-01-01

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation. PMID:24247240

Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER.

PubMed

Sobering, Andrew K; Watanabe, Reika; Romeo, Martin J; Yan, Benjamin C; Specht, Charles A; Orlean, Peter; Riezman, Howard; Levin, David E

2004-05-28

The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes. Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins. We also show that GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT. We propose that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes.

New Method for Measuring the Anchoring Energy of Strongly-Bound Membrane-Associated Proteins [Method for measuring the anchoring energy of strongly-bound membrane-associated proteins].

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kent, Michael S.; La Bauve, Elisa; Vernon, Briana C.

Here, we describe a new method to measure the activation energy required to remove a strongly-bound membrane-associated protein from a lipid membrane (anchoring energy). It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method was used to determine anchoring energy for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. We also measured the binding energy of sE at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipidmore » bilayer. The anchoring energy (37 +/- 1.7 kcal/mol, 20% PG) was found to be much larger than the binding energy (7.8 +/- 0.3 kcal/mol for 30% PG, or est. 7.0 kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH 5.5, but assembles into trimers after associating with membranes. But, trimerization alone is insufficient to account for the observed difference in energies, and we conclude that some energy dissipation occurs during the release process. This new method to determine anchoring energy should be useful to understand the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.« less

New Method for Measuring the Anchoring Energy of Strongly-Bound Membrane-Associated Proteins [Method for measuring the anchoring energy of strongly-bound membrane-associated proteins].

DOE PAGES

Kent, Michael S.; La Bauve, Elisa; Vernon, Briana C.; ...

2016-02-01

Here, we describe a new method to measure the activation energy required to remove a strongly-bound membrane-associated protein from a lipid membrane (anchoring energy). It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method was used to determine anchoring energy for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. We also measured the binding energy of sE at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipidmore » bilayer. The anchoring energy (37 +/- 1.7 kcal/mol, 20% PG) was found to be much larger than the binding energy (7.8 +/- 0.3 kcal/mol for 30% PG, or est. 7.0 kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH 5.5, but assembles into trimers after associating with membranes. But, trimerization alone is insufficient to account for the observed difference in energies, and we conclude that some energy dissipation occurs during the release process. This new method to determine anchoring energy should be useful to understand the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.« less

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering

PubMed Central

Blaževitš, Olga; Mideksa, Yonatan G.; Šolman, Maja; Ligabue, Alessio; Ariotti, Nicholas; Nakhaeizadeh, Hossein; Fansa, Eyad K.; Papageorgiou, Anastassios C.; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Abankwa, Daniel

2016-01-01

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling. PMID:27087647

The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3.

PubMed

Liger, D; Nizard, P; Gaillard, C; vanderSpek, J C; Murphy, J R; Pitard, B; Gillet, D

1998-11-01

We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.

HKUST-1 Membranes Anchored on Porous Substrate by Hetero MIL-110 Nanorod Array Seeds.

PubMed

Mao, Yiyin; Cao, Wei; Li, Junwei; Sun, Luwei; Peng, Xinsheng

2013-09-02

Great anchors and seeds: Hetero-seeding growth processes and anchored nanorod arrays were successfully utilized in the synthesis of HKUST-1 membranes. These arrays were firmly anchored on porous substrates by using a MIL-110 nanorod array as both the anchor and seed. The resulting HKUST-1 membranes demonstrated good separation factors for binary gases exceeding the Knudson selectivity. Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration*

PubMed Central

Haeussler, Dagmar J.; Pimentel, David R.; Hou, Xiuyun; Burgoyne, Joseph R.; Cohen, Richard A.; Bachschmid, Markus M.

2013-01-01

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling. PMID:23548900

Pressure modulation of Ras-membrane interactions and intervesicle transfer.

PubMed

Kapoor, Shobhna; Werkmüller, Alexander; Goody, Roger S; Waldmann, Herbert; Winter, Roland

2013-04-24

Proteins attached to the plasma membrane frequently encounter mechanical stresses, including high hydrostatic pressure (HHP) stress. Signaling pathways involving membrane-associated small GTPases (e.g., Ras) have been identified as critical loci for pressure perturbation. However, the impact of mechanical stimuli on biological outputs is still largely terra incognita. The present study explores the effect of HHP on the membrane association, dissociation, and intervesicle transfer process of N-Ras by using a FRET-based assay to obtain the kinetic parameters and volumetric properties along the reaction path of these processes. Notably, membrane association is fostered upon pressurization. Conversely, depending on the nature and lateral organization of the lipid membrane, acceleration or retardation is observed for the dissociation step. In addition, HHP can be inferred as a positive regulator of N-Ras clustering, in particular in heterogeneous membranes. The susceptibility of membrane interaction to pressure raises the idea of a role of lipidated signaling molecules as mechanosensors, transducing mechanical stimuli to chemical signals by regulating their membrane binding and dissociation. Finally, our results provide first insights into the influence of pressure on membrane-associated Ras-controlled signaling events in organisms living under extreme environmental conditions such as those that are encountered in the deep sea and sub-seafloor environments, where pressures reach the kilobar (100 MPa) range.

The Tumor Suppressor DiRas3 Forms a Complex with H-Ras and C-RAF Proteins and Regulates Localization, Dimerization, and Kinase Activity of C-RAF*

PubMed Central

Baljuls, Angela; Beck, Matthias; Oenel, Ayla; Robubi, Armin; Kroschewski, Ruth; Hekman, Mirko; Rudel, Thomas; Rapp, Ulf R.

2012-01-01

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity. PMID:22605333

H-Ras Modulates N-Methyl-d-aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity*

PubMed Central

Thornton, Claire; Yaka, Rami; Dinh, Son; Ron, Dorit

2005-01-01

Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hip-pocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction. PMID:12695509

Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching

NASA Astrophysics Data System (ADS)

Vinnakota, Kalyan C.; Mitchell, David A.; Deschenes, Robert J.; Wakatsuki, Tetsuro; Beard, Daniel A.

2010-06-01

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane-localized fluorophore with the cytosol during fluorescence recovery after photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma membrane-localized wild-type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 µm × 1 µm bleach region-of-interest (ROI) and a 0.5 µm × 0.5 µm bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in the yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane-associated fluorophores using FRAP on commercial confocal laser scanning microscopes.

TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro.

PubMed

Tullis, K; Olsen, H; Bombick, D W; Matsumura, F; Jankun, J

1992-01-01

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21ras protein, or indirectly measured by the specific binding of 3H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P less than 0.05, Student t test) levels of hepatic p21ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the difference became statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21ras protein which did not become statistically significant even at 24 hr host-dosing. TCDD evoked increases in hepatic p21ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a 3H-GTP binding protein with MW of approximately 21 Kd. 3H-GTP binding in total hepatic membranes was also elevated (P less than 0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of approximately 21 Kd.

Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

PubMed

Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

2017-09-16

Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

PubMed

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

PubMed

Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

2015-12-15

Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

PubMed

Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

1992-05-15

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

Golgi retention of a trans-Golgi membrane protein, galactosyltransferase, requires cysteine and histidine residues within the membrane-anchoring domain.

PubMed Central

Aoki, D; Lee, N; Yamaguchi, N; Dubois, C; Fukuda, M N

1992-01-01

Galactosyltransferase (GT; UDPgalactose:beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha subunit (hCG alpha) as a reporter. COS-1 cells transfected with pGT-hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, delta tail and delta stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29----Ser29 and His32----Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31----Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32----Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and

Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

PubMed

Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

2013-09-17

Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

H-Ras Exerts Opposing Effects on Type I Interferon Responses Depending on Its Activation Status.

PubMed

Chen, Guann-An; Lin, Yun-Ru; Chung, Hai-Ting; Hwang, Lih-Hwa

2017-01-01

Using shRNA high-throughput screening, we identified H-Ras as a regulator of antiviral activity, whose depletion could enhance Sindbis virus replication. Further analyses indicated that depletion of H-Ras results in a robust increase in vesicular stomatitis virus infection and a decrease in Sendai virus (SeV)-induced retinoic acid-inducible gene-I-like receptor (RLR) signaling. Interestingly, however, ectopic expression of wild-type H-Ras results in a biphasic mode of RLR signaling regulation: while low-level expression of H-Ras enhances SeV-induced RLR signaling, high-level expression of H-Ras significantly inhibits this signaling. The inhibitory effects correlate with the activation status of H-Ras. As a result, oncogenic H-Ras, H-RasV12, strongly inhibits SeV-induced IFN-β promoter activity and type I interferon signaling. Conversely, the positive effects exerted by H-Ras on RLR signaling are independent of its signaling activity, as a constitutively inactive form of H-Ras, H-RasN17, also positively regulates RLR signaling. Mechanistically, we demonstrate that depletion of H-Ras reduces the formation of MAVS-TNF receptor-associated factor 3 signaling complexes. These results reveal that the H-Ras protein plays a role in promoting MAVS signalosome assembly in the mitochondria, whereas oncogenic H-Ras exerts a negative effect on type I IFN responses.

K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D.

PubMed

Parker, Jillian A; Volmar, Alicia Y; Pavlopoulos, Spiro; Mattos, Carla

2018-06-05

Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCH 2 p, with K-Ras in the state 1 conformation. Signatures of conformational states obtained by one-dimensional proton NMR confirm that K-Ras has a more substantial population of state 1 in solution than H-Ras, which predominantly favors state 2. The oncogenic mutant K-RasG12D favors state 2, changing the balance of conformational states in favor of interactions with effector proteins. Differences in the population of conformational states between K-Ras and H-Ras, as well as between K-Ras and its mutants, can provide a structural basis for focused targeting of the K-Ras isoform in cancer-specific strategies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cluster Formation of Anchored Proteins Induced by Membrane-Mediated Interaction

PubMed Central

Li, Shuangyang; Zhang, Xianren; Wang, Wenchuan

2010-01-01

Abstract Computer simulations were used to study the cluster formation of anchored proteins in a membrane. The rate and extent of clustering was found to be dependent upon the hydrophobic length of the anchored proteins embedded in the membrane. The cluster formation mechanism of anchored proteins in our work was ascribed to the different local perturbations on the upper and lower monolayers of the membrane and the intermonolayer coupling. Simulation results demonstrated that only when the penetration depth of anchored proteins was larger than half the membrane thickness, could the structure of the lower monolayer be significantly deformed. Additionally, studies on the local structures of membranes indicated weak perturbation of bilayer thickness for a shallowly inserted protein, while there was significant perturbation for a more deeply inserted protein. The origin of membrane-mediated protein-protein interaction is therefore due to the local perturbation of the membrane thickness, and the entropy loss—both of which are caused by the conformation restriction on the lipid chains and the enhanced intermonolayer coupling for a deeply inserted protein. Finally, in this study we addressed the difference of cluster formation mechanisms between anchored proteins and transmembrane proteins. PMID:20513399

Unimpeded permeation of water through biocidal graphene oxide sheets anchored on to 3D porous polyolefinic membranes

NASA Astrophysics Data System (ADS)

Mural, Prasanna Kumar S.; Jain, Shubham; Kumar, Sachin; Madras, Giridhar; Bose, Suryasarathi

2016-04-01

3D porous membranes were developed by etching one of the phases (here PEO, polyethylene oxide) from melt-mixed PE/PEO binary blends. Herein, we have systematically discussed the development of these membranes using X-ray micro-computed tomography. The 3D tomograms of the extruded strands and hot-pressed samples revealed a clear picture as to how the morphology develops and coarsens over a function of time during post-processing operations like compression molding. The coarsening of PE/PEO blends was traced using X-ray micro-computed tomography and scanning electron microscopy (SEM) of annealed blends at different times. It is now understood from X-ray micro-computed tomography that by the addition of a compatibilizer (here lightly maleated PE), a stable morphology can be visualized in 3D. In order to anchor biocidal graphene oxide sheets onto these 3D porous membranes, the PE membranes were chemically modified with acid/ethylene diamine treatment to anchor the GO sheets which were further confirmed by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and surface Raman mapping. The transport properties through the membrane clearly reveal unimpeded permeation of water which suggests that anchoring GO on to the membranes does not clog the pores. Antibacterial studies through the direct contact of bacteria with GO anchored PE membranes resulted in 99% of bacterial inactivation. The possible bacterial inactivation through physical disruption of the bacterial cell wall and/or reactive oxygen species (ROS) is discussed herein. Thus this study opens new avenues in designing polyolefin based antibacterial 3D porous membranes for water purification.3D porous membranes were developed by etching one of the phases (here PEO, polyethylene oxide) from melt-mixed PE/PEO binary blends. Herein, we have systematically discussed the development of these membranes using X-ray micro-computed tomography. The 3D tomograms of the extruded strands and

Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function▿

PubMed Central

Plowman, Sarah J.; Ariotti, Nicholas; Goodall, Andrew; Parton, Robert G.; Hancock, John F.

2008-01-01

The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades. PMID:18458061

Ras trafficking, localization and compartmentalized signalling

PubMed Central

Prior, Ian A.; Hancock, John F.

2012-01-01

Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes. PMID:21924373

Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

NASA Astrophysics Data System (ADS)

Li, Jing; Liu, Xiuhua; Tian, Falin; Yue, Tongtao; Zhang, Xianren; Cao, Dapeng

2018-05-01

GPI-Anchored proteins (GPI-APs) can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

PubMed Central

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M.; Abel, Steven M.; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S.; Hansen, Scott D.; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K.; Kuriyan, John; Groves, Jay T.

2014-01-01

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. PMID:24994643

Unimpeded permeation of water through biocidal graphene oxide sheets anchored on to 3D porous polyolefinic membranes.

PubMed

Mural, Prasanna Kumar S; Jain, Shubham; Kumar, Sachin; Madras, Giridhar; Bose, Suryasarathi

2016-04-21

3D porous membranes were developed by etching one of the phases (here PEO, polyethylene oxide) from melt-mixed PE/PEO binary blends. Herein, we have systematically discussed the development of these membranes using X-ray micro-computed tomography. The 3D tomograms of the extruded strands and hot-pressed samples revealed a clear picture as to how the morphology develops and coarsens over a function of time during post-processing operations like compression molding. The coarsening of PE/PEO blends was traced using X-ray micro-computed tomography and scanning electron microscopy (SEM) of annealed blends at different times. It is now understood from X-ray micro-computed tomography that by the addition of a compatibilizer (here lightly maleated PE), a stable morphology can be visualized in 3D. In order to anchor biocidal graphene oxide sheets onto these 3D porous membranes, the PE membranes were chemically modified with acid/ethylene diamine treatment to anchor the GO sheets which were further confirmed by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and surface Raman mapping. The transport properties through the membrane clearly reveal unimpeded permeation of water which suggests that anchoring GO on to the membranes does not clog the pores. Antibacterial studies through the direct contact of bacteria with GO anchored PE membranes resulted in 99% of bacterial inactivation. The possible bacterial inactivation through physical disruption of the bacterial cell wall and/or reactive oxygen species (ROS) is discussed herein. Thus this study opens new avenues in designing polyolefin based antibacterial 3D porous membranes for water purification.

CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

PubMed Central

Zheng, Ze-Yi; Cheng, Chiang-Min; Fu, Xin-Rong; Chen, Liuh-Yow; Xu, Lizhong; Terrillon, Sonia; Wong, Stephen T.; Bar-Sagi, Dafna; Songyang, Zhou; Chang, Eric C.

2011-01-01

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling. PMID:22231449

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

PubMed

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M; Abel, Steven M; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S; Hansen, Scott D; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K; Kuriyan, John; Groves, Jay T

2014-07-04

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. Copyright © 2014, American Association for the Advancement of Science.

The Function of Embryonic Stem Cell-expressed RAS (E-RAS), a Unique RAS Family Member, Correlates with Its Additional Motifs and Its Structural Properties.

PubMed

Nakhaei-Rad, Saeideh; Nakhaeizadeh, Hossein; Kordes, Claus; Cirstea, Ion C; Schmick, Malte; Dvorsky, Radovan; Bastiaens, Philippe I H; Häussinger, Dieter; Ahmadian, Mohammad Reza

2015-06-19

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Ras, an Actor on Many Stages

PubMed Central

Arozarena, Imanol; Calvo, Fernando; Crespo, Piero

2011-01-01

Among the wealth of information that we have gathered about Ras in the past decade, the introduction of the concept of space in the field has constituted a major revolution that has enabled many pieces of the Ras puzzle to fall into place. In the early days, it was believed that Ras functioned exclusively at the plasma membrane. Today, we know that within the plasma membrane, the 3 Ras isoforms—H-Ras, K-Ras, and N-Ras—occupy different microdomains and that these isoforms are also present and active in endomembranes. We have also discovered that Ras proteins are not statically associated with these localizations; instead, they traffic dynamically between compartments. And we have learned that at these localizations, Ras is under site-specific regulatory mechanisms, distinctively engaging effector pathways and switching on diverse genetic programs to generate different biological responses. All of these processes are possible in great part due to the posttranslational modifications whereby Ras proteins bind to membranes and to regulatory events such as phosphorylation and ubiquitination that Ras is subject to. As such, space and these control mechanisms act in conjunction to endow Ras signals with an enormous signal variability that makes possible its multiple biological roles. These data have established the concept that the Ras signal, instead of being one single, homogeneous entity, results from the integration of multiple, site-specified subsignals, and Ras has become a paradigm of how space can differentially shape signaling. PMID:21779492

Simulations of a Membrane-Anchored Peptide: Structure, Dynamics, and Influence on Bilayer Properties

PubMed Central

Jensen, Morten Ø.; Mouritsen, Ole G.; Peters, Günther H.

2004-01-01

A three-dimensional structure of a model decapeptide is obtained by performing molecular dynamics simulations of the peptide in explicit water. Interactions between an N-myristoylated form of the folded peptide anchored to dipalmitoylphosphatidylcholine fluid phase lipid membranes are studied at different applied surface tensions by molecular dynamics simulations. The lipid membrane environment influences the conformational space explored by the peptide. The overall secondary structure of the anchored peptide is found to deviate at times from its structure in aqueous solution through reversible conformational transitions. The peptide is, despite the anchor, highly mobile at the membrane surface with the peptide motion along the bilayer normal being integrated into the collective modes of the membrane. Peptide anchoring moderately alters the lateral compressibility of the bilayer by changing the equilibrium area of the membrane. Although membrane anchoring moderately affects the elastic properties of the bilayer, the model peptide studied here exhibits conformational flexibility and our results therefore suggest that peptide acylation is a feasible way to reinforce peptide-membrane interactions whereby, e.g., the lifetime of receptor-ligand interactions can be prolonged. PMID:15189854

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

PubMed

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2

PubMed Central

Cho, Kwang-jin; Casteel, Darren E.; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A.; Kim, Choel; Capon, Robert J.; Lacey, Ernest; Cunha, Shane R.; Gorfe, Alemayehu A.

2016-01-01

K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function. PMID:27697864

Aurora kinase A interacts with H-Ras and potentiates Ras-MAPK signaling | Office of Cancer Genomics

Cancer.gov

In cancer, upregulated Ras promotes cellular transformation and proliferation in part through activation of oncogenic Ras-MAPK signaling. While directly inhibiting Ras has proven challenging, new insights into Ras regulation through protein-protein interactions may offer unique opportunities for therapeutic intervention. Here we report the identification and validation of Aurora kinase A (Aurora A) as a novel Ras binding protein. We demonstrate that the kinase domain of Aurora A mediates the interaction with the N-terminal domain of H-Ras.

Plant glycosylphosphatidylinositol (GPI) anchored proteins at the plasma membrane-cell wall nexus.

PubMed

Yeats, Trevor H; Bacic, Antony; Johnson, Kim L

2018-04-18

Approximately 1% of plant proteins are predicted to be post-translationally modified with a glycosylphosphatidylinositol (GPI) anchor that tethers the polypeptide to the outer leaflet of the plasma membrane. While the synthesis and structure of GPI anchors is largely conserved across eukaryotes, the repertoire of functional domains present in the GPI-anchored proteome has diverged substantially. In plants, this includes a large fraction of the GPI-anchored proteome being further modified with plant-specific arabinogalactan (AG) O-glycans. The importance of the GPI-anchored proteome to plant development is underscored by the fact that GPI biosynthetic null mutants exhibit embryo lethality. Mutations in genes encoding specific GPI-anchored proteins (GAPs) further supports their contribution to diverse biological processes occurring at the interface of the plasma membrane and cell wall, including signaling, cell wall metabolism, cell wall polymer cross-linking, and plasmodesmatal transport. Here, we review the literature concerning plant GPI-anchored proteins in the context of their potential to act as molecular hubs that mediate interactions between the plasma membrane and the cell wall and their potential to transduce the signal into the protoplast and thereby activate signal transduction pathways. This article is protected by copyright. All rights reserved.

Downregulation of Ras C-terminal processing by JNK inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mouri, Wataru; Department of Neurosurgery, Yamagata University School of Medicine, Yamagata 990-9585; Biology Division, National Cancer Center Research Institute, Tokyo 104-0045

2008-06-27

After translation, Ras proteins undergo a series of modifications at their C-termini. This post-translational C-terminal processing is essential for Ras to become functional, but it remains unknown whether and how Ras C-terminal processing is regulated. Here we show that the C-terminal processing and subsequent plasma membrane localization of H-Ras as well as the activation of the downstream signaling pathways by H-Ras are prevented by JNK inhibition. Conversely, JNK activation by ultraviolet irradiation resulted in promotion of C-terminal processing of H-Ras. Furthermore, increased cell density promoted C-terminal processing of H-Ras most likely through an autocrine/paracrine mechanism, which was also blocked undermore » JNK-inhibited condition. Ras C-terminal processing was sensitive to JNK inhibition in the case of H- and N-Ras but not K-Ras, and in a variety of cell types. Thus, our results suggest for the first time that Ras C-terminal processing is a regulated mechanism in which JNK is involved.« less

Geranylgeranyl Diphosphate Synthase Modulates Fetal Lung Branching Morphogenesis Possibly through Controlling K-Ras Prenylation.

PubMed

Jia, Wen-Jun; Jiang, Shan; Tang, Qiao-Li; Shen, Di; Xue, Bin; Ning, Wen; Li, Chao-Jun

2016-06-01

G proteins play essential roles in regulating fetal lung development, and any defects in their expression or function (eg, activation or posttranslational modification) can lead to lung developmental malformation. Geranylgeranyl diphosphate synthase (GGPPS) can modulate protein prenylation that is required for protein membrane-anchoring and activation. Here, we report that GGPPS regulates fetal lung branching morphogenesis possibly through controlling K-Ras prenylation during fetal lung development. GGPPS was continuously expressed in lung epithelium throughout whole fetal lung development. Specific deletion of geranylgeranyl diphosphate synthase 1 (Ggps1) in lung epithelium during fetal lung development resulted in neonatal respiratory distress syndrome-like disease. The knockout mice died at postnatal day 1 of respiratory failure, and the lungs showed compensatory pneumonectasis, pulmonary atelectasis, and hyaline membranes. Subsequently, we proved that lung malformations in Ggps1-deficient mice resulted from the failure of fetal lung branching morphogenesis. Further investigation revealed Ggps1 deletion blocked K-Ras geranylgeranylation and extracellular signal-related kinase 1 or 2/mitogen-activated protein kinase signaling, which in turn disturbed fibroblast growth factor 10 regulation on fetal lung branching morphogenesis. Collectively, our data suggest that GGPPS is essential for maintaining fetal lung branching morphogenesis, which is possibly through regulating K-Ras prenylation. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

PubMed Central

Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-01-01

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway.

PubMed

Hein, Zeynep; Hooper, Nigel M; Naim, Hassan Y

2009-01-15

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPDeltaGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPDeltaGPI was completely soluble. The lack of association of MDPDeltaGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.

Calcium Currents Are Enhanced by α2δ-1 Lacking Its Membrane Anchor*

PubMed Central

Kadurin, Ivan; Alvarez-Laviada, Anita; Ng, Shu Fun Josephine; Walker-Gray, Ryan; D'Arco, Marianna; Fadel, Michael G.; Pratt, Wendy S.; Dolphin, Annette C.

2012-01-01

The accessory α2δ subunits of voltage-gated calcium channels are membrane-anchored proteins, which are highly glycosylated, possess multiple disulfide bonds, and are post-translationally cleaved into α2 and δ. All α2δ subunits have a C-terminal hydrophobic, potentially trans-membrane domain and were described as type I transmembrane proteins, but we found evidence that they can be glycosylphosphatidylinositol-anchored. To probe further the function of membrane anchoring in α2δ subunits, we have now examined the properties of α2δ-1 constructs truncated at their putative glycosylphosphatidylinositol anchor site, located before the C-terminal hydrophobic domain (α2δ-1ΔC-term). We find that the majority of α2δ-1ΔC-term is soluble and secreted into the medium, but unexpectedly, some of the protein remains associated with detergent-resistant membranes, also termed lipid rafts, and is extrinsically bound to the plasma membrane. Furthermore, heterologous co-expression of α2δ-1ΔC-term with CaV2.1/β1b results in a substantial enhancement of the calcium channel currents, albeit less than that produced by wild-type α2δ-1. These results call into question the role of membrane anchoring of α2δ subunits for calcium current enhancement. PMID:22869375

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

PubMed Central

Iwig, Jeffrey S; Vercoulen, Yvonne; Das, Rahul; Barros, Tiago; Limnander, Andre; Che, Yan; Pelton, Jeffrey G; Wemmer, David E; Roose, Jeroen P; Kuriyan, John

2013-01-01

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI: http://dx.doi.org/10.7554/eLife.00813.001 PMID:23908768

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

PubMed

Martín-Sánchez, Paloma; Luengo, Alicia; Griera, Mercedes; Orea, María Jesús; López-Olañeta, Marina; Chiloeches, Antonio; Lara-Pezzi, Enrique; de Frutos, Sergio; Rodríguez-Puyol, Manuel; Calleros, Laura; Rodríguez-Puyol, Diego

2018-02-01

Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs. We have demonstrated that H- ras gene deletion produces mice hypotension via a soluble guanylate cyclase-protein kinase G (PKG)-dependent mechanism. In this study, we analyzed the consequences of H- ras deletion on cardiac remodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Left ventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H -ras -/- ) and control wild-type (H -ras +/+ ) mice, as were extracellular matrix protein expression. Increased cardiac PKG-Iβ protein expression in H -ras -/- mice suggests the involvement of this protein in heart protection. Ex vivo experiments on cardiac explants could support this mechanism, as PKG blockade blunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H -ras -/- mice. Genetic modulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3β-dependent activation of the transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iβ overexpression in H -ras -/- mouse embryonic fibroblasts. This study demonstrates that H- ras deletion protects against AngII-induced cardiac remodeling, possibly via a mechanism in which PKG-Iβ overexpression could play a partial role, and points to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.-Martín-Sánchez, P., Luengo, A., Griera, M., Orea, M. J., López-Olañeta, M., Chiloeches, A., Lara-Pezzi, E., de Frutos, S., Rodríguez-Puyol, M., Calleros, L., Rodríguez-Puyol, D. H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Iβ pathway activation.

Analysis of the signal for attachment of a glycophospholipid membrane anchor

PubMed Central

1989-01-01

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophospholipid (GPI) membrane anchor. To define this signal we deleted portions of the DAF COOH terminus and expressed the mutant cDNAs it CV1 origin-deficient SV-40 cells. Our results show that the COOH-terminal hydrophobic domain (17 residues) is absolutely required for GPI anchor attachment. However, when fused to the COOH terminus of a secreted protein this hydrophobic domain is insufficient to direct attachment of a GPI anchor. Additional specific information located within the adjacent 20 residues appears to be necessary. We speculate that by analogy with signal sequences for membrane translocation, GPI anchor attachment requires both a COOH- terminal hydrophobic domain (the GPI signal) as well as a suitable cleavage/attachment site located NH2 terminal to the signal. PMID:2466848

Trafficking and Membrane Organization of GPI-Anchored Proteins in Health and Diseases.

PubMed

Paladino, Simona; Lebreton, Stéphanie; Zurzolo, Chiara

2015-01-01

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of lipid-anchored proteins attached to the membranes by a glycolipid anchor that is added, as posttranslation modification, in the endoplasmic reticulum. GPI-APs are expressed at the cell surface of eukaryotes where they play diverse vital functions. Like all plasma membrane proteins, GPI-APs must be correctly sorted along the different steps of the secretory pathway to their final destination. The presence of both a glycolipid anchor and a protein portion confers special trafficking features to GPI-APs. Here, we discuss the recent advances in the field of GPI-AP trafficking, focusing on the mechanisms regulating their biosynthetic pathway and plasma membrane organization. We also discuss how alterations of these mechanisms can result in different diseases. Finally, we will examine the strict relationship between the trafficking and function of GPI-APs in epithelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

A 26-week carcinogenicity study of 2-amino-3-methylimidazo[4,5-f]quinoline in rasH2 mice.

PubMed

Okamura, Miwa; Moto, Mitsuyoshi; Muguruma, Masako; Ito, Tadashi; Jin, Meilan; Kashida, Yoko; Mitsumori, Kunitoshi

2006-01-01

To evaluate the carcinogenic susceptibility of rasH2 mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 7-week-old rasH2 mice and their wild-type littermates (non-Tg mice) of both the sexes were fed a diet containing 0 or 300 ppm IQ for 26 weeks. Microscopical examinations revealed that the proliferative lesions of the forestomach, including squamous cell hyperplasias, papillomas, and carcinomas, were frequently encountered in male and female rasH2 mice fed with IQ. In non-Tg mice, no significant differences in the incidence of forestomach lesions were observed between the 0 ppm and 300 ppm groups. Histopathological changes such as periportal hepatocellular hypertrophy and oval cell proliferation in the liver were more apparent in female rasH2 and non-Tg mice than in males, and the incidence of hepatocellular altered foci significantly increased in female rasH2 mice in the 300 ppm group as compared to that in the 0 ppm group. These results suggest that the carcinogenic potential of IQ can be detected in rasH2 mice by a 26-week, short-term carcinogenicity test.

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus.

PubMed

Meier, Elizabeth L; Razavi, Shiva; Inoue, Takanari; Goley, Erin D

2016-07-01

In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis. © 2016 John Wiley & Sons Ltd.

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells.

PubMed

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival.

Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

DOE PAGES

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; ...

2015-06-16

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas G12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

PubMed Central

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

2015-01-01

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

Ras Dimer Formation as a New Signaling Mechanism and Potential Cancer Therapeutic Target

PubMed Central

Chen, Mo; Peters, Alec; Huang, Tao; Nan, Xiaolin

2016-01-01

The K-, N-, and HRas small GTPases are key regulators of cell physiology and are frequently mutated in human cancers. Despite intensive research, previous efforts to target hyperactive Ras based on known mechanisms of Ras signaling have been met with little success. Several studies have provided compelling evidence for the existence and biological relevance of Ras dimers, establishing a new mechanism for regulating Ras activity in cells additionally to GTP-loading and membrane localization. Existing data also start to reveal how Ras proteins dimerize on the membrane. We propose a dimer model to describe Ras-mediated effector activation, which contrasts existing models of Ras signaling as a monomer or as a 5-8 membered multimer. We also discuss potential implications of this model in both basic and translational Ras biology. PMID:26423697

The cell cycle regulator ecdysoneless cooperates with H-Ras to promote oncogenic transformation of human mammary epithelial cells

PubMed Central

Bele, Aditya; Mirza, Sameer; Zhang, Ying; Ahmad Mir, Riyaz; Lin, Simon; Kim, Jun Hyun; Gurumurthy, Channabasavaiah Basavaraju; West, William; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

The mammalian ortholog of Drosophila ecdysoneless (Ecd) gene product regulates Rb-E2F interaction and is required for cell cycle progression. Ecd is overexpressed in breast cancer and its overexpression predicts shorter survival in patients with ErbB2-positive tumors. Here, we demonstrate Ecd knock down (KD) in human mammary epithelial cells (hMECs) induces growth arrest, similar to the impact of Ecd Knock out (KO) in mouse embryonic fibroblasts. Furthermore, whole-genome mRNA expression analysis of control vs. Ecd KD in hMECs demonstrated that several of the top 40 genes that were down-regulated were E2F target genes. To address the role of Ecd in mammary oncogenesis, we overexpressed Ecd and/or mutant H-Ras in hTERT-immortalized hMECs. Cell cycle analyses revealed hMECs overexpressing Ecd+Ras showed incomplete arrest in G1 phase upon growth factor deprivation, and more rapid cell cycle progression in growth factor-containing medium. Analyses of cell migration, invasion, acinar structures in 3-D Matrigel and anchorage-independent growth demonstrated that Ecd+Ras-overexpressing cells exhibit substantially more dramatic transformed phenotype as compared to cells expressing vector, Ras or Ecd. Under conditions of nutrient deprivation, Ecd+Ras-overexpressing hMECs exhibited better survival, with substantial upregulation of the autophagy marker LC3 both at the mRNA and protein levels. Significantly, while hMECs expressing Ecd or mutant Ras alone did not form tumors in NOD/SCID mice, Ecd+Ras-overexpressing hMECs formed tumors, clearly demonstrating oncogenic cooperation between Ecd and mutant Ras. Collectively, we demonstrate an important co-oncogenic role of Ecd in the progression of mammary oncogenesis through promoting cell survival. PMID:25616580

Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor.

PubMed

El Khattabi, M; Ockhuijsen, C; Bitter, W; Jaeger, K E; Tommassen, J

1999-06-01

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.

Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids.

PubMed Central

Sipos, G; Puoti, A; Conzelmann, A

1994-01-01

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X-->PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins. Images PMID:8026463

A cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing.

PubMed

Ke, Guoliang; Zhu, Zhi; Wang, Wei; Zou, Yuan; Guan, Zhichao; Jia, Shasha; Zhang, Huimin; Wu, Xuemeng; Yang, Chaoyong James

2014-09-10

Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid-DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid-DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid-DNA probe showed sensitive and reversible response to pH change in the range of 6.0-8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid-DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid-DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies.

MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9.

PubMed

Chai, Sunghee; Cambronne, Xiaolu A; Eichhorn, Stephen W; Goodman, Richard H

2013-10-29

MicroRNA-134 (miR-134) serves as a widely accepted model for microRNA function in synaptic plasticity. In this model, synaptic activity stimulates miR-134 expression, which then regulates dendrite growth and spine formation. By using a ratiometric microRNA sensor, we found, unexpectedly, that miR-134 activity in cortical neurons was restricted to interneurons. Using an assay designed to trap microRNA-mRNA complexes, we determined that miR-134 interacted directly with the mRNA encoding the palmitoylation enzyme, DHHC9. This enzyme is known to palmitoylate H-Ras, a modification required for proper membrane trafficking. Treatment with bicuculline, a GABAA receptor antagonist, decreased DHHC9 expression in somatostatin-positive interneurons and membrane localization of an H-Ras reporter in a manner that depended on miR-134. Thus, although miR-134 has been proposed to affect all types of neurons, we showed that functionally active miR-134 is produced in only a selected population of neurons where it influences the expression of targets, such as DHHC9, that regulate membrane targeting of critical signaling molecules.

Enhanced vapor transport in membrane distillation via functionalized carbon nanotubes anchored into electrospun nanofibres

PubMed Central

Kyoungjin An, Alicia; Lee, Eui-Jong; Guo, Jiaxin; Jeong, Sanghyun; Lee, Jung-Gil; Ghaffour, Noreddine

2017-01-01

To ascertain membrane distillation (MD) as an emerging desalination technology to meet the global water challenge, development of membranes with ideal material properties is crucial. Functionalized carbon nanotubes (CNTs) were anchored to nanofibres of electrospun membranes. Covalent modification and fluorination of CNTs improved their dispersibility and interfacial interaction with the polymer membrane, resulting in well-aligned CNTs inside crystalline fibres with superhydrophobicity. Consideration for the chemical/physical properties of the CNT composite membranes and calculation of their theoretical fluxes revealed the mechanism of MD: CNTs facilitated the repulsive force for Knudsen and molecular diffusions, reduced the boundary-layer effect in viscous flow, and assisted surface diffusion, allowing for fast vapor transport with anti-wetting. This study shows that the role of CNTs and an optimal composite ratio can be used to reduce the gap between theoretical and experimental approaches to desalination. PMID:28134288

Enhanced vapor transport in membrane distillation via functionalized carbon nanotubes anchored into electrospun nanofibres

NASA Astrophysics Data System (ADS)

Kyoungjin An, Alicia; Lee, Eui-Jong; Guo, Jiaxin; Jeong, Sanghyun; Lee, Jung-Gil; Ghaffour, Noreddine

2017-01-01

To ascertain membrane distillation (MD) as an emerging desalination technology to meet the global water challenge, development of membranes with ideal material properties is crucial. Functionalized carbon nanotubes (CNTs) were anchored to nanofibres of electrospun membranes. Covalent modification and fluorination of CNTs improved their dispersibility and interfacial interaction with the polymer membrane, resulting in well-aligned CNTs inside crystalline fibres with superhydrophobicity. Consideration for the chemical/physical properties of the CNT composite membranes and calculation of their theoretical fluxes revealed the mechanism of MD: CNTs facilitated the repulsive force for Knudsen and molecular diffusions, reduced the boundary-layer effect in viscous flow, and assisted surface diffusion, allowing for fast vapor transport with anti-wetting. This study shows that the role of CNTs and an optimal composite ratio can be used to reduce the gap between theoretical and experimental approaches to desalination.

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

PubMed Central

Grabocka, Elda; Pylayeva-Gupta, Yuliya; Jones, Mathew JK; Lubkov, Veronica; Yemanaberhan, Eyoel; Taylor, Laura; Jeng, Hao Hsuan; Bar-Sagi, Dafna

2014-01-01

SUMMARY Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anti-cancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways, and consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo. PMID:24525237

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

PubMed

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells

NASA Technical Reports Server (NTRS)

Scott, D. K.; Weaver, W. R.; Clohisy, J. C.; Brakenhoff, K. D.; Kahn, A. J.; Partridge, N. C.

1992-01-01

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS).

Syndecan-1 alterations during the tumorigenic progression of human colonic Caco-2 cells induced by human Ha-ras or polyoma middle T oncogenes.

PubMed Central

Levy, P.; Munier, A.; Baron-Delage, S.; Di Gioia, Y.; Gespach, C.; Capeau, J.; Cherqui, G.

1996-01-01

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras and pp60c-src in human colonic Caco-2 cells is associated with specific alterations of syndecan-1, a membrane-anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated (Val 12) human Ha-ras gene or with the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines (1) contained smaller amounts of membrane-anchored PGs; (2) exhibited decreased syndecan-1 expression at the protein but not the mRNA level; (3) synthesized 35S-labelled syndecan-1 with decreased specific activity; (4) produced a syndecan-1 ectodomain with a lower molecular mass and reduced GAG chain size and sulphation; and (5) expressed heparanase degradative activity. These results show that the dramatic activation of the tumorigenic potential induced by oncogenic p21ras or Py-MT/pp60c-src in Caco-2 cells is associated with marked alterations of syndecan-1 expression at the translational and post-translational levels. Images Figure 2 PMID:8695359

Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane-anchored EGFP.

PubMed

Deng, Yaqi; Kim, BongWoo; He, Xuelian; Kim, Sunja; Lu, Changqing; Wang, Haibo; Cho, Ssang-Goo; Hou, Yiping; Li, Jianrong; Zhao, Xianghui; Lu, Q Richard

2014-04-01

Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane-anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt-Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin-induced demyelination animal model. Taken together, the membrane-anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries.

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras.

PubMed

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-08-09

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras

PubMed Central

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-01-01

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients. PMID:27437771

Tg.rasH2 Mice and not CByB6F1 Mice Should Be Used for 28-Day Dose Range Finding Studies Prior to 26-Week Tg.rasH2 Carcinogenicity Studies.

PubMed

Paranjpe, Madhav G; Belich, Jessica; Vidmar, Tom J; Elbekai, Reem H; McKeon, Marie; Brown, Caren

Our recent retrospective analysis of data, collected from 29 Tg.rasH2 mouse carcinogenicity studies, determined how successful the strategy of choosing the high dose for the 26-week studies was based on the estimated maximum tolerated dose (EMTD) derived from earlier 28-day dose range finding (DRF) studies conducted in CByB6F1 mice. Our analysis demonstrated that the high doses applied at EMTD in the 26-week Tg.rasH2 studies failed to detect carcinogenic effects. To investigate why the dose selection process failed in the 26-week carcinogenicity studies, the initial body weights, terminal body weights, body weight gains, food consumption, and mortality from the first 4 weeks of 26-week studies with Tg.rasH2 mice were compared with 28-day DRF studies conducted with CByB6F1 mice. Both the 26-week and the earlier respective 28-day studies were conducted with the exact same vehicle, test article, and similar dose levels. The analysis of our results further emphasizes that the EMTD and subsequent lower doses, determined on the basis of the 28-day studies in CByB6F1 mice, may not be an accurate strategy for selecting appropriate dose levels for the 26-week carcinogenicity studies in Tg.rasH2 mice. Based on the analysis presented in this article, we propose that the Tg.rasH2 mice and not the CByB6F1 mice should be used in future DRF studies. The Tg.rasH2 mice demonstrate more toxicity than the CByB6F1 mice, possibly because of their smaller size compared to CByB6F1 mice. Also, the Tg.rasH2 males appear to be more sensitive than the female Tg.rasH2 mice.

Toward the Structure of Dynamic Membrane-Anchored Actin Networks

PubMed Central

Weber, Igor

2007-01-01

In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

PubMed

Weikl, Thomas R; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

2016-09-02

The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

PubMed Central

Weikl, Thomas R.; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

2016-01-01

ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

The possible mechanism of enhanced carcinogenesis induced by genotoxic carcinogens in rasH2 mice.

PubMed

Okamura, Miwa; Unami, Akira; Moto, Mitsuyoshi; Muguruma, Masako; Ito, Tadashi; Jin, Meilan; Oishi, Yuji; Kashida, Yoko; Mitsumori, Kunitoshi

2007-01-08

Microarray and RT-PCR analyses were performed for the transgene and Ras-related genes in forestomach squamous cell carcinomas (SCCs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rasH2 mice; these results were compared with our previous molecular data of N-ethyl-N-nitrosourea-induced forestomach SCCs and urethane-induced lung adenomas in rasH2 mice. Overexpression of the transgene was detected in the DMBA-induced SCCs, suggesting that the transgene plays an important role in enhanced carcinogenesis in rasH2 mice. In addition, the mouse endogenous ras genes were up-regulated in the DMBA-induced SCCs, and are probably involved in the tumorigenesis of forestomach SCCs. Genes such as osteopontin, Cks1b, Tpm1, Reck, gelsolin, and amphiregulin that were commonly altered in these three different carcinogen-induced tumors may contribute to the development of tumors in rasH2 mice.

NSAID-derived gamma-secretase modulators. Part III: Membrane anchoring.

PubMed

Baumann, Stefanie; Höttecke, Nicole; Schubenel, Robert; Baumann, Karlheinz; Schmidt, Boris

2009-12-15

Selective lowering of Abeta(42) levels with small-molecule substrate targeting gamma-secretase modulators (sGSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. Here we present N-substituted carbazole- and O-substituted fenofibrate-derived sGSMs and their activity data. Seven out of 19 screened compounds exhibited promising activity against Abeta(42) secretion at a low micromolar level. We presume that the sGSMs interact with lys624 at the membrane interface and that the lipophilic substituents anchor the compound orientation in the membrane.

Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

PubMed

Barz, W P; Walter, P

1999-04-01

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

PubMed Central

Barz, Wolfgang P.; Walter, Peter

1999-01-01

Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δ cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins. PMID:10198056

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Han-Soo; Kim, Ju Won; Gang, Jingu

2006-09-15

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain themore » differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.« less

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

PubMed Central

Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

2013-01-01

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

Lipid transfer proteins do their thing anchored at membrane contact sites… but what is their thing?

PubMed

Wong, Louise H; Levine, Tim P

2016-04-15

Membrane contact sites are structures where two organelles come close together to regulate flow of material and information between them. One type of inter-organelle communication is lipid exchange, which must occur for membrane maintenance and in response to environmental and cellular stimuli. Soluble lipid transfer proteins have been extensively studied, but additional families of transfer proteins have been identified that are anchored into membranes by transmembrane helices so that they cannot diffuse through the cytosol to deliver lipids. If such proteins target membrane contact sites they may be major players in lipid metabolism. The eukaryotic family of so-called Lipid transfer proteins Anchored at Membrane contact sites (LAMs) all contain both a sterol-specific lipid transfer domain in the StARkin superfamily (related to StART/Bet_v1), and one or more transmembrane helices anchoring them in the endoplasmic reticulum (ER), making them interesting subjects for study in relation to sterol metabolism. They target a variety of membrane contact sites, including newly described contacts between organelles that were already known to make contact by other means. Lam1-4p target punctate ER-plasma membrane contacts. Lam5p and Lam6p target multiple contacts including a new category: vacuolar non-NVJ cytoplasmic ER (VancE) contacts. These developments confirm previous observations on tubular lipid-binding proteins (TULIPs) that established the importance of membrane anchored proteins for lipid traffic. However, the question remaining to be solved is the most difficult of all: are LAMs transporters, or alternately are they regulators that affect traffic more indirectly? © 2016 Authors; published by Portland Press Limited.

Inhibition of Oncogenic functionality of STAT3 Protein by Membrane Anchoring

NASA Astrophysics Data System (ADS)

Liu, Baoxu; Fletcher, Steven; Gunning, Patrick; Gradinaru, Claudiu

2009-03-01

Signal Transducer and Activator of Transcription 3 (STAT3) protein plays an important role in oncogenic processes. A novel molecular therapeutic approach to inhibit the oncogenic functionality of STAT3 is to design a prenylated small peptide sequence which could sequester STAT3 to the plasma membrane. We have also developed a novel fluorescein derivative label (F-NAc), which is much more photostable compared to the popular fluorescein label FITC. Remarkably, the new dye shows fluorescent properties that are invariant over a wide pH range, which is advantageous for our application. We have shown that F-NAc is suitable for single-molecule measurements and its properties are not affected by ligation to biomolecules. The membrane localization via high-affinity prenylated small-molecule binding agents is studied by encapsulating FNAc-labeled STAT3 and inhibitors within a liposome model cell system. The dynamics of the interaction between the protein and the prenylated ligands is investigated at single molecule level. The efficiency and stability of the STAT3 anchoring in lipid membranes are addressed via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope.

Involvement of H- and N-Ras isoforms in transforming growth factor-{beta}1-induced proliferation and in collagen and fibronectin synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Instituto 'Reina Sofia' de Investigacion Nefrologica, Universidad de Salamanca, 37007 Salamanca

2006-07-01

Transforming growth factor {beta}1 (TGF-{beta}1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-{beta} and Ras signaling pathways are closely related: TGF-{beta}1 overcomes Ras mitogenic effects and Ras counteracts TGF-{beta} signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-{beta}1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras {sup -/-}/N-ras {sup -/-}) isoforms andmore » from heterozygote mice (H-ras {sup +/-}/N-ras {sup +/-}). ECM synthesis is increased in basal conditions in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts, this increase being higher after stimulation with TGF-{beta}1. TGF-{beta}1-induced fibroblast proliferation is smaller in H-ras {sup -/-}/N-ras {sup -/-} than in H-ras {sup +/-}/N-ras {sup +/-} fibroblasts. Erk activation is decreased in H-ras {sup -/-}/N-ras {sup -/-} fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras.« less

Rab-NANOPS: FRET biosensors for Rab membrane nanoclustering and prenylation detection in mammalian cells.

PubMed

Najumudeen, Arafath Kaja; Guzmán, Camilo; Posada, Itziar M D; Abankwa, Daniel

2015-01-01

Rab proteins constitute the largest subfamily of Ras-like small GTPases. They are central to vesicular transport and organelle definition in eukaryotic cells. Unlike their Ras counterparts, they are not a hallmark of cancer. However, a number of diseases, including cancer, show a misregulation of Rab protein activity. As for all membrane-anchored signaling proteins, correct membrane organization is critical for Rabs to operate. In this chapter, we provide a detailed protocol for the use of a flow cytometry-based Fluorescence Resonance Energy Transfer (FRET)-biosensors assay, which allows to detect changes in membrane anchorage, subcellular distribution, and of the nanoscale organization of Rab-GTPases in mammalian cell lines. This assay is high-throughput amenable and can therefore be utilized in chemical-genomic and drug discovery efforts.

Novel revertants of H-ras oncogene-transformed R6-PKC3 cells.

PubMed Central

Krauss, R S; Guadagno, S N; Weinstein, I B

1992-01-01

Rat 6 fibroblasts that overproduce protein kinase C beta 1 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) (W.-L. W. Hsiao, G. M. Housey, M. D. Johnson, and I. B. Weinstein, Mol. Cell. Biol. 9:2641-2647, 1989). Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-1a and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-1a line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-1a and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation. Images PMID:1535685

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

PubMed

Ge, Y; Li, Z H; Marshall, M S; Broxmeyer, H E; Lu, L

1998-06-01

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

In TCR-Stimulated T-cells, N-ras Regulates Specific Genes and Signal Transduction Pathways

PubMed Central

Lynch, Stephen J.; Zavadil, Jiri; Pellicer, Angel

2013-01-01

It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4+ T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types. PMID:23755101

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

PubMed Central

Goodwin, J. Shawn; Drake, Kimberly R.; Rogers, Carl; Wright, Latasha; Lippincott-Schwartz, Jennifer; Philips, Mark R.; Kenworthy, Anne K.

2005-01-01

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes. PMID:16027222

¹H, ¹³C and ¹⁵N resonance assignment for the human K-Ras at physiological pH.

PubMed

Vo, Uybach; Embrey, Kevin J; Breeze, Alexander L; Golovanov, Alexander P

2013-10-01

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

Resistance and resilience of small-scale recirculating aquaculture systems (RAS) with or without algae to pH perturbation

PubMed Central

Giatsis, Christos; Md Yusoff, Fatimah; Verreth, Johan; Verdegem, Marc

2018-01-01

The experimental set-up of this study mimicked recirculating aquaculture systems (RAS) where water quality parameters such as dissolved oxygen, pH, temperature, and turbidity were controlled and wastes produced by fish and feeding were converted to inorganic forms. A key process in the RAS was the conversion of ammonia to nitrite and nitrite to nitrate through nitrification. It was hypothesized that algae inclusion in RAS would improve the ammonia removal from the water; thereby improving RAS water quality and stability. To test this hypothesis, the stability of the microbiota community composition in a freshwater RAS with (RAS+A) or without algae (RAS-A) was challenged by introducing an acute pH drop (from pH 7 to 4 during three hours) to the system. Stigeoclonium nanum, a periphytic freshwater microalga was used in this study. No significant effect of the algae presence was found on the resistance to the acute pH drop on ammonia conversion to nitrite and nitrite conversion to nitrate. Also the resilience of the ammonia conversion to the pH drop disruption was not affected by the addition of algae. This could be due to the low biomass of algae achieved in the RAS. However, with regard to the conversion step of nitrite to nitrate, RAS+A was significantly more resilient than RAS-A. In terms of overall bacterial communities, the composition and predictive function of the bacterial communities was significantly different between RAS+A and RAS-A. PMID:29659617

Nanoclustering as a dominant feature of plasma membrane organization.

PubMed

Garcia-Parajo, Maria F; Cambi, Alessandra; Torreno-Pina, Juan A; Thompson, Nancy; Jacobson, Ken

2014-12-01

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent. © 2014. Published by The Company of Biologists Ltd.

High-Resolution FRET Microscopy of Cholera Toxin B-Subunit and GPI-anchored Proteins in Cell Plasma Membranes

PubMed Central

Kenworthy, Anne K.; Petranova, Nadezda; Edidin, Michael

2000-01-01

“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface. PMID:10793141

Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buhrman, Greg; O; #8242

2012-09-17

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the 'off' and 'on' allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond themore » active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.« less

Lipid Domain Structure of the Plasma Membrane Revealed by Patching of Membrane Components

PubMed Central

Harder, Thomas; Scheiffele, Peter; Verkade, Paul; Simons, Kai

1998-01-01

Lateral assemblies of glycolipids and cholesterol, “rafts,” have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T–lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components. PMID:9585412

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

PubMed

Kearney, Bradley M; Johnson, Christian W; Roberts, Daniel M; Swartz, Paul; Mattos, Carla

2014-02-06

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. Copyright © 2013 Elsevier Ltd. All rights reserved.

Membrane anchoring γ-secretase modulators with terpene-derived moieties.

PubMed

Naumann, Eva Christine; Göring, Stefan; Ogorek, Isabella; Weggen, Sascha; Schmidt, Boris

2013-07-01

Modulation of γ-secretase activity is a promising therapeutic strategy for the treatment of Alzheimer's disease. Herein we report on the synthesis of carprofen- and tocopherol-derived small-molecule modulators carrying terpene moieties as lipophilic membrane anchors. Additionally, these modulators are equipped with an acidic moiety, which contributes to the desired modulatory effect on the γ-secretase with decreased formation of Aβ42 and increased Aβ38 production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

PubMed Central

Mayor, S; Sabharanjak, S; Maxfield, F R

1998-01-01

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins. PMID:9707422

Graded inhibition of oncogenic Ras-signaling by multivalent Ras-binding domains

PubMed Central

2014-01-01

Background Ras is a membrane-associated small G-protein that funnels growth and differentiation signals into downstream signal transduction pathways by cycling between an inactive, GDP-bound and an active, GTP-bound state. Aberrant Ras activity as a result of oncogenic mutations causes de novo cell transformation and promotes tumor growth and progression. Results Here, we describe a novel strategy to block deregulated Ras activity by means of oligomerized cognate protein modules derived from the Ras-binding domain of c-Raf (RBD), which we named MSOR for multivalent scavengers of oncogenic Ras. The introduction of well-characterized mutations into RBD was used to adjust the affinity and hence the blocking potency of MSOR towards activated Ras. MSOR inhibited several oncogenic Ras-stimulated processes including downstream activation of Erk1/2, induction of matrix-degrading enzymes, cell motility and invasiveness in a graded fashion depending on the oligomerization grade and the nature of the individual RBD-modules. The amenability to accurate experimental regulation was further improved by engineering an inducible MSOR-expression system to render the reversal of oncogenic Ras effects controllable. Conclusion MSOR represent a new tool for the experimental and possibly therapeutic selective blockade of oncogenic Ras signals. PMID:24383791

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains.

PubMed

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

Two Membrane-Anchored Aspartic Proteases Contribute to Pollen and Ovule Development1[OPEN

PubMed Central

Gao, Hui; Zhang, Yinghui; Wang, Wanlei; Zhao, Keke; Liu, Chunmei; Bai, Lin; Li, Rui

2017-01-01

Aspartic proteases are a class of proteolytic enzymes with conserved aspartate residues, which are implicated in protein processing, maturation, and degradation. Compared with yeast and animals, plants possess a larger aspartic protease family. However, little is known about most of these enzymes. Here, we characterized two Arabidopsis (Arabidopsis thaliana) putative glycosylphosphatidylinositol (GPI)-anchored aspartic protease genes, A36 and A39, which are highly expressed in pollen and pollen tubes. a36 and a36 a39 mutants display significantly reduced pollen activity. Transmission electron microscopy and terminal-deoxynucleotidyl transferase-mediated nick end labeling assays further revealed that the unviable pollen in a36 a39 may undergo unanticipated apoptosis-like programmed cell death. The degeneration of female gametes also occurred in a36 a39. Aniline Blue staining, scanning electron microscopy, and semi in vitro guidance assays indicated that the micropylar guidance of pollen tubes is significantly compromised in a36 a39. A36 and A39 that were fused with green fluorescent protein are localized to the plasma membrane and display punctate cytosolic localization and colocalize with the GPI-anchored protein COBRA-LIKE10. Furthermore, in a36 a39, the abundance of highly methylesterified homogalacturonans and xyloglucans was increased significantly in the apical pollen tube wall. These results indicate that A36 and A39, two putative GPI-anchored aspartic proteases, play important roles in plant reproduction in Arabidopsis. PMID:27872247

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

PubMed Central

Martin, Erik W.; Buzza, Marguerite S.; Driesbaugh, Kathryn H.; Liu, Shihui; Fortenberry, Yolanda M.; Leppla, Stephen H.; Antalis, Toni M.

2015-01-01

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent. PMID:26392335

Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

USDA-ARS?s Scientific Manuscript database

Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

Secreted Glioblastoma Nanovesicles Contain Intracellular Signaling Proteins and Active Ras Incorporated in a Farnesylation-dependent Manner*

PubMed Central

Luhtala, Natalie; Aslanian, Aaron; Yates, John R.; Hunter, Tony

2017-01-01

Glioblastomas (GBMs) are malignant brain tumors with a median survival of less than 18 months. Redundancy of signaling pathways represented within GBMs contributes to their therapeutic resistance. Exosomes are extracellular nanovesicles released from cells and present in human biofluids that represent a possible biomarker of tumor signaling state that could aid in personalized treatment. Herein, we demonstrate that mouse GBM cell-derived extracellular nanovesicles resembling exosomes from an H-RasV12 myr-Akt mouse model for GBM are enriched for intracellular signaling cascade proteins (GO: 0007242) and Ras protein signal transduction (GO: 0007265), and contain active Ras. Active Ras isolated from human and mouse GBM extracellular nanovesicles lysates using the Ras-binding domain of Raf also coprecipitates with ESCRT (endosomal sorting complex required for transport)-associated exosome proteins Vps4a and Alix. Although we initially hypothesized a role for active Ras protein signaling in exosome biogenesis, we found that GTP binding of K-Ras was dispensable for its packaging within extracellular nanovesicles and for the release of Alix. By contrast, farnesylation of K-Ras was required for its packaging within extracellular nanovesicles, yet expressing a K-Ras farnesylation mutant did not decrease the number of nanovesicles or the amount of Alix protein released per cell. Overall, these results emphasize the primary importance of membrane association in packaging of extracellular nanovesicle factors and indicate that screening nanovesicles within human fluids could provide insight into tissue origin and the wiring of signaling proteins at membranes to predict onset and behavior of cancer and other diseases linked to deregulated membrane signaling states. PMID:27909058

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains

PubMed Central

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS. PMID:27924253

Activation of Bmp2-Smad1 Signal and Its Regulation by Coordinated Alteration of H3K27 Trimethylation in Ras-Induced Senescence

PubMed Central

Kaneda, Atsushi; Fujita, Takanori; Anai, Motonobu; Yamamoto, Shogo; Nagae, Genta; Morikawa, Masato; Tsuji, Shingo; Oshima, Masanobu; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence. PMID:22072987

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

PubMed

Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

2011-02-11

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

PubMed

Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

2013-03-01

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus.

PubMed

Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

2016-04-21

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.

Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus

PubMed Central

Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

2016-01-01

Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses. PMID:27110810

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans

PubMed Central

Masoudi, Neda; Fancsalszky, Luca; Pourkarimi, Ehsan; Vellai, Tibor; Alexa, Anita; Reményi, Attila; Gartner, Anton; Mehta, Anil; Takács-Vellai, Krisztina

2013-01-01

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis. PMID:23900546

Polyaspartic acid-anchored mesoporous silica nanoparticles for pH-responsive doxorubicin release

PubMed Central

Zhan, Guiting; Yi, Ping; Yang, Hai; Gan, Lu; Yang, Xiangliang

2018-01-01

Background Nanotechnology-based drug delivery systems exhibit promising therapeutic efficacy in cancer chemotherapy. However, ideal nano drug carriers are supposed to be sufficiently internalized into cancer cells and then release therapeutic cargoes in response to certain intracellular stimuli, which has never been an easy task to achieve. Objective This study is to design mesoporous silica nanoparticles (MSNs)-based pH-responsive nano drug delivery system that is effectively internalized into cancer cells and then release drug in response to lysosomal/endosomal acidified environment. Methods We synthesized MSNs by sol-gel method. Doxorubicin (DOX) was encapsulated into the pores as a model drug. Polyaspartic acid (PAsA) was anchored on the surface of mesoporous MSNs (P-MSNs) as a gatekeeper via amide linkage and endowed MSNs with positive charge. Results In vitro release analysis demonstrated enhanced DOX release from DOX-loaded PAsA-anchored MSNs (DOX@P-MSNs) under endosomal/lysosomal acidic pH condition. Moreover, more DOX@P-MSNs were internalized into HepG2 cells than DOX-loaded MSNs (DOX@MSNs) and free DOX revealed by flow cytometry. Likewise, confocal microscopic images revealed that DOX@P-MSNs effectively released DOX and translocated to the nucleus. Much stronger cytotoxicity of DOX@P-MSNs against HepG2 cells was observed compared with DOX@MSNs and free DOX. Conclusion DOX@P-MSNs were successfully fabricated and achieved pH-responsive DOX release. We anticipated this nanotherapeutics might be suitable contenders for future in vivo cancer chemotherapeutic applications. PMID:29497295

Membrane-Induced Structural Rearrangement and Identification of a Novel Membrane Anchor in Talin F2F3

PubMed Central

Arcario, Mark J.; Tajkhorshid, Emad

2014-01-01

Experimental challenges associated with characterization of the membrane-bound form of talin have prevented us from understanding the molecular mechanism of its membrane-dependent integrin activation. Here, utilizing what we believe to be a novel membrane mimetic model, we present a reproducible model of membrane-bound talin observed across multiple independent simulations. We characterize both local and global membrane-induced structural transitions that successfully reconcile discrepancies between biochemical and structural studies and provide insight into how talin might modulate integrin function. Membrane binding of talin, captured in unbiased simulations, proceeds through three distinct steps: initial electrostatic recruitment of the F2 subdomain to anionic lipids via several basic residues; insertion of an initially buried, conserved hydrophobic anchor into the membrane; and association of the F3 subdomain with the membrane surface through a large, interdomain conformational change. These latter two steps, to our knowledge, have not been observed or described previously. Electrostatic analysis shows talin F2F3 to be highly polarized, with a highly positive underside, which we attribute to the initial electrostatic recruitment, and a negative top face, which can help orient the protein optimally with respect to the membrane, thereby reducing the number of unproductive membrane collision events. PMID:25418091

Expression of chimeric ras protein with OmpF signal peptide in Escherichia coli: localization of OmpF fusion protein in the inner membrane.

PubMed

Yamamoto, T; Okawa, N; Endo, T; Kaji, A

1991-08-01

The ras gene was fused with the DNA sequence of OmpF signal peptide or with the DNA sequence of OmpF signal peptide plus the amino terminal portion of the OmpF gene. They were placed in plasmids together with the bacteriophage lambda PL promoter. These plasmids were introduced into Escherichia coli strain K-12 and the OmpF signal peptide fusion proteins were expressed. These fusion proteins were identified as 29.0 and 30.0 kDa proteins. However, processed products of these proteins were not found in the extract. The fusion proteins were localized mostly in the cytoplasm and the inner membrane, but none of them was secreted into the periplasmic space. On the other hand, the ras protein alone was found in the cytoplasm and not in the inner membrane. Viable counts of E. coli harbouring these plasmids decreased when these fused proteins were induced. Induction of the ras protein alone did not harm cells. These observations suggest that insertion of the heterologous proteins into the inner membrane may cause the bactericidal effect.

SLP-65 signal transduction requires Src homology 2 domain-mediated membrane anchoring and a kinase-independent adaptor function of Syk.

PubMed

Abudula, Abulizi; Grabbe, Annika; Brechmann, Markus; Polaschegg, Christian; Herrmann, Nadine; Goldbeck, Ingo; Dittmann, Kai; Wienands, Jürgen

2007-09-28

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

p21ras independent down-regulation of ras-induced increases in natural antibody binding during tumor progression.

PubMed

Tough, D F; Feng, X; Chow, D A

1995-01-01

Selective outgrowth of v-H-ras-infected 10T1/2 cells based on the cointroduction of a gene for resistance to geneticin (G418), yielded cells which exhibited an increased capacity to bind polyclonal serum natural antibody (NAb). This demonstrated an NAb-susceptible phase of tumor development which would be a basic requirement for NAb-mediated surveillance of tumors. The ras-oncogene dependence of the high-NAb-binding phenotype provided a model for assessing NAb resistance against ras transformants in vivo and for a comparative analysis of phenotypic and genetic alterations contributing to the progression of ras transformants. Variants were developed through in vitro and in vivo models of tumor progression. T24-H-ras and v-H-ras transformants were isolated in vitro through more rigorous growth conditions, focus formation in the presence of untransformed cells with no selecting drug. These clones expressed p21ras but exhibited little or no increase in NAb binding. Variants recovered following growth from intravenous or threshold subcutaneous (s.c.) inocula of high-NAb-binding ras transformants in syngeneic C3H/HeN mice exhibited decreases in NAb binding but no uniform change in p21ras. Concurring inverse correlations between NAb binding and s.c. tumorigenicity were exhibited by the T24-H-ras transformant clones, the ras transformants grown in vivo, and the v-H-ras-transformed clones isolated in the presence versus the absence of untransformed cells. This consistent inverse correlation, together with the reduced NAb binding of the ras transformants grown in vivo, provides strong evidence that NAb participates in the defense against ras-transformed cells in vivo. The lack of any direct correlation between p21ras expression and the reduction in NAb binding or the increase in tumorigenicity of cells generated through progression in vivo suggested the regulatory action of additional genes. Hybridization studies between high- and low-NAb-binding clones implicated the

Discovery of Dual-Action Membrane-Anchored Modulators of Incretin Receptors

PubMed Central

Fortin, Jean-Philippe; Chinnapen, Daniel; Beinborn, Martin; Lencer, Wayne; Kopin, Alan S.

2011-01-01

Background The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function. Methodology/Principal Findings Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G7]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G7 into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G7]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes. Conclusions These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target. PMID:21935440

Inhibition of RAS activation due to a homozygous ezrin variant in patients with profound intellectual disability.

PubMed

Riecken, Lars Björn; Tawamie, Hasan; Dornblut, Carsten; Buchert, Rebecca; Ismayel, Amina; Schulz, Alexander; Schumacher, Johannes; Sticht, Heinrich; Pohl, Katja J; Cui, Yan; Reis, André; Morrison, Helen; Abou Jamra, Rami

2015-02-01

Gain-of-function alterations in several components and modulators of the Ras-MAPK pathway lead to dysregulation of the pathway and cause a broad spectrum of autosomal dominant developmental disorders, collectively known as RASopathies. These findings demonstrate the importance of tight multilevel Ras regulation to safeguard signaling output and prevent aberrant activity. We have recently identified ezrin as a novel regulatory element required for Ras activation. Homozygosity mapping and exome sequencing have now revealed the first presumably disease-causing variant in the coding gene EZR in two siblings with a profound intellectual disability. Localization and membrane targeting of the altered ezrin protein appeared normal but molecular modeling suggested protein interaction surfaces to be disturbed. Functional analysis revealed that the altered ezrin protein is no longer able to bind Ras and facilitate its activation. Furthermore, expression of the altered ezrin protein in different cell lines resulted in abnormal cellular processes, including reduced proliferation and neuritogenesis, thus revealing a possible mechanism for its phenotype in humans. To our knowledge, this is the first report of an autosomal recessively inherited loss-of-function mutation causing reduced Ras activity and thus extends and complements the pathogenicity spectrum of known Ras-MAPK pathway disturbances. © 2014 WILEY PERIODICALS, INC.

Ras-related tumorigenesis is suppressed by BNIP3-mediated autophagy through inhibition of cell proliferation.

PubMed

Wu, Shan-Ying; Lan, Sheng-Hui; Cheng, Da-En; Chen, Wei-Kai; Shen, Cheng-Huang; Lee, Ying-Ray; Zuchini, Roberto; Liu, Hsiao-Sheng

2011-12-01

Autophagy plays diverse roles in Ras-related tumorigenesis. H-ras(val12) induces autophagy through multiple signaling pathways including Raf-1/ERK pathway, and various ERK downstream molecules of autophagy have been reported. In this study, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) is identified as a downstream transducer of the Ras/Raf/ERK signaling pathway to induce autophagy. BNIP3 was upregulated by H-ras(val12) at the transcriptional level to compete with Beclin 1 for binding with Bcl-2. H-ras(val12)-induced autophagy suppresses cell proliferation demonstrated both in vitro and in vivo by expression of ectopic BNIP3, Atg5, or interference RNA of BNIP3 (siBNIP3) and Atg5 (shAtg5) using mouse NIH3T3 and embryo fibroblast cells. H-ras(val12) induces different autophagic responses depending on the duration of Ras overexpression. After a short time (48 hours) of Ras overexpression, autophagy inhibits cell proliferation. In contrast, a longer time (2 weeks) of Ras overexpression, cell proliferation was enhanced by autophagy. Furthermore, overexpression of mutant Ras, BNIP3, and LC3-II was detected in bladder cancer T24 cells and the tumor parts of 75% of bladder cancer specimens indicating a positive correlation between autophagy and tumorigenesis. Taken together, our mouse model demonstrates a balance between BNIP3-mediated autophagy and H-ras(val12)-induced tumor formation and reveals that H-ras(val12) induces autophagy in a BNIP3-dependent manner, and the threshold of autophagy plays a decisive role in H-ras(val12)-induced tumorigenesis. Our findings combined with others' reports suggest a new therapeutic strategy against Ras-related tumorigenesis by negative or positive regulation of autophagic activity, which is determined by the level of autophagy and tumor progression stages.

The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serasinghe, Madhavika N.; Mitochondrial Research and Innovation Group, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; Yoon, Yisang

2008-11-15

Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six {alpha}-helices ({alpha}1-{alpha}6) out of which {alpha}2-{alpha}5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found thatmore » hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the {alpha}1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that {alpha}1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the {alpha}5 helix and the linker between {alpha}3 and {alpha}4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by {alpha}1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.« less

Drugging the undruggable Ras: mission possible?

PubMed Central

Cox, Adrienne D.; Fesik, Stephen W.; Kimmelman, Alec C.; Luo, Ji; Der, Channing J.

2015-01-01

Despite more than three decades of intensive effort, no effective pharmacologic inhibitors of the Ras oncoproteins have reached the clinic, prompting the widely held perception that Ras proteins are “undruggable”. However, there is renewed hope that this is not the case. In this review, we summarize the progress and promise of five key directions. First, we focus on the prospects of direct inhibitors of Ras. Second, we revisit the issue of whether blocking Ras membrane association is a viable approach. Third, we assess the status of targeting Ras downstream effector signalling, arguably the most favourable current direction. Fourth, we address whether the search for synthetic lethal interactors of mutant RAS still holds promise. Finally, Ras-mediated changes in cell metabolism have recently been described. Can these changes be exploited for new therapeutic directions? We conclude with perspectives on how additional complexities, not yet fully understood, may impact each of these approaches. PMID:25323927

Temporal dissection of K-ras(G12D) mutant in vitro and in vivo using a regulatable K-ras(G12D) mouse allele.

PubMed

Wang, Zuoyun; Feng, Yan; Bardeesy, Nabeel; Bardessy, Nabeel; Wong, Kwok-Kin; Liu, Xin-Yuan; Ji, Hongbin

2012-01-01

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-ras(G12D) (ER-K-ras(G12D)) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-ras(G12D) independent of the gender status. Furthermore, tamoxifen-inducible activation of K-ras(G12D) promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-ras(G12D) in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy

PubMed Central

Ramos-Kuri, Manuel; Rapti, Kleopatra; Mehel, Hind; Zhang, Shihong; Dhandapany, Perundurai S.; Liang, Lifan; García-Carrancá, Alejandro; Bobe, Regis; Fischmeister, Rodolphe; Adnot, Serge; Lebeche, Djamel; Hajjar, Roger J.; Lipskaia, Larissa; Chemaly, Elie R.

2015-01-01

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy. PMID:26260012

Estrogen-anchored pH-sensitive liposomes as nanomodule designed for site-specific delivery of doxorubicin in breast cancer therapy.

PubMed

Paliwal, Shivani R; Paliwal, Rishi; Pal, Harish C; Saxena, Ajeet K; Sharma, Pradyumana R; Gupta, Prem N; Agrawal, Govind P; Vyas, Suresh P

2012-01-01

The present investigation reports the development of nanoengineered estrogen receptor (ER) targeted pH-sensitive liposome for the site-specific intracellular delivery of doxorubicin (DOX) for breast cancer therapy. Estrone, a bioligand, was anchored on the surface of pH-sensitive liposome for drug targeting to ERs. The estrone-anchored pH-sensitive liposomes (ES-pH-sensitive-SL) showed fusogenic potential at acidic pH (5.5). In vitro cytotoxicity studies carried out on ER-positive MCF-7 breast carcinoma cells revealed that ES-pH-sensitive-SL formulation was more cytotoxic than non-pH-sensitive targeted liposomes (ES-SL). The flow cytometry analysis confirmed significant enhanced uptake (p < 0.05) of ES-pH-sensitive-SL by MCF-7 cells. Intracellular delivery and nuclear localization of the DOX was confirmed by fluorescence microscopy. The mechanism for higher cytotoxicity shown by estrone-anchored pH-sensitive liposomal-DOX was elucidated using reactive oxygen species (ROS) determination. The in vivo biodistribution studies and antitumor activities of formulations were evaluated on tumor bearing female Balb/c mice followed by intravenous administration. The ES-pH-sensitive-SL efficiently suppressed the breast tumor growth in comparison to both ES-SL and free DOX. Serum enzyme activities such as LDH and CPK levels were assayed for the evaluation of DOX induced cardiotoxicity. The ES-pH-sensitive-SL accelerated the intracellular trafficking of encapsulated DOX, thus increasing the therapeutic efficacy. The findings support that estrone-anchored pH-sensitive liposomes could be one of the promising nanocarriers for the targeted intracellular delivery of anticancer agents to breast cancer with reduced systemic side effects.

High Background Incidence of Spontaneous Subcapsular Adrenal Gland Hyperplasia of Tg.rasH2 Mice Used in 26-week Carcinogenicity Studies.

PubMed

Boyle, Molly H; Paranjpe, Madhav G; Creasy, Dianne M

2018-06-01

The Tg.rasH2 model was accepted by regulatory agencies worldwide for 26-week carcinogenicity assays as an alternative to the standard 2-year assays in conventional mice in 2003. Several references documenting spontaneous nonneoplastic findings and incidences of spontaneous tumors in the Tg.rasH2 mice have been published. The purpose of this publication is to add adrenal gland subcapsular hyperplasia to the database pertaining to spontaneous lesions noted in Tg.rasH2 mice, review physiology related to this finding, and discuss its significance. The incidence of spontaneous subcapsular adrenal gland hyperplasia was determined in control Tg.rasH2 mice from nine 26-week carcinogenicity studies carried out within the last 5 years at two contract research organizations. Incidence of this finding ranged from 56% to 79% in males and 88% to 100% in females, with an incidence average of 62% in males and 93% in females. Adrenal gland subcapsular hyperplasia is a common finding in male and female Tg.rasH2 mice that did not progress to neoplasia in Tg.rasH2 mice. In general, it tends to be more frequent and severe in females in comparison to males.

Mutation-Specific RAS Oncogenicity Explains N-RAS Codon 61 Selection in Melanoma

PubMed Central

Burd, Christin E.; Liu, Wenjin; Huynh, Minh V.; Waqas, Meriam A.; Gillahan, James E.; Clark, Kelly S.; Fu, Kailing; Martin, Brit L.; Jeck, William R.; Souroullas, George P.; Darr, David B.; Zedek, Daniel C.; Miley, Michael J.; Baguley, Bruce C.; Campbell, Sharon L.

2014-01-01

N-RAS mutation at codon 12, 13 or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an N-RasQ61R knock-in allele to similarly designed K-RasG12D and N-RasG12D alleles. With concomitant p16INK4a inactivation, K-RasG12D or N-RasQ61R expression efficiently promoted melanoma in vivo, whereas N-RasG12D did not. Additionally, N-RasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of N-RasQ61R and N-RasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, N-RasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity and increased stability when compared to N-RasG12D. This work identifies a faithful model of human N-RAS mutant melanoma, and suggests that the increased melanomagenecity of N-RasQ61R over N-RasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. PMID:25252692

Ras and relatives--job sharing and networking keep an old family together.

PubMed

Ehrhardt, Annette; Ehrhardt, Götz R A; Guo, Xuecui; Schrader, John W

2002-10-01

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

New insights into the targeting of a sub-set of tail-anchored proteins to the outer mitochondrial membrane

USDA-ARS?s Scientific Manuscript database

Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins that are defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Nout-Cin orientation. The molecular mechanisms by which TA p...

Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jemmerson, R.; Low, M.G.

1987-09-08

Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast,more » treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.« less

Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

PubMed Central

Grover, Rahul; Fischer, Janine; Schwarz, Friedrich W.; Walter, Wilhelm J.; Schwille, Petra; Diez, Stefan

2016-01-01

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. PMID:27803325

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

PubMed

Fiordalisi, James J; Holly, Stephen P; Johnson, Ronald L; Parise, Leslie V; Cox, Adrienne D

2002-03-29

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first

The Tip of the Four N-Terminal α-Helices of Clostridium sordellii Lethal Toxin Contains the Interaction Site with Membrane Phosphatidylserine Facilitating Small GTPases Glucosylation

PubMed Central

Varela Chavez, Carolina; Haustant, Georges Michel; Baron, Bruno; England, Patrick; Chenal, Alexandre; Pauillac, Serge; Blondel, Arnaud; Popoff, Michel-Robert

2016-01-01

Clostridium sordellii lethal toxin (TcsL) is a powerful virulence factor responsible for severe toxic shock in man and animals. TcsL belongs to the large clostridial glucosylating toxin (LCGT) family which inactivates small GTPases by glucosylation with uridine-diphosphate (UDP)-glucose as a cofactor. Notably, TcsL modifies Rac and Ras GTPases, leading to drastic alteration of the actin cytoskeleton and cell viability. TcsL enters cells via receptor-mediated endocytosis and delivers the N-terminal glucosylating domain (TcsL-cat) into the cytosol. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to the lipid membrane. We have previously reported that the N-terminal four helical bundle structure (1–93 domain) recognizes a broad range of lipids, but that TcsL-cat specifically binds to PS and phosphatidic acid. Here, we show using mutagenesis that the PS binding site is localized on the tip of the four-helix bundle which is rich in positively-charged amino acids. Residues Y14, V15, F17, and R18 on loop 1, between helices 1 and 2, in coordination with R68 from loop 3, between helices 3 and 4, form a pocket which accommodates L-serine. The functional PS-binding site is required for TcsL-cat binding to the plasma membrane and subsequent cytotoxicity. TcsL-cat binding to PS facilitates a high enzymatic activity towards membrane-anchored Ras by about three orders of magnitude as compared to Ras in solution. The PS-binding site is conserved in LCGTs, which likely retain a common mechanism of binding to the membrane for their full activity towards membrane-bound GTPases. PMID:27023605

Inhibition of Ras for cancer treatment: the search continues

PubMed Central

Baines, Antonio T.; Xu, Dapeng; Der, Channing J.

2012-01-01

Background The RAS oncogenes (HRAS, NRAS and KRAS) comprise the most frequently mutated class of oncogenes in human cancers (33%), stimulating intensive effort in developing anti-Ras inhibitors for cancer treatment. Discussion Despite intensive effort, to date no effective anti-Ras strategies have successfully made it to the clinic. We present an overview of past and ongoing strategies to inhibit oncogenic Ras in cancer. Conclusions Since approaches to directly target mutant Ras have not been successful, most efforts have focused on indirect approaches to block Ras membrane association or downstream effector signaling. While inhibitors of effector signaling are currently under clinical evaluation, genome-wide unbiased genetic screens have identified novel directions for future anti-Ras drug discovery. PMID:22004085

The C-terminal ester of membrane anchored peptide ion channels affects anion transport.

PubMed

Djedovic, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Schlesinger, Paul H; Gokel, George W

2003-12-07

Five heptapeptide derivatives, [CH3(CH2)17]2NCOCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR, in which R = ethyl, 2-propyl, heptyl, benzyl, and cyclohexylmethyl, were found to transport chloride anion through a phospholipid bilayer to varying extents dependent on the identity of R. It was concluded that the R group is a membrane anchor for the synthetic chloride channels.

Differentiated State of Initiating Tumor Cells Is Key to Distinctive Immune Responses Seen in H-RasG12V-Induced Squamous Tumors.

PubMed

Podolsky, Michael A; Bailey, Jacob T; Gunderson, Andrew J; Oakes, Carrie J; Breech, Kyle; Glick, Adam B

2017-03-01

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-Ras G12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1 -/- background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1 +/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1 +/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198-210. ©2017 AACR . ©2017 American Association for Cancer Research.

CuSO4/H2O2-Triggered Polydopamine/Poly(sulfobetaine methacrylate) Coatings for Antifouling Membrane Surfaces.

PubMed

Zhang, Chao; Li, Hao-Nan; Du, Yong; Ma, Meng-Qi; Xu, Zhi-Kang

2017-02-07

Mussel-inspired polydopamine (PDA) coatings have been broadly exploited for constructing functional membrane surfaces. One-step codeposition of PDA with antifouling polymers, especially zwitterionic polymers, has been regarded as a promising strategy for fabricating antifouling membrane surfaces. However, one challenge is that the codeposition is usually a slow process over 10 h or even several days. Herein, we report that CuSO 4 /H 2 O 2 is able to notably accelerate the codeposition process of PDA with poly(sulfobetaine methacrylate) (PSBMA). In our case, PSBMA is facilely anchored to the polypropylene microporous membrane (PPMM) surfaces within 1 h with the assistance of PDA because of its strong interfacial adhesion. The PDA/PSBMA-coated PPMMs show excellent surface hydrophilicity, high water permeation flux (7506 ± 528 L/m 2 ·h at 0.1 MPa), and an outstanding antifouling property. Moreover, the antifouling property is maintained after the membranes are treated with acid and alkali solutions as well as organic solvents. To recap, it provides a facile, universal, and time-saving strategy for exploiting high-efficiency and durable antifouling membrane surfaces.

Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

PubMed

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

Targeting the RAS oncogene

PubMed Central

Takashima, Asami

2013-01-01

Introduction The Ras proteins (K-Ras, N-Ras, H-Ras) are GTPases that function as molecular switches for a variety of critical cellular activities and their function is tightly and temporally regulated in normal cells. Oncogenic mutations in the RAS genes, which create constitutively-active Ras proteins, can result in uncontrolled proliferation or survival in tumor cells. Areas covered The paper discusses three therapeutic approaches targeting the Ras pathway in cancer: 1) Ras itself, 2) Ras downstream pathways, and 3) synthetic lethality. The most adopted approach is targeting Ras downstream signaling, and specifically the PI3K-AKT-mTOR and Raf-MEK pathways, as they are frequently major oncogenic drivers in cancers with high Ras signaling. Although direct targeting of Ras has not been successful clinically, newer approaches being investigated in preclinical studies, such as RNA interference-based and synthetic lethal approaches, promise great potential for clinical application. Expert opinion The challenges of current and emerging therapeutics include the lack of “tumor specificity” and their limitation to those cancers which are “dependent” upon aberrant Ras signaling for survival. While the newer approaches have the potential to overcome these limitations, they also highlight the importance of robust preclinical studies and bidirectional translational research for successful clinical development of Ras-related targeted therapies. PMID:23360111

TMPRSS2, a novel membrane-anchored mediator in cancer pain

PubMed Central

Lam, David K.; Dang, Dongmin; Flynn, Andrea N.; Hardt, Markus; Schmidt, Brian L.

2016-01-01

More than half of all cancer patients will suffer significant pain during the course of their disease. The strategic localization of TMPRSS2, a membrane-bound serine protease, on the cancer cell surface may allow it to mediate signal transduction between the cancer cell and its extracellular environment. Here we show TMPRSS2 expression is not only dramatically increased in the primary cancers of patients but TMPRSS2-immunopositivity is also directly correlated with cancer pain severity in these patients. TMPRSS2 induced proteolytic activity, activated trigeminal neurons, and produced marked mechanical hyperalgesia when administered into the hindpaw of wild-type mice but not in PAR2-deficient mice. Co-culture of human cancer cells with murine trigeminal neurons demonstrated co-localization of TMPRSS2 with PAR2. These results point to a novel role for a cell membrane-anchored mediator in cancer pain, as well as pain in general. PMID:25734995

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Gastroprotective effects of several H2RAs on ibuprofen-induced gastric ulcer in rats.

PubMed

Liu, Jing; Sun, Dan; He, Jinfeng; Yang, Chengli; Hu, Tingting; Zhang, Lijing; Cao, Hua; Tong, Ai-Ping; Song, Xiangrong; Xie, Yongmei; He, Gu; Guo, Gang; Luo, Youfu; Cheng, Ping; Zheng, Yu

2016-03-15

Ibuprofen is the first line of treatment for osteoarthritis and arthritis. The main side effects of ibuprofen especially in long-term treatment include gastric ulcer, duodenal ulcer and indigestion etc. Therefore, screening drugs with effective gastric protective effects and low toxicity for combination therapy with ibuprofen is necessary. The mechanism of gastric damage induced by ibuprofen is still unclear, however, cell damage caused by reactive oxygen species (ROS) is considered as the main reason. Preliminary screening of literature with the criteria of low toxicity led to four histamine-2 receptor antagonists (H2RAs): nizatidine, famotidine, lafutidine, and roxatidine acetate, which were selected for further investigation. These drugs were evaluated systemically by examining the gastric ulcer index, lipid peroxidation (LPO), membrane permeability, toxicity to main organs, and the influence on the activity of antioxidant enzymes, and myeloperoxidase (MPO). Nizatidine was found to be the best gastric protective agent. It exhibited excellent protective effect by increasing antioxidant enzyme activity, decreasing MPO activity, reducing LPO, and membrane permeability. Combination treatment with nizatidine and ibuprofen did not show any significant toxicity. Nizatidine was considered as a good option for combination therapy with ibuprofen especially for diseases that require long-term treatment such as arthritis and osteoarthritis. Copyright © 2016 Elsevier Inc. All rights reserved.

Atomistic-level study of the interactions between hIAPP protofibrils and membranes: Influence of pH and lipid composition.

PubMed

Qian, Zhenyu; Zou, Yu; Zhang, Qingwen; Chen, Peijie; Ma, Buyong; Wei, Guanghong; Nussinov, Ruth

2018-02-09

The pathology of type 2 diabetes mellitus is associated with the aggregation of human islet amyloid polypeptide (hIAPP) and aggregation-mediated membrane disruption. The interactions of hIAPP aggregates with lipid membrane, as well as the effects of pH and lipid composition at the atomic level, remain elusive. Herein, using molecular dynamics simulations, we investigate the interactions of hIAPP protofibrillar oligomers with lipids, and the membrane perturbation that they induce, when they are partially inserted in an anionic dipalmitoyl-phosphatidylglycerol (DPPG) membrane or a mixed dipalmitoyl-phosphatidylcholine (DPPC)/DPPG (7:3) lipid bilayer under acidic/neutral pH conditions. We observed that the tilt angles and insertion depths of the hIAPP protofibril are strongly correlated with the pH and lipid composition. At neutral pH, the tilt angle and insertion depth of hIAPP protofibrils at a DPPG bilayer reach ~52° and ~1.62 nm with respect to the membrane surface, while they become ~77° and ~1.75 nm at a mixed DPPC/DPPG membrane. The calculated tilt angle of hIAPP at DPPG membrane is consistent with a recent chiral sum frequency generation spectroscopic study. The acidic pH induces a smaller tilt angle of ~40° and a shallower insertion depth (~1.24 nm) of hIAPP at the DPPG membrane surface, mainly due to protonation of His18 near the turn region. These differences mainly result from a combination of distinct electrostatic, van der Waals, hydrogen bonding and salt-bridge interactions between hIAPP and lipid bilayers. The hIAPP-membrane interaction energy analysis reveals that besides charged residues K1, R11 and H18, aromatic residues Phe15 and Phe23 also exhibit strong interactions with lipid bilayers, revealing the crucial role of aromatic residues in stabilizing the membrane-bound hIAPP protofibrils. hIAPP-membrane interactions disturb the lipid ordering and the local bilayer thickness around the peptides. Our results provide atomic-level information

The AAA protein Msp1 mediates clearance of excess tail-anchored proteins from the peroxisomal membrane

PubMed Central

Weir, Nicholas R; Kamber, Roarke A; Martenson, James S

2017-01-01

Msp1 is a conserved AAA ATPase in budding yeast localized to mitochondria where it prevents accumulation of mistargeted tail-anchored (TA) proteins, including the peroxisomal TA protein Pex15. Msp1 also resides on peroxisomes but it remains unknown how native TA proteins on mitochondria and peroxisomes evade Msp1 surveillance. We used live-cell quantitative cell microscopy tools and drug-inducible gene expression to dissect Msp1 function. We found that a small fraction of peroxisomal Pex15, exaggerated by overexpression, is turned over by Msp1. Kinetic measurements guided by theoretical modeling revealed that Pex15 molecules at mitochondria display age-independent Msp1 sensitivity. By contrast, Pex15 molecules at peroxisomes are rapidly converted from an initial Msp1-sensitive to an Msp1-resistant state. Lastly, we show that Pex15 interacts with the peroxisomal membrane protein Pex3, which shields Pex15 from Msp1-dependent turnover. In sum, our work argues that Msp1 selects its substrates on the basis of their solitary membrane existence. PMID:28906250

Hydration of sulfonated polyimide membranes. I. Na + and NH +(C 2H 5) 3 homopolymers

NASA Astrophysics Data System (ADS)

Jamróz, Dorota; Maréchal, Yves

2004-05-01

Hydration of Na + and HN +(C 2H 5) 3 sulfonated polyimide membranes is probed by infrared spectrometry using a recently described method. These membranes consist of identical sulfonated repeat unit (homopolymers) and form a good starting point to study more elaborated membranes, composed of these units plus similar ones with no sulfonic groups (block copolymers). These latter membranes may be used in fuel cells and will be described in a forthcoming article. We first identify the bands of hydrophilic groups, which will be anchor points for hydration. We then define three 'elementary hydration spectra' onto which all hydration spectra can be decomposed. Their analysis allows us to measure the water uptake of these membranes as a function of the hygrometry of the ambient atmosphere and to determine the structures of the dried membranes and their hydration mechanisms in terms of chemical reactions.

Depletion of Pokemon gene inhibits hepatocellular carcinoma cell growth through inhibition of H-ras.

PubMed

Zhang, Quan-Le; Tian, De-An; Xu, Xiang-Jiang

2011-01-01

Pokemon is a transcription repressor which plays a critical role in cell transformation and malignancy. However, little is known about its effect on the development and progression of hepatocellular carcinoma (HCC). The aim of this study was to investigate the expression of Pokemon in human HCC tissues and the biological behavior of Pokemon in HCC cells in which it is overexpressed. We also explored the expression of potential downstream cofactors of Pokemon. Reverse transcription polymerase chain reaction and Western blot analysis were used to investigate the expression of Pokemon in tissues of 30 HCC patients. We then examined cell proliferation or apoptosis and β-catenin or H-ras expression in Pokemon-depleted HepG(2) cells using DNA vector-based RNA interference technology. Pokemon was markedly expressed in 22/30 (73.3%) HCC tissues, with expression levels higher than in adjacent normal liver tissues (p < 0.05); expression is correlated with tumor size. In contrast, depletion of Pokemon inhibited proliferation of HepG(2) or induced apoptosis. Also, H-ras expression decreased to a large extent. Pokemon exerts its oncogenic activity in the development of HCC by promoting cancer cell growth and reducing apoptosis, and the effect may be mediated by H-ras. Copyright © 2011 S. Karger AG, Basel.

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

PubMed

Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2018-05-01

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.

PubMed

Philips, M R; Pillinger, M H; Staud, R; Volker, C; Rosenfeld, M G; Weissmann, G; Stock, J B

1993-02-12

In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.

Phosphatidylkojibiosyl Diglyceride: metabolism and function as an anchor in bacterial cell membrane.

PubMed

Pieringer, R A

1975-07-01

The recently discovered phosphoglycolipid, phosphatidylkojibiosyl diglyceride (PKD), was first observed as a biosynthetic by-product of glycosyl diglyceride metabolism in Streptococcus faecalis (faecium) ATCC 9790. Its structure is 1, 2-diacyl-3-O-alpha-Dglucopyranosyl-6'-O-phosphoryl- [1'', 2''-diacyl-3''-O-sn-glycerol]-alpha-D-glucopyranosyl)-sn-glycerol. The biosynthesis of phosphatidyl-kojibiosyl diglyceride occurs by a novel transphosphatidylation reaction in which a phosphatidyl glycerol to the primary alcohol function at the 6 position of the internal glucose of kojibiosyl diglyceride. The reaction is catalyzed by a membrane-derived enzyme. Phosphatidyl-kojibiosyl diglyceride is bound covalently through a phosphodiester bond to the polyglycerol phosphate moiety of membrane lipoteichoic acid from S. faecalis. Phosphatidylkojibiosyl diglyceride has four nonpolar long chain fatty acyl groups and appears to have the necessary physico-chemical properties to anchor the long hydrophilic glycerol phosphate polymer of lipoteichoic acid to the hydrophobic enviroment of the membrane of S. faecalis and probably other gram-positive bacteria as well.

Cancer stem cell drugs target K-ras signaling in a stemness context

PubMed Central

Najumudeen, A K; Jaiswal, A; Lectez, B; Oetken-Lindholm, C; Guzmán, C; Siljamäki, E; Posada, I M D; Lacey, E; Aittokallio, T; Abankwa, D

2016-01-01

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC. PMID:26973241

Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V

PubMed Central

Schmees, C.; Villaseñor, R.; Zheng, W.; Ma, H.; Zerial, M.; Heldin, C.-H.; Hellberg, C.

2012-01-01

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis. PMID:22573884

Anti-cancer peptides from ras-p21 and p53 proteins.

PubMed

Pincus, Matthew R; Fenelus, Maly; Sarafraz-Yazdi, Ehsan; Adler, Victor; Bowne, Wilbur; Michl, Josef

2011-01-01

We have employed computer-based molecular modeling approaches to design peptides from the ras-p21 and p53 proteins that either induce tumor cell reversion to the untransformed phenotype or induce tumor cell necrosis without affecting normal cells. For rasp21, we have computed and superimposed the average low energy structures for the wild-type protein and oncogenic forms of this protein and found that specific domains change conformation in the oncogenic proteins. We have synthesized peptides corresponding to these and found that ras peptides, 35-47 (PNC-7) and 96-110 (PNC-2), block oncogenic ras-p21-induced oocyte maturation but have no effect on insulin-induced oocyte maturation that requires activation of endogenous wild-type ras-p21. These results show signal transduction pathway differences between oncogenic and activated wild-type ras-p21. Both peptides, attached to a membrane-penetrating peptide (membrane residency peptide or MRP), either induce phenotypic reversion to the untransformed phenotype or tumor cell necrosis of several ras-transformed cell lines, but have no effect on the growth of normal cells. Using other computational methods, we have designed two peptides, PNC-27 and 28, containing HDM-2-protein-binding domain sequences from p53 linked on their C-termini to the MRP that induce pore formation in the membranes of a wide range of cancer cells but not any normal cells tested. This is due to the expression of HDM-2 in the cancer cell membrane that does not occur in normal cells. These peptides eradicate a highly malignant tumor in nude mice with no apparent side effects. Both ras and p53 peptides show promise as anti-tumor agents in humans.

Molecular Details of α-Synuclein Membrane Association Revealed by Neutrons and Photons

PubMed Central

Jiang, Zhiping; Hess, Sara K.; Heinrich, Frank; Lee, Jennifer C.

2015-01-01

α-Synuclein (α-syn) is an abundant neuronal protein associated with Parkinson’s disease that is disordered in solution, but exists in equilibrium between a bent- and an elongated-helix on negatively charged membranes. Here, neutron reflectometry (NR) and fluorescence spectroscopy were employed to uncover molecular details of the interaction between α-syn and two anionic lipids, phosphatidic acid (PA) and phosphatidylserine (PS). Both NR and site-specific Trp measurements indicate that penetration depth of α-syn is similar for either PA- or PS-containing membranes (~9–11 Å from bilayer center) even though there is a preference for α-syn binding to PA. However, closer examination of the individual Trp quenching profiles by brominated lipids reveal differences into local membrane interactions especially at position 39 where conformational heterogeneity was observed. The data also indicate that while W94 penetrates the bilayer as deeply as W4, W94 resides in a more polar surrounding. Taken together, we suggest the N- and C-terminal regions near positions 4 and 94 are anchored to the membrane, while the putative linker spanning residue 39 samples multiple conformations, which are sensitive to the chemical nature of the membrane surface. This flexibility may enable α-syn to bind diverse biomembranes in vivo. PMID:25790164

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability.

PubMed

Suffrian, K; Schulz, K G; Gutowska, M A; Riebesell, U; Bleich, M

2011-05-01

• To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. • Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). • Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). • The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

Activation of RAS family genes in urothelial carcinoma.

PubMed

Boulalas, I; Zaravinos, A; Karyotis, I; Delakas, D; Spandidos, D A

2009-05-01

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

RAS - Screens & Assays - Drug Discovery

Cancer.gov

The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

PPIs Prevent Aspirin-Induced Gastrointestinal Bleeding Better than H2RAs. A Systematic Review and Meta-analysis.

PubMed

Szabó, Imre L; Mátics, Robert; Hegyi, Peter; Garami, Andras; Illés, Anita; Sarlós, Patricia; Bajor, Judit; Szűcs, Akos; Mosztbacher, Dora; Márta, Katalin; Szemes, Kata; Csekő, Kata; Kővári, Balint; Rumbus, Zoltan; Vincze, Áron

2017-12-01

Aspirin is one of the most widely used medication for its analgesic and anti-platelet properties and thus a major cause for gastrointestinal (GI) bleeding. This study compared the preventive effect of histamine-2 receptor antagonists (H2RAs) and proton-pump inhibitors (PPIs) against chronic low-dose aspirin (LDA)-related GI bleeding and ulcer formation. Electronic databases of Pubmed, Embase and Cochrane Central Register of Controlled Trials were searched for human observations (randomised controlled trials and observational studies) comparing the long term effects of PPIs and H2RAs treatment in the prevention of GI bleeding or ulcer formation in patients on chronic LDA treatment listed up till September 30, 2016. Two independent authors searched databases using PICO questions (aspirin, H2RA, PPI, GI bleeding or ulcer), and reviewed abstracts and articles for comprehensive studies keeping adequate study quality. Data of weighted odds ratios were statistically evaluated using Comprehensive Metaanalysis (Biostat, Inc., Engelwood, MJ, USA), potential bias was checked. Nine studies for GI bleeding and eight studies for ulcer formation were found meeting inclusion criteria, altogether 1,879 patients were included into review. The H2RAs prevented less effectively LDA-related GI bleeding (OR= 2.102, 95% CI: 1.008-4.385, p<0.048) and ulcer formation (OR= 2.257, 95% CI: 1.277-3.989, p<0.005) than PPIs. The meta-analysis showed that H2RAs were less effective in the prevention of LDA-related GI bleeding and ulcer formation suggesting the preferable usage of PPIs in case of tolerance.

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Yijun; Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan; Pattnaik, Asit K.

2012-03-01

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating thatmore » the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.« less

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

PubMed

Parrell, Daniel; Zhang, Yang; Olenic, Sandra; Kroos, Lee

2017-10-01

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli , we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli , including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His 6 -MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His 6 -MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL. IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are

Using membrane composition to fine-tune the pKa of an optical liposome pH sensor.

PubMed

Clear, Kasey J; Virga, Katelyn; Gray, Lawrence; Smith, Bradley D

2016-04-14

Liposomes containing membrane-anchored pH-sensitive optical probes are valuable sensors for monitoring pH in various biomedical samples. The dynamic range of the sensor is maximized when the probe p K a is close to the expected sample pH. While some biomedical samples are close to neutral pH there are several circumstances where the pH is 1 or 2 units lower. Thus, there is a need to fine-tune the probe p K a in a predictable way. This investigation examined two lipid-conjugated optical probes, each with appended deep-red cyanine dyes containing indoline nitrogen atoms that are protonated in acid. The presence of anionic phospholipids in the liposomes stabilized the protonated probes and increased the probe p K a values by < 1 unit. The results show that rational modification of the membrane composition is a general non-covalent way to fine-tune the p K a of an optical liposome sensor for optimal pH sensing performance.

Stability and dynamics of membrane-spanning DNA nanopores

NASA Astrophysics Data System (ADS)

Maingi, Vishal; Burns, Jonathan R.; Uusitalo, Jaakko J.; Howorka, Stefan; Marrink, Siewert J.; Sansom, Mark S. P.

2017-03-01

Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine.

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals

PubMed Central

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-01-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. PMID:27083698

Novel applications for glycosylphosphatidylinositol-anchored proteins in pharmaceutical and industrial biotechnology.

PubMed

Müller, Günter

2011-04-01

Glycosylphosphatidylinositol (GPI)-anchored proteins have been regarded as typical cell surface proteins found in most eukaryotic cells from yeast to man. They are embedded in the outer plasma membrane leaflet via a carboxy-terminally linked complex glycolipid GPI structure. The amphiphilic nature of the GPI anchor, its compatibility with the function of the attached protein moiety and the capability of GPI-anchored proteins for spontaneous insertion into and transfer between artificial and cellular membranes initially suggested their potential for biotechnological applications. However, these expectations have been hardly fulfilled so far. Recent developments fuel novel hopes with regard to: (i) Automated online expression, extraction and purification of therapeutic proteins as GPI-anchored proteins based on their preferred accumulation in plasma membrane lipid rafts, (ii) multiplex custom-made protein chips based on GPI-anchored cell wall proteins in yeast, (iii) biomaterials and biosensors with films consisting of sets of distinct GPI-anchored binding-proteins or enzymes for sequential or combinatorial catalysis, and (iv) transport of therapeutic proteins across or into relevant tissue cells, e.g., enterocytes or adipocytes. Latter expectations are based on the demonstrated translocation of GPI-anchored proteins from plasma membrane lipid rafts to cytoplasmic lipid droplets and eventually further into microvesicles which upon release from donor cells transfer their GPI-anchored proteins to acceptor cells. The value of these technologies, which are all based on the interaction of GPI-anchored proteins with membranes and surfaces, for the engineering, production and targeted delivery of biomolecules for a huge variety of therapeutic and biotechnological purposes should become apparent in the near future.

Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae.

PubMed

Kitagaki, Hiroshi; Wu, Hong; Shimoi, Hitoshi; Ito, Kiyoshi

2002-11-01

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.

Deletion of H-Ras decreases renal fibrosis and myofibroblast activation following ureteral obstruction in mice.

PubMed

Grande, M Teresa; Fuentes-Calvo, Isabel; Arévalo, Miguel; Heredia, Fabiana; Santos, Eugenio; Martínez-Salgado, Carlos; Rodríguez-Puyol, Diego; Nieto, M Angela; López-Novoa, José M

2010-03-01

Tubulointerstitial fibrosis is characterized by the presence of myofibroblasts that contribute to extracellular matrix accumulation. These cells may originate from resident fibroblasts, bone-marrow-derived cells, or renal epithelial cells converting to a mesenchymal phenotype. Ras GTPases are activated during renal fibrosis and play crucial roles in regulating both cell proliferation and TGF-beta-induced epithelial-mesenchymal transition. Here we set out to assess the contribution of Ras to experimental renal fibrosis using the well-established model of unilateral ureteral obstruction. Fifteen days after obstruction, both fibroblast proliferation and inducers of epithelial-mesenchymal transition were lower in obstructed kidneys of H-ras knockout mice and in fibroblast cell lines derived from these mice. Interestingly, fibronectin, collagen I accumulation, overall interstitial fibrosis, and the myofibroblast population were also lower in the knockout than in the wild-type mice. As expected, we found lower levels of activated Akt in the kidneys and cultured fibroblasts of the knockout. Whether Ras inhibition will turn out to prevent progression of renal fibrosis will require more direct studies.

Vibrational Stark effect spectroscopy reveals complementary electrostatic fields created by protein-protein binding at the interface of Ras and Ral.

PubMed

Walker, David M; Hayes, Ellen C; Webb, Lauren J

2013-08-07

Electrostatic fields at the interface of the GTPase H-Ras (Ras) docked with the Ras binding domain of the protein Ral guanine nucleoside dissociation stimulator (Ral) were measured with vibrational Stark effect (VSE) spectroscopy. Nine residues on the surface of Ras that participate in the protein-protein interface were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe into the protein-protein interface. The absorption energy of the nitrile was measured both on the surface of Ras in its monomeric state, then after incubation with the Ras binding domain of Ral to form the docked complex. Boltzmann-weighted structural snapshots of the nitrile-labeled Ras protein were generated both in monomeric and docked configurations from molecular dynamics simulations using enhanced sampling of the cyanocysteine side chain's χ2 dihedral angle. These snapshots were used to determine that on average, most of the nitrile probes were aligned along the Ras surface, parallel to the Ras-Ral interface. The average solvent-accessible surface areas (SASA) of the cyanocysteine side chain were found to be <60 Å(2) for all measured residues, and was not significantly different whether the nitrile was on the surface of the Ras monomer or immersed in the docked complex. Changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and found a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface. These data are used to rationalize several puzzling features of the Ras-Ral interface.

Inhibitors of Ras-SOS Interactions.

PubMed

Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

2016-04-19

Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS

DOE PAGES

Huang, William Y. C.; Yan, Qingrong; Lin, Wan-Chen; ...

2016-07-01

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kineticsmore » and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. In conclusion, the generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.« less

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS

PubMed Central

Huang, William Y. C.; Yan, Qingrong; Lin, Wan-Chen; Chung, Jean K.; Hansen, Scott D.; Christensen, Sune M.; Tu, Hsiung-Lin; Kuriyan, John; Groves, Jay T.

2016-01-01

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes. PMID:27370798

Phosphotyrosine-mediated LAT assembly on membranes drives kinetic bifurcation in recruitment dynamics of the Ras activator SOS.

PubMed

Huang, William Y C; Yan, Qingrong; Lin, Wan-Chen; Chung, Jean K; Hansen, Scott D; Christensen, Sune M; Tu, Hsiung-Lin; Kuriyan, John; Groves, Jay T

2016-07-19

The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.

Structure of the glycosyl-phosphatidylinositol membrane anchor of acetylcholinesterase from the electric organ of the electric-fish, Torpedo californica.

PubMed Central

Mehlert, A; Varon, L; Silman, I; Homans, S W; Ferguson, M A

1993-01-01

The structure of the glycan moiety of the glycosyl-phosphatidylinositol (GPI) membrane anchor from Torpedo californica (electric fish) electric-organ acetylcholinesterase was solved using n.m.r., methylation analysis and chemical and enzymic micro-sequencing. Two structures were found to be present: Glc alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol and Glc alpha 1-2Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule. PMID:8257440

Acid-triggered membrane insertion of Pseudomonas exotoxin A involves an original mechanism based on pH-regulated tryptophan exposure.

PubMed

Méré, Jocelyn; Morlon-Guyot, Juliette; Bonhoure, Anne; Chiche, Laurent; Beaumelle, Bruno

2005-06-03

Exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain. This process is a prerequisite for PE translocation to the cytosol where it inactivates protein synthesis. Although hydrophobic helices enable membrane insertion of related bacterial toxins such as diphtheria toxin, the PE translocation domain is devoid of hydrophobic stretches and the structural features triggering acid-induced membrane insertion of PE are not known. Here we have identified a molecular device that enables PE membrane insertion. This process is promoted by exposure of a key tryptophan residue. At neutral pH, this Trp is buried in a hydrophobic pocket closed by the smallest alpha-helix of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. This tryptophan-based membrane insertion system is surprisingly similar to the membrane-anchoring mechanism of human annexin-V and could be used by other proteins as well.

Nickel Ferrite Nanoparticles Anchored onto Silica Nanofibers for Designing Magnetic and Flexible Nanofibrous Membranes.

PubMed

Hong, Feifei; Yan, Chengcheng; Si, Yang; He, Jianxin; Yu, Jianyong; Ding, Bin

2015-09-16

Many applications proposed for magnetic silica nanofibers require their assembly into a cellular membrane structure. The feature to keep structure stable upon large deformation is crucial for a macroscopic porous material which functions reliably. However, it remains a key issue to realize robust flexibility in two-dimensional (2D) magnetic silica nanofibrous networks. Here, we report that the combination of electrospun silica nanofibers with zein dip-coating can lead to the formation of flexible, magnetic, and hierarchical porous silica nanofibrous membranes (SNM). The 290 nm diameter silica nanofibers act as templates for the uniform anchoring of nickel ferrite nanoparticles (size of 50 nm). Benefiting from the homogeneous and stable nanofiber-nanoparticle composite structure, the resulting magnetic SNM can maintain their structure integrity under repeated bending as high as 180° and can facilely recover. The unique hierarchical structure also provides this new class of silica membrane with integrated properties of ultralow density, high porosity, large surface area, good magnetic responsiveness, robust dye adsorption capacity, and effective emulsion separation performance. Significantly, the synthesis of such fascinating membranes may provide new insight for further application of silica in a self-supporting, structurally adaptive, and 2D membrane form.

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling.

PubMed

Channavajhala, Padma L; Wu, Leeying; Cuozzo, John W; Hall, J Perry; Liu, Wei; Lin, Lih-Ling; Zhang, Yuhua

2003-11-21

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.

Pulmonary Delivery of Anti-Tubercular Drugs Using Ligand Anchored pH Sensitive Liposomes for the Treatment of Pulmonary Tuberculosis.

PubMed

Bhardwaj, Ankur; Grobler, Anne; Rath, Goutam; Goyal, Amit Kumar; Jain, Amit Kumar; Mehta, Abhinav

2016-01-01

Mycobacterium tuberculosis (M. TB) remains the prime cause of bacterial mortality and morbidity world-wide. Therefore, effective delivery and targeting of drug to the cellular tropics is essentially required to generate significant results for tuberculosis treatment. The aim of the present study was to develop and characterize ligand anchored pH sensitive liposomes (TPSL) as dry powder inhaler for the targeted delivery of drugs in the target site i.e. lungs. Ligand anchored PSL (TPSL) was prepared by thin film hydration for the combined delivery of Isoniazid (INH) and Ciprofloxacin HCl (CIP HCl) using 4-aminophenyl-α-D mannopyranoside (Man) as surface functionalized ligand and characterized using different parameters. It was observed that size of the ligand anchored liposomes (TPSL) was slightly more than the non-ligand anchored liposomes (PSL). Drug release was studied at different pH for 24 hrs and it was observed that liposomes exhibited slow release at alkaline pH (58-64%) as compared to macrophage pH (81-87%) where it increased dramatically due to the destabilization of pH sensitive liposome (PSL). In vitro cellular uptake study showed that much higher concentration was achieved in the alveolar macrophage using ligand anchored liposomes as compared to its counterpart. In vivo study showed that maximum drug accumulation was achieved in the lung by delivering drug using ligand anchored PSL as compared to conventional PSL. It was concluded that ligand anchored pH sensitive liposome is one of the promising systems for the targeted drug therapy in pulmonary tuberculosis.

A cell-surface-anchored ratiometric i-motif sensor for extracellular pH detection.

PubMed

Ying, Le; Xie, Nuli; Yang, Yanjing; Yang, Xiaohai; Zhou, Qifeng; Yin, Bincheng; Huang, Jin; Wang, Kemin

2016-06-14

A FRET-based sensor is anchored on the cell surface through streptavidin-biotin interactions. Due to the excellent properties of the pH-sensitive i-motif structure, the sensor can detect extracellular pH with high sensitivity and excellent reversibility.

TRIM67 Protein Negatively Regulates Ras Activity through Degradation of 80K-H and Induces Neuritogenesis*

PubMed Central

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-01-01

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis. PMID:22337885

TRIM67 protein negatively regulates Ras activity through degradation of 80K-H and induces neuritogenesis.

PubMed

Yaguchi, Hiroaki; Okumura, Fumihiko; Takahashi, Hidehisa; Kano, Takahiro; Kameda, Hiroyuki; Uchigashima, Motokazu; Tanaka, Shinya; Watanabe, Masahiko; Sasaki, Hidenao; Hatakeyama, Shigetsugu

2012-04-06

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.

Dynamic studies of H-Ras•GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution

PubMed Central

Vo, Uybach; Vajpai, Navratna; Embrey, Kevin J.; Golovanov, Alexander P.

2016-01-01

The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide exchange is completed has not been described to date. Here we show that solution NMR spectra of H-Ras∙GTPγS mixed with a functional fragment of Sos (SosCat) at a 2:1 ratio are consistent with the formation of a rather dynamic assembly. H-Ras∙GTPγS binding was in fast exchange on the NMR timescale and retained a significant degree of molecular tumbling independent of SosCat, while SosCat also tumbled largely independently of H-Ras. Estimates of apparent molecular weight from both NMR data and SEC-MALS revealed that, at most, only one H-Ras∙GTPγS molecule appears stably bound to Sos. The weak transient interaction between Sos and the second H-Ras∙GTPγS may provide a necessary mechanism for complex dissociation upon the completion of the native GDP → GTP exchange reaction, but also explains measurable GTP → GTP exchange activity of Sos routinely observed in in vitro assays that use fluorescently-labelled analogs of GTP. Overall, the data presents the first dynamic snapshot of Ras functional cycle as controlled by Sos. PMID:27412770

Dynamic studies of H-Ras•GTPγS interactions with nucleotide exchange factor Sos reveal a transient ternary complex formation in solution.

PubMed

Vo, Uybach; Vajpai, Navratna; Embrey, Kevin J; Golovanov, Alexander P

2016-07-14

The cycling between GDP- and GTP- bound forms of the Ras protein is partly regulated by the binding of Sos. The structural/dynamic behavior of the complex formed between activated Sos and Ras at the point of the functional cycle where the nucleotide exchange is completed has not been described to date. Here we show that solution NMR spectra of H-Ras∙GTPγS mixed with a functional fragment of Sos (Sos(Cat)) at a 2:1 ratio are consistent with the formation of a rather dynamic assembly. H-Ras∙GTPγS binding was in fast exchange on the NMR timescale and retained a significant degree of molecular tumbling independent of Sos(Cat), while Sos(Cat) also tumbled largely independently of H-Ras. Estimates of apparent molecular weight from both NMR data and SEC-MALS revealed that, at most, only one H-Ras∙GTPγS molecule appears stably bound to Sos. The weak transient interaction between Sos and the second H-Ras∙GTPγS may provide a necessary mechanism for complex dissociation upon the completion of the native GDP → GTP exchange reaction, but also explains measurable GTP → GTP exchange activity of Sos routinely observed in in vitro assays that use fluorescently-labelled analogs of GTP. Overall, the data presents the first dynamic snapshot of Ras functional cycle as controlled by Sos.

RhVI1 is a membrane-anchored vacuolar invertase highly expressed in Rosa hybrida L. petals.

PubMed

Farci, Domenica; Collu, Gabriella; Kirkpatrick, Joanna; Esposito, Francesca; Piano, Dario

2016-05-01

Invertases are a widespread group of enzymes that catalyse the conversion of sucrose into fructose and glucose. Plants invertases and their substrates are essential factors that play an active role in primary metabolism and in cellular differentiation and by these activities they sustain development and growth. Being naturally present in multiple isoforms, invertases are known to be highly differentiated and tissue specific in such a way that every isoform is characteristic of a specific part of the plant. In this work, we report the identification of the invertase RhVI1 that was found to be highly expressed in rose petals. A characterization of this protein revealed that RhVI1 is a glycosylated membrane-anchored protein associated with the cytosolic side of the vacuolar membrane which occurs in vivo in a monomeric form. Purification yields have shown that the levels of expression decreased during the passage of petals from buds to mature and pre-senescent flowers. Moreover, the activity assay indicates RhVI1 to be an acidic vacuolar invertase. The physiological implications of these findings are discussed, suggesting a possible role of this protein during anthesis. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Does Harvey-Ras gene expression lead to oral squamous cell carcinoma? A clinicopathological aspect

PubMed Central

Krishna, Akhilesh; Singh, Shraddha; Singh, Vineeta; Kumar, Vijay; Singh, Uma Shankar; Sankhwar, Satya Narayan

2018-01-01

Background: Harvey-Ras (H-Ras) is an important guanosine triphosphatase protein for the regulation of cellular growth and survival. Altered Ras signaling has been observed in different types of cancer either by gene amplification and/or mutation. The H-Ras oncogene mutations are well reported, but expression of the H-Ras gene is still unknown. Objective: This study aimed to examine both protein and messenger-RNA (mRNA) expressions of H-Ras in oral squamous cell carcinoma (OSCC) and analyzed the association with risk habits and the clinicopathological profile of cases. Methodology: A total of 65 tissue specimens of OSCC (case group) and equal number of normal tissues (control group) were included in this study. H-Ras protein and mRNA expressions were analyzed using immunohistochemical and quantitative real time-polymerase chain reaction techniques, respectively. Results: The H-Ras protein was significantly overexpressed in the oral carcinoma group compared to the normal group (P = 0.03). Most of the OSCC cases showed positive staining with moderate expression, while negative and moderate staining was high in the control group. The majority of H-Ras positive cases were found in individuals with multiple risk habits including tobacco chewing. The risk of H-Ras positivity was 1.46 times higher in smokers than non-smokers. H-Ras positivity increased in cases affected with buccal mucosa site and higher grade of carcinoma. Relative mRNA level of H-Ras was significantly elevated in oral carcinoma as compared with the control group (P ≤ 0.001). Protein and mRNA levels of H-Ras in case group was poorly correlated. Conclusion: H-Ras oncogene expression was markedly higher in oral carcinoma, and it can be a prognostic marker and target for an effective molecular therapy. PMID:29731559

Genetic and pharmacological suppression of oncogenic mutations in RAS genes of yeast and humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schafer, W.R.; Sterne, R.; Thorner, J.

1989-07-28

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast-a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay.more » The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations. 50 refs., 3 figs., 3 tabs.« less

Anchored but not internalized: shape dependent endocytosis of nanodiamond

NASA Astrophysics Data System (ADS)

Zhang, Bokai; Feng, Xi; Yin, Hang; Ge, Zhenpeng; Wang, Yanhuan; Chu, Zhiqin; Raabova, Helena; Vavra, Jan; Cigler, Petr; Liu, Renbao; Wang, Yi; Li, Quan

2017-04-01

Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.

Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

PubMed Central

Benanti, Jennifer A.; Galloway, Denise A.

2004-01-01

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated β-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS. PMID:15024073

A Glycosylphosphatidylinositol Anchor Is Required for Membrane Localization but Dispensable for Cell Wall Association of Chitin Deacetylase 2 in Cryptococcus neoformans

PubMed Central

Gilbert, Nicole M.; Baker, Lorina G.; Specht, Charles A.; Lodge, Jennifer K.

2012-01-01

ABSTRACT Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. PMID:22354955

Clustering of Rac1: Selective Lipid Sorting Drives Signaling.

PubMed

Maxwell, Kelsey N; Zhou, Yong; Hancock, John F

2018-02-01

The ability of lipid-anchored small GTPases to form nanometer-scale lipid domains on the cell plasma membrane (PM) is precipitating exciting new insights into membrane-anchored protein regulation. A recent article by Remorino et al. demonstrates that Rac1, similar to Ras, forms nanoclusters on the PM. The implications of these findings are discussed herein. Copyright © 2017. Published by Elsevier Ltd.

Transcriptional activity of the homopurine-homopyrimidine repeat of the c-Ki-ras promoter is independent of its H-forming potential.

PubMed Central

Raghu, G; Tevosian, S; Anant, S; Subramanian, K N; George, D L; Mirkin, S M

1994-01-01

The mouse c-Ki-ras protooncogene promoter contains an unusual DNA element consisting of a 27 bp-long homopurine-homopyrimidine mirror repeat (H-motif) adjacent to a d(C-G)5 repeat. We have previously shown that in vitro these repeats may adopt H and Z conformations, respectively, causing nuclease and chemical hypersensitivity. Here we have studied the functional role of these DNA stretches using fine deletion analysis of the promoter and a transient transcription assay in vivo. We found that while the H-motif is responsible for approximately half of the promoter activity in both mouse and human cell lines, the Z-forming sequence exhibits little, if any, such activity. Mutational changes introduced within the homopurine-homopyrimidine stretch showed that its sequence integrity, rather than its H-forming potential, is responsible for its effect on transcription. Electrophoretic mobility shift assays revealed that the putative H-motif tightly binds several nuclear proteins, one of which is likely to be transcription factor Sp1, as determined by competition experiments. Southwestern hybridization studies detected two major proteins specifically binding to the H-motif: a 97 kD protein which presumably corresponds to Sp1 and another protein of 60 kD in human and 64 kD in mouse cells. We conclude that the homopurine-homopyrimidine stretch is required for full transcriptional activity of the c-Ki-ras promoter and at least two distinct factors, Sp1 and an unidentified protein, potentially contribute to the positive effect on transcription. Images PMID:8078760

Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking.

PubMed

Mayor, S; Rothberg, K G; Maxfield, F R

1994-06-24

Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

PubMed

Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

1997-04-29

Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

Plasma membrane vesicles decorated with glycolipid-anchored antigens and adjuvants via protein transfer as an antigen delivery platform for inhibition of tumor growth.

PubMed

Patel, Jaina M; Vartabedian, Vincent F; Bozeman, Erica N; Caoyonan, Brianne E; Srivatsan, Sanjay; Pack, Christopher D; Dey, Paulami; D'Souza, Martin J; Yang, Lily; Selvaraj, Periasamy

2016-01-01

Antigen delivered within particulate materials leads to enhanced antigen-specific immunity compared to soluble administration of antigen. However, current delivery approaches for antigen encapsulated in synthetic particulate materials are limited by the complexity of particle production that affects stability and immunogenicity of the antigen. Herein, we describe a protein delivery system that utilizes plasma membrane vesicles (PMVs) derived from biological materials such as cultured cells or isolated tissues and a simple protein transfer technology. We show that these particulate PMVs can be easily modified within 4 h by a protein transfer process to stably incorporate a glycosylphosphatidylinositol (GPI)-anchored form of the breast cancer antigen HER-2 onto the PMV surface. Immunization of mice with GPI-HER-2-modified-PMVs induced strong HER-2-specific antibody responses and protection from tumor challenge in two different breast cancer models. Further incorporation of the immunostimulatory molecules IL-12 and B7-1 onto the PMVs by protein transfer enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. Since protein antigens can be easily converted to GPI-anchored forms, these results demonstrate that isolated plasma membrane vesicles can be modified with desired antigens along with immunostimulatory molecules by protein transfer and used as a vaccine delivery vehicle to elicit potent antigen-specific immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene.

PubMed

Goodrow, T; Reynolds, S; Maronpot, R; Anderson, M

1990-08-01

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a

Quantitative determination of the lateral density and intermolecular correlation between proteins anchored on the membrane surfaces using grazing incidence small-angle X-ray scattering and grazing incidence X-ray fluorescence.

PubMed

Abuillan, Wasim; Vorobiev, Alexei; Hartel, Andreas; Jones, Nicola G; Engstler, Markus; Tanaka, Motomu

2012-11-28

As a physical model of the surface of cells coated with densely packed, non-crystalline proteins coupled to lipid anchors, we functionalized the surface of phospholipid membranes by coupling of neutravidin to biotinylated lipid anchors. After the characterization of fine structures perpendicular to the plane of membrane using specular X-ray reflectivity, the same membrane was characterized by grazing incidence small angle X-ray scattering (GISAXS). Within the framework of distorted wave Born approximation and two-dimensional Percus-Yevick function, we can analyze the form and structure factors of the non-crystalline, membrane-anchored proteins for the first time. As a new experimental technique to quantify the surface density of proteins on the membrane surface, we utilized grazing incidence X-ray fluorescence (GIXF). Here, the mean intermolecular distance between proteins from the sulfur peak intensities can be calculated by applying Abelé's matrix formalism. The characteristic correlation distance between non-crystalline neutravidin obtained by the GISAXS analysis agrees well with the intermolecular distance calculated by GIXF, suggesting a large potential of the combination of GISAXS and GIXF in probing the lateral density and correlation of non-crystalline proteins displayed on the membrane surface.

Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

PubMed Central

Schneider, Falk; Waithe, Dominic; Clausen, Mathias P.; Galiani, Silvia; Koller, Thomas; Ozhan, Gunes; Eggeling, Christian; Sezgin, Erdinc

2017-01-01

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signaling and are suggested to be strongly associated with the actin cytoskeleton. Here we use superresolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live-cell plasma membrane and in actin cytoskeleton–free, cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids is abolished in the GPMVs, whereas transient nanodomain incorporation of ganglioside lipid GM1 is apparent in both the live-cell membrane and GPMVs. For GPI-APs, we detect two molecular pools in living cells; one pool shows high mobility with transient incorporation into nanodomains, and the other pool forms immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules and highlight a powerful experimental approach to decipher specific influences on molecular plasma membrane dynamics. PMID:28404749

Andrographolide Sensitizes Ras-Transformed Cells to Radiation in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hung, Shih-Kai; Department of Radiation Oncology, Buddhist Dalin Tzu Chi General Hospital, Chiayi, Taiwan; Tzu Chi University School of Medicine, Hualian, Taiwan

2010-07-15

Purpose: Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization. Methods and Materials: An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-{kappa}B) and Akt inmore » andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting. Results: Concurrent andrographolide treatment (10 {mu}M, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 {+-} 2.5 days) compared with radiation alone (22 {+-} 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-{kappa}B activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-{kappa}B activity. Conclusion: Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-{kappa}B activity. These observations indicate that andrographolide

K-Ras protein as a drug target.

PubMed

McCormick, Frank

2016-03-01

K-Ras proteins are major drivers of human cancers, playing a direct causal role in about one million cancer cases/year. In cancers driven by mutant K-Ras, the protein is locked in the active, GTP-bound state constitutively, through a defect in the off-switch mechanism. As such, the mutant protein resembles the normal K-Ras protein from a structural perspective, making therapeutic attack extremely challenging. K-Ras is a member of a large family of related proteins, which share very similar GDP/GTP-binding domains, making specific therapies more difficult. Furthermore, Ras proteins lack pockets to which small molecules can bind with high affinity, with a few interesting exceptions. However, new insights into the structure and function of K-Ras proteins reveal opportunities for intervention that were not appreciated many years ago, when efforts were launched to develop K-Ras therapies. Furthermore, K-Ras undergoes post-translational modification and interactions with cellular signaling proteins that present additional therapeutic opportunities, such as specific binding to calmodulin and regulation of non-canonical Wnt signaling.

Tracking Glideosome-associated protein 50 reveals the development and organization of the inner membrane complex of Plasmodium falciparum.

PubMed

Yeoman, Jeffrey A; Hanssen, Eric; Maier, Alexander G; Klonis, Nectarios; Maco, Bohumil; Baum, Jake; Turnbull, Lynne; Whitchurch, Cynthia B; Dixon, Matthew W A; Tilley, Leann

2011-04-01

The most deadly of the human malaria parasites, Plasmodium falciparum, has different stages specialized for invasion of hepatocytes, erythrocytes, and the mosquito gut wall. In each case, host cell invasion is powered by an actin-myosin motor complex that is linked to an inner membrane complex (IMC) via a membrane anchor called the glideosome-associated protein 50 (PfGAP50). We generated P. falciparum transfectants expressing green fluorescent protein (GFP) chimeras of PfGAP50 (PfGAP50-GFP). Using immunoprecipitation and fluorescence photobleaching, we show that C-terminally tagged PfGAP50-GFP can form a complex with endogenous copies of the linker protein PfGAP45 and the myosin A tail domain-interacting protein (MTIP). Full-length PfGAP50-GFP is located in the endoplasmic reticulum in early-stage parasites and then redistributes to apical caps during the formation of daughter merozoites. In the final stage of schizogony, the PfGAP50-GFP profile extends further around the merozoite surface. Three-dimensional (3D) structured illumination microscopy reveals the early-stage IMC as a doubly punctured flat ellipsoid that separates to form claw-shaped apposed structures. A GFP fusion of PfGAP50 lacking the C-terminal membrane anchor is misdirected to the parasitophorous vacuole. Replacement of the acid phosphatase homology domain of PfGAP50 with GFP appears to allow correct trafficking of the chimera but confers a growth disadvantage.

Ras mutations are rare in solitary cold and toxic thyroid nodules.

PubMed

Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

Trend analysis of body weight parameters, mortality, and incidence of spontaneous tumors in Tg.rasH2 mice.

PubMed

Paranjpe, Madhav G; Denton, Melissa D; Vidmar, Tom; Elbekai, Reem H

2014-01-01

Carcinogenicity studies have been performed in conventional 2-year rodent studies for at least 3 decades, whereas the short-term carcinogenicity studies in transgenic mice, such as Tg.rasH2, have only been performed over the last decade. In the 2-year conventional rodent studies, interlinked problems, such as increasing trends in the initial body weights, increased body weight gains, high incidence of spontaneous tumors, and low survival, that complicate the interpretation of findings have been well established. However, these end points have not been evaluated in the short-term carcinogenicity studies involving the Tg.rasH2 mice. In this article, we present retrospective analysis of data obtained from control groups in 26-week carcinogenicity studies conducted in Tg.rasH2 mice since 2004. Our analysis showed statistically significant decreasing trends in initial body weights of both sexes. Although the terminal body weights did not show any significant trends, there was a statistically significant increasing trend toward body weight gains, more so in males than in females, which correlated with increasing trends in the food consumption. There were no statistically significant alterations in mortality trends. In addition, the incidence of all common spontaneous tumors remained fairly constant with no statistically significant differences in trends. © The Author(s) 2014.

Levels of H-ras codon 61 CAA to AAA mutation: response to 4-ABP-treatment and Pms2-deficiency.

PubMed

Parsons, Barbara L; Delongchamp, Robert R; Beland, Frederick A; Heflich, Robert H

2006-01-01

DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA-->AAA mutation was examined in Pms2 knockout mice (Pms2-/-, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 micromol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA-->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65x10(-5) to 2.91x10(-5) and from 3.40x10(-5) to 4.70x10(-5), respectively. Pooling data from 4-ABP-treated and control mice, the approximately 2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P=0.0207) and consistent with what has been reported previously in terms of induction of G:C-->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P=0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26x10(-5) and 1.30x10(-5), respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA-->AAA mutational response induced by 4-ABP.

New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

PubMed Central

Marty, Naomi J.; Teresinski, Howard J.; Hwang, Yeen Ting; Clendening, Eric A.; Gidda, Satinder K.; Sliwinska, Elwira; Zhang, Daiyuan; Miernyk, Ján A.; Brito, Glauber C.; Andrews, David W.; Dyer, John M.; Mullen, Robert T.

2014-01-01

Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well-understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins. PMID:25237314

Evidence for carboxyl-terminal processing and glycolipid-anchoring of human carcinoembryonic antigen.

PubMed

Takami, N; Misumi, Y; Kuroki, M; Matsuoka, Y; Ikehara, Y

1988-09-05

We have investigated the post-translational modification of carcinoembryonic antigen (CEA) for membrane-anchoring in QGP-1 cells derived from a human pancreatic carcinoma. Pulse-chase experiments with [3H]leucine demonstrated that CEA was initially synthesized as a precursor form with Mr 150,000 having N-linked high-mannose-type oligosaccharides, which was then converted to a mature form with Mr 200,000 containing the complex type sugar chains. The mature protein thus labeled was found to be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that CEA is a phosphatidylinositol-linked membrane protein. This was confirmed by metabolic incorporation into CEA of 3H-labeled compounds such as ethanolamine, myo-inositol, palmitic acid, and stearic acid. The 3H-labeled fatty acids incorporated were specifically removed from the protein by nitrous acid deamination as well as by phosphatidylinositol-specific phospholipase C treatment. Since the available cDNA sequence predicts that CEA contains a single methionine residue only in its carboxyl-terminal hydrophobic domain, processing of the carboxyl terminus was examined by pulse-chase experiments with [35S]methionine. It was found that CEA with Mr 150,000 was initially labeled with [35S]methionine but its radioactivity was immediately lost with chase. Taken together, these results suggest that CEA is anchored to the membrane by simultaneously occurring proteolysis of the carboxyl terminus and replacement by the glycophospholipid immediately after the synthesis.

Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging.

PubMed

Yasuda, Ryohei; Harvey, Christopher D; Zhong, Haining; Sobczyk, Aleksander; van Aelst, Linda; Svoboda, Karel

2006-02-01

To understand the biochemical signals regulated by neural activity, it is necessary to measure protein-protein interactions and enzymatic activity in neuronal microcompartments such as axons, dendrites and their spines. We combined two-photon excitation laser scanning with fluorescence lifetime imaging to measure fluorescence resonance energy transfer at high resolutions in brain slices. We also developed sensitive fluorescent protein-based sensors for the activation of the small GTPase protein Ras with slow (FRas) and fast (FRas-F) kinetics. Using FRas-F, we found in CA1 hippocampal neurons that trains of back-propagating action potentials rapidly and reversibly activated Ras in dendrites and spines. The relationship between firing rate and Ras activation was highly nonlinear (Hill coefficient approximately 5). This steep dependence was caused by a highly cooperative interaction between calcium ions (Ca(2+)) and Ras activators. The Ras pathway therefore functions as a supersensitive threshold detector for neural activity and Ca(2+) concentration.

The role of d-allo-isoleucine in the deposition of the anti-Leishmania peptide bombinin H4 as revealed by 31P solid-state NMR, VCD spectroscopy, and MD simulation.

PubMed

Mijiddorj, Batsaikhan; Kaneda, Shiho; Sato, Hisako; Kitahashi, Yuki; Javkhlantugs, Namsrai; Naito, Akira; Ueda, Kazuyoshi; Kawamura, Izuru

2018-07-01

Bombinin H4 is an antimicrobial peptide that was isolated from the toad Bombina variegata. Bombinin H family peptides are active against gram-positive, gram-negative bacteria, and fungi as well as the parasite Leishmania. Among them, bombinin H4 (H4), which contains d-allo-isoleucine (d-allo-Ile) as the second residue in its sequence, is the most active, and its l-isomer is bombinin H2 (H2). H4 has a significantly lower LC50 than H2 against Leishmania. However, the atomic-level mechanism of the membrane interaction and higher activity of H4 has not been clarified. In this work, we investigated the behavior of the conformations and interactions of H2 and H4 with the Leishmania membrane using 31 P solid-state nuclear magnetic resonance (NMR), vibrational circular dichroism (VCD) spectroscopy, and molecular dynamics (MD) simulations. The generation of isotropic 31 P NMR signals depending on the peptide concentration indicated the abilities of H2 and H4 to exert antimicrobial activity via membrane disruption. The VCD experiment and density functional theory calculation confirmed the different stability and conformations of the N-termini of H2 and H4. MD simulations revealed that the N-terminus of H4 is more stable than that of H2 in the membrane, in line with the VCD experiment data. VCD and MD analyses demonstrated that the first l-Ile and second d-allo-Ile of H4 tend to take a cis conformation. These residues function as an anchor and facilitate the easy winding of the helical conformation of H4 in the membrane. It may assist to quickly reach to the threshold concentration of H4 on the Leishmania membrane. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca. Copyright © 2018 Elsevier B.V. All rights reserved.

Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

PubMed Central

Mayor, S; Maxfield, F R

1995-01-01

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined. Images PMID:7579703

ACB-PCR measurement of spontaneous and furan-induced H-ras codon 61 CAA to CTA and CAA to AAA mutation in B6C3F1 mouse liver.

PubMed

Banda, Malathi; Recio, Leslie; Parsons, Barbara L

2013-10-01

Furan is a rodent liver carcinogen, but the mode of action for furan hepatocarcinogenicity is unclear. H-ras codon 61 mutations have been detected in spontaneous liver tumors of B6C3F1 mice, and the fraction of liver tumors carrying H-ras codon 61 CAA to AAA mutation increased in furan-treated mice. Allele-specific competitive blocker PCR (ACB-PCR) has been used previously to quantify early, carcinogen-induced increases in tumor-associated mutations. The present pilot study investigated whether furan drives clonal expansion of pre-existing H-ras mutant cells in B6C3F1 mouse liver. H-ras codon 61 CAA to CTA and CAA to AAA mutations were measured in DNA isolated from liver tissue of female mice treated with 0, 1, 2, 4, or 8 mg furan/kg body weight, five days per week for three weeks, using five mice per treatment group. Spontaneous levels of mutation were low, with two of five control mice having an H-ras codon 61 CTA or AAA mutant fraction (MF) greater than 10(-5) . Several furan-treated mice had H-ras codon 61 AAA or CTA MFs greater than those measured in control mice and lower bound estimates of induced MF were calculated. However, no statistically-significant differences were observed between treatment groups. Therefore, while sustained exposure to furan is carcinogenic, at the early stage of carcinogenesis examined in this study (three weeks), there was not a significant expansion of H-ras mutant cells. Copyright © 2013 Wiley Periodicals, Inc.

Twenty-six-week oral carcinogenicity study of 3-monochloropropane-1,2-diol in CB6F1-rasH2 transgenic mice.

PubMed

Lee, Byoung-Seok; Park, Sang-Jin; Kim, Yong-Bum; Han, Ji-Seok; Jeong, Eun Ju; Son, Hwa-Young; Moon, Kyoung-Sik

2017-01-01

The carcinogenic potential of 3-monochloro-1,2-propanediol (3-MCPD) was evaluated in a short-term carcinogenicity testing study using CB6F1 rasH2-Tg (rasH2-Tg) mice. 3-MCPD is found in many foods and food ingredients as a result of storage or processing and is regarded as a carcinogen since it is known to induce Leydig cell and kidney tumors in rats. Male and female rasH2-Tg mice were administered 3-MCPD once daily by oral gavage at doses of 0, 10, 20, and 40 mg/kg body weight (bw) per day for 26 weeks. As a positive control, N-methyl-N-nitrosourea (MNU) was administered as a single intraperitoneal injection (75 mg/kg). In 3-MCPD-treated mice, there was no increase in the incidence of neoplastic lesions compared to the incidence in vehicle control mice. However, 3-MCPD treatment resulted in an increased incidence of tubular basophilia in the kidneys and germ cell degeneration in the testes, with degenerative germ cell debris in the epididymides of males at 20 and 40 mg/kg bw per day. In 3-MCPD-treated females, vacuolation of the brain and spinal cord was observed at 40 mg/kg bw per day; however, only one incidence of vacuolation was observed in males. Forestomach and cutaneous papilloma and/or carcinoma and lymphoma were observed in most rasH2 mice receiving MNU treatment. We concluded that 3-MCPD did not show carcinogenic potential in the present study using rasH2-Tg mice. The findings of this study suggest that the carcinogenic potential of 3-MCPD is species specific.

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity

PubMed Central

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane. PMID:19531344

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

PubMed

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H-Ras transformed human mammary epithelial cells.

PubMed

Hahn, Young-Il; Kim, Su-Jung; Choi, Bu-Young; Cho, Kyung-Cho; Bandu, Raju; Kim, Kwang Pyo; Kim, Do-Hee; Kim, Wonki; Park, Joon Sung; Han, Byung Woo; Lee, Jeewoo; Na, Hye-Kyung; Cha, Young-Nam; Surh, Young-Joon

2018-04-23

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin is an anti-inflammatory natural compound isolated from the turmeric (Curcuma longa L., Zingiberaceae) that has been extensively used in a traditional medicine over the centuries. In the present study, we have found that curcumin inhibits STAT3 signaling that is persistently overactivated in H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of this transcription factor. We identified the cysteine residue 259 of STAT3 as a putative site for curcumin binding. Site-directed mutation of this cysteine residue abolished curcumin-induced inactivation of STAT3 and apoptosis in H-Ras MCF10A cells. The α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its binding to STAT3 in H-Ras MCF10A cells. Tetrahydrocurcumin that lacks such electrophilic moiety failed to interact with STAT3 and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate aberrant activation of STAT3 through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis.

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

NASA Astrophysics Data System (ADS)

Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

2017-01-01

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

Ras mutation cooperates with β-catenin activation to drive bladder tumourigenesis.

PubMed

Ahmad, I; Patel, R; Liu, Y; Singh, L B; Taketo, M M; Wu, X-R; Leung, H Y; Sansom, O J

2011-03-03

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras(Q61L) or K-Ras(G12D)) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.

Differential insertion of GPI-anchored GFPs into lipid rafts of live cells.

PubMed

Legler, Daniel F; Doucey, Marie-Agnès; Schneider, Pascal; Chapatte, Laurence; Bender, Florent C; Bron, Claude

2005-01-01

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

RAS diseases in children

PubMed Central

Niemeyer, Charlotte M.

2014-01-01

RAS genes encode a family of 21 kDa proteins that are an essential hub for a number of survival, proliferation, differentiation and senescence pathways. Signaling of the RAS-GTPases through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade is essential in development. A group of genetic syndromes, named “RASopathies”, had been identified which are caused by heterozygosity for germline mutations in genes that encode protein components of the RAS/MAPK pathway. Several of these clinically overlapping disorders, including Noonan syndrome, Noonan-like CBL syndrome, Costello syndrome, cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type I, and Legius syndrome, predispose to cancer and abnormal myelopoiesis in infancy. This review focuses on juvenile myelomonocytic leukemia (JMML), a malignancy of early childhood characterized by initiating germline and/or somatic mutations in five genes of the RAS/MAPK pathway: PTPN11, CBL, NF-1, KRAS and NRAS. Natural courses of these five subtypes differ, although hematopoietic stem cell transplantation remains the only curative therapy option for most children with JMML. With whole-exome sequencing studies revealing few secondary lesions it will be crucial to better understand the RAS/MAPK signaling network with its crosstalks and feed-back loops to carefully design early clinical trials with novel pharmacological agents in this still puzzling leukemia. PMID:25420281

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

PubMed

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd

Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

PubMed Central

Bhagatji, Pinkesh; Leventis, Rania; Comeau, Jonathan; Refaei, Mohammad

2009-01-01

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein. PMID:19687251

NMR 1H,13C, 15N backbone and 13C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP.

PubMed

Sharma, Alok K; Lee, Seung-Joo; Rigby, Alan C; Townson, Sharon A

2018-05-02

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here 1 H N, 15 N, and 13 C resonance assignments for the 19.3 kDa (aa 1-169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS G12C-GDP ), using heteronuclear, multidimensional NMR spectroscopy. Backbone 1 H- 15 N correlations have been assigned for all non-proline residues, except for the first methionine residue.

The RasGAP Gene, RASAL2, is a Tumor and Metastasis Suppressor

PubMed Central

McLaughlin, Sara Koenig; Olsen, Sarah Naomi; Dake, Benjamin; De Raedt, Thomas; Lim, Elgene; Bronson, Roderick Terry; Beroukhim, Rameen; Polyak, Kornelia; Brown, Myles; Kuperwasser, Charlotte; Cichowski, Karen

2013-01-01

SUMMARY RAS genes are commonly mutated in cancer; however, RAS mutations are rare in breast cancer, despite the fact that Ras and ERK are frequently hyperactivated. Here we report that the RasGAP gene, RASAL2, functions as a tumor and metastasis suppressor. RASAL2 is mutated or suppressed in human breast cancer and RASAL2 ablation promotes tumor growth, progression, and metastasis in mouse models. In human breast cancer RASAL2-loss is associated with metastatic disease, low RASAL2 levels correlate with recurrence of luminal B tumors, and RASAL2 ablation promotes metastasis of luminal mouse tumors. Additional data reveal a broader role for RASAL2 inactivation in other tumor-types. These studies highlight the expanding role of RasGAPs and reveal an alternative mechanism of activating Ras in cancer. PMID:24029233

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

PubMed Central

Lee, Young Kwang; Low-Nam, Shalini T.; Chung, Jean K.; Hansen, Scott D.; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T.

2017-01-01

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification. PMID:28452363

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

PubMed

Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

2017-04-28

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

PubMed

Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

2018-01-01

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

Establishing a laboratory animal model from a transgenic animal: RasH2 mice as a model for carcinogenicity studies in regulatory science.

PubMed

Urano, K; Tamaoki, N; Nomura, T

2012-01-01

Transgenic animal models have been used in small numbers in gene function studies in vivo for a period of time, but more recently, the use of a single transgenic animal model has been approved as a second species, 6-month alternative (to the routine 2-year, 2-animal model) used in short-term carcinogenicity studies for generating regulatory application data of new drugs. This article addresses many of the issues associated with the creation and use of one of these transgenic models, the rasH2 mouse, for regulatory science. The discussion includes strategies for mass producing mice with the same stable phenotype, including constructing the transgene, choosing a founder mouse, and controlling both the transgene and background genes; strategies for developing the model for regulatory science, including measurements of carcinogen susceptibility, stability of a large-scale production system, and monitoring for uniform carcinogenicity responses; and finally, efficient use of the transgenic animal model on study. Approximately 20% of mouse carcinogenicity studies for new drug applications in the United States currently use transgenic models, typically the rasH2 mouse. The rasH2 mouse could contribute to animal welfare by reducing the numbers of animals used as well as reducing the cost of carcinogenicity studies. A better understanding of the advantages and disadvantages of the transgenic rasH2 mouse will result in greater and more efficient use of this animal model in the future.

[Farnesyl transferase inhibitors (anti-Ras). A new class of anticancer agents].

PubMed

Levy, R

Ras genes are frequently activated in human tumours. The role of their product, the P21 proteins, in the transduction of the mitogenic signal makes them attractive targets for an anti-neoplastic therapy. The p21 ras proteins are linked to the plasma membrane and transformed into an active form for signal transmission. Their effect is to mediate the effects of growth factors. Two drug families, the Benzodiazepine peptidomimetics and the CAAX tetrapeptides which inhibit the farnesylation of P21-Ras proteins abolish the transforming properties of mutated P21. These promising drugs could rapidly have clinical applications. They have been shown to be highly active at precise concentrations on ras-transformed cells but at the same concentrations are not toxic for untransformed cells. They do not effect other similar enzyme systems within the cell, underlining their selective capacity. Theoretically anti-ras therapy could only suspend cell transformation although it might be possible that if given long enough, a lethal threshold could be reached.

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype.

PubMed

Lee, H; Li, D; Prior, T; Casto, B C; Weghorst, C M; Shuler, C F; Milo, G E

1997-10-01

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells' ability to exhibit a malignant potential in nude mice.

Allosteric modulation of Ras positions Q61 for a direct role in catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buhrman, Greg; Holzapfel, Genevieve; Fetics, Susan

2010-11-03

Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the {gamma}-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild-type Rasmore » with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the {gamma}-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca{sup 2+} and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.« less

The bridging conformations of double-end anchored polymer-surfactants destabilize a hydrogel of lipid membranes

NASA Astrophysics Data System (ADS)

Slack, N. L.; Davidson, P.; Chibbaro, M. A.; Jeppesen, C.; Eiselt, P.; Warriner, H. E.; Schmidt, H.-W.; Pincus, P.; Safinya, C. R.

2001-10-01

Double-end-anchored poly-ethylene-glycol-surfactants (DEA-PEG-surfactants) induce the gelation of lyotropic lamellar Lα phases stabilized by undulation forces. The physical hydrogel (Lα,g) derives its viscoelasticity from the proliferation of defects at a mesoscopic level. The DEA-PEG-surfactants assume both looping and bridging conformations. The existence of novel bridging conformations is indicated by the coexistence of two lamellar phases and the limited swelling of the Lα and Lα,g phases. Modeling of the polymer decorated membranes demonstrates the existence of bridging and yields a rapidly decreasing density of bridging conformations with increasing interlayer spacing.

[3H]aniracetam binds to specific recognition sites in brain membranes.

PubMed

Fallarino, F; Genazzani, A A; Silla, S; L'Episcopo, M R; Camici, O; Corazzi, L; Nicoletti, F; Fioretti, M C

1995-08-01

[3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

PubMed Central

Sasaki, Atsuo T.; Chun, Cheryl; Takeda, Kosuke; Firtel, Richard A.

2004-01-01

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal. PMID:15534002

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

NASA Astrophysics Data System (ADS)

Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

2016-02-01

Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP-GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

GPI-anchored protein organization and dynamics at the cell surface

PubMed Central

Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

2016-01-01

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. PMID:26394904

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis.

PubMed

East, Alexandra; Mechaly, Ariel E; Huysmans, Gerard H M; Bernarde, Cédric; Tello-Manigne, Diana; Nadeau, Nathalie; Pugsley, Anthony P; Buschiazzo, Alejandro; Alzari, Pedro M; Bond, Peter J; Francetic, Olivera

2016-01-05

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching.

PubMed

Harrison, Rane A; Lu, Jia; Carrasco, Martin; Hunter, John; Manandhar, Anuj; Gondi, Sudershan; Westover, Kenneth D; Engen, John R

2016-11-20

Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogen-deuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Deconstruction of the Ras switching cycle through saturation mutagenesis

PubMed Central

Bandaru, Pradeep; Shah, Neel H; Bhattacharyya, Moitrayee; Barton, John P; Kondo, Yasushi; Cofsky, Joshua C; Gee, Christine L; Chakraborty, Arup K; Kortemme, Tanja; Ranganathan, Rama; Kuriyan, John

2017-01-01

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins. DOI: http://dx.doi.org/10.7554/eLife.27810.001 PMID:28686159

Ras history

PubMed Central

2010-01-01

Although the roots of Ras sprouted from the rich history of retrovirus research, it was the discovery of mutationally activated RAS genes in human cancer in 1982 that stimulated an intensive research effort to understand Ras protein structure, biochemistry and biology. While the ultimate goal has been developing anti-Ras drugs for cancer treatment, discoveries from Ras have laid the foundation for three broad areas of science. First, they focused studies on the origins of cancer to the molecular level, with the subsequent discovery of genes mutated in cancer that now number in the thousands. Second, elucidation of the biochemical mechanisms by which Ras facilitates signal transduction established many of our fundamental concepts of how a normal cell orchestrates responses to extracellular cues. Third, Ras proteins are also founding members of a large superfamily of small GTPases that regulate all key cellular processes and established the versatile role of small GTP-binding proteins in biology. We highlight some of the key findings of the last 28 years. PMID:21686117

Loss of protein phosphatase 6 in mouse keratinocytes enhances K-rasG12D -driven tumor promotion.

PubMed

Kurosawa, Koreyuki; Inoue, Yui; Kakugawa, Yoichiro; Yamashita, Yoji; Kanazawa, Kosuke; Kishimoto, Kazuhiro; Nomura, Miyuki; Momoi, Yuki; Sato, Ikuro; Chiba, Natsuko; Suzuki, Mai; Ogoh, Honami; Yamada, Hidekazu; Miura, Koh; Watanabe, Toshio; Tanuma, Nobuhiro; Tachi, Masahiro; Shima, Hiroshi

2018-05-14

Here, we address the function of protein phosphatase 6 (PP6) loss on K-ras-initiated tumorigenesis in keratinocytes. To do so, we developed tamoxifen-inducible double mutant (K-ras G12D -expressing and Ppp6c-deficient) mice in which K-ras G12D expression is driven by the cytokeratin 14 (K14) promoter. Doubly-mutant mice showed early onset tumor formation in lip, nipples, external genitalia, anus and palms, and had to be sacrificed by three weeks after induction by tamoxifen, while comparably-treated K-ras G12D -expressing mice did not. HE-staining of lip tumors before euthanasia revealed that all were papillomas, some containing focal squamous cell carcinoma. Immunohistochemical analysis of lip of doubly-mutant versus K-ras G12D mice revealed that cell proliferation and cell size increased approximately two-fold relative to K-ras G12D -expressing mutants, and epidermal thickness of lip tissue greatly increased relative to that seen in K-ras G12D only mice. Moreover, AKT phosphorylation increased in K-ras G12D -expressing/Ppp6c-deficient cells, as did phosphorylation of the downstream effectors 4EBP1, S6, and GSK3, suggesting that protein synthesis and survival signals are enhanced in lip tissues of doubly-mutant mice. Finally, increased numbers of K14-positive cells were present in the suprabasal layer of doubly-mutant mice, indicating abnormal keratinocyte differentiation, and γH2AX-positive cells accumulated, indicating perturbed DNA repair. Taken together, Ppp6c deficiency enhances K-ras G12D -dependent tumor promotion. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Alpha lipoic acid selectively inhibits proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells.

PubMed

Yamasaki, Masao; Iwase, Masahiro; Kawano, Kazuo; Sakakibara, Yoichi; Suiko, Masahito; Nishiyama, Kazuo

2012-05-01

Here, we focused on the effects of racemic α-lipoic acid on proliferation and adhesion properties of 3Y1 rat fibroblasts and the v-H-ras-transformed derivative, HR-3Y1-2 cells. Racemic α-lipoic acid inhibited proliferation of HR-3Y1-2 but not 3Y1 cells at 0.3 and 1.0 mM. R-(+)-α-lipoic acid also inhibited proliferation of HR-3Y1-2 cells equivalent to that of racemic α-lipoic acid. In addition, racemic α-lipoic acid decreased intracellular reactive oxygen species levels in HR-3Y1 cells but not 3Y1 cells. Next, we evaluated the effects of racemic α-lipoic acid on cell adhesion to fibronectin. The results indicated that racemic α-lipoic acid decreased adhesive ability of HR-3Y1-2 cells to fibronectin-coated plates. As blocking antibody experiment revealed that β1-integrin plays a key role in cell adhesion in this experimental system, the effects of racemic α-lipoic acid on the expression of β1-integrin were examined. The results indicated that racemic α-lipoic acid selectively downregulated the expression of cell surface β1-integrin expression in HR-3Y1-2 cells. Intriguingly, exogenous hydrogen peroxide upregulated cell surface β1-integrin expression in 3Y1 cells. Taken together, these data suggest that reduction of intracellular reactive oxygen species levels by α-lipoic acid could be an effective means of ameliorating abnormal growth and adhesive properties in v-H-ras transformed cells.

15 CFR Appendix C to Subpart H of... - No-Anchoring Seagrass Protection Zones in Tomales Bay

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false No-Anchoring Seagrass Protection Zones in Tomales Bay C Appendix C to Subpart H of Part 922 Commerce and Foreign Trade Regulations Relating... of the Farallones National Marine Sanctuary Pt. 922, Subpt. H, App. C Appendix C to Subpart H of Part...

Anchorage Behaviors of Frictional Tieback Anchors in Silty Sand

NASA Astrophysics Data System (ADS)

Hsu, Shih-Tsung; Hsiao, Wen-Ta; Chen, Ke-Ting; Hu, Wen-Chi; Wu, Ssu-Yi

2017-06-01

Soil anchors are extensively used in geotechnical applications, most commonly serve as tieback walls in deep excavations. To investigate the anchorage mechanisms of this tieback anchor, a constitutive model that considers both strain hardening and softening and volume dilatancy entitled SHASOVOD model, and FLAC3D software are used to perform 3-D numerical analyses. The results from field anchor tests are compared with those calculated by numerical analyses to enhance the applicability of the numerical method. After the calibration, this research carried out the parameter studies by numerical analyses. The numerical results reveal that whether the yield of soil around an anchor develops to ground surface and/or touches the diaphragm wall depending on the overburden depth H and the embedded depth Z of an anchor, this study suggests the minimum overburden and embedded depths to avoid the yield of soils develop to ground surface and/or touch the diaphragm wall. When the embedded depth, overburden depth or fixed length of an anchor increases, the anchorage capacity also increases. Increasing fixed length should be the optimum method to increase the anchorage capacity for fixed length less than 20m. However, when the fixed length of an anchor exceeds 30 m, the increasing rate of anchorage capacity per fixed length decreases, and progressive yield occurs obviously between the fixed length and surrounding soil.

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

PubMed

Coyle, Scott M; Lim, Wendell A

2016-01-14

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.

Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK

PubMed Central

Wang, Qin; Downey, Gregory P.; McCulloch, Christopher A.

2011-01-01

In connective tissue cells, IL-1-induced ERK activation leading to matrix metalloproteinase (MMP)-3 expression is dependent on cooperative interactions between focal adhesions and the endoplasmic reticulum (ER). As Ras can be activated on the ER, we investigated the role of Ras in IL-1 signaling and focal adhesion formation. We found that constitutively active H-Ras, K-Ras or N-Ras enhanced focal adhesion maturation and β1-integrin activation. IL-1 promoted the accumulation of Ras isoforms in ER and focal adhesion fractions, as shown in cells cotransfected with GFP-tagged Ras isoforms and YFP-ER protein and by analysis of subcellular fractions enriched for ER or focal adhesion proteins. Dominant-negative H-Ras or K-Ras reduced accumulation of H-Ras and K-Ras in focal adhesions induced by IL-1 and also blocked ERK activation and focal adhesion maturation. Ras-GRF was enriched constitutively in focal adhesion fractions and was required for Ras recruitment to focal adhesions. We conclude that Ras activation and IL-1 signaling are interactive processes that regulate the maturation of focal adhesions, which, in turn, is required for ERK activation.—Wang, Q., Downey, G. P., McCulloch, C. A. Focal adhesions and Ras are functionally and spatially integrated to mediate IL-1 activation of ERK. PMID:21719512

RAS signaling and anti-RAS therapy: lessons learned from genetically engineered mouse models, human cancer cells, and patient-related studies.

PubMed

Fang, Bingliang

2016-01-01

Activating mutations of oncogenic RAS genes are frequently detected in human cancers. The studies in genetically engineered mouse models (GEMMs) reveal that Kras-activating mutations predispose mice to early onset tumors in the lung, pancreas, and gastrointestinal tract. Nevertheless, most of these tumors do not have metastatic phenotypes. Metastasis occurs when tumors acquire additional genetic changes in other cancer driver genes. Studies on clinical specimens also demonstrated that KRAS mutations are present in premalignant tissues and that most of KRAS mutant human cancers have co-mutations in other cancer driver genes, including TP53, STK11, CDKN2A, and KMT2C in lung cancer; APC, TP53, and PIK3CA in colon cancer; and TP53, CDKN2A, SMAD4, and MED12 in pancreatic cancer. Extensive efforts have been devoted to develop therapeutic agents that target enzymes involved in RAS posttranslational modifications, that inhibit downstream effectors of RAS signaling pathways, and that kill RAS mutant cancer cells through synthetic lethality. Recent clinical studies have revealed that sorafenib, a pan-RAF and VEGFR inhibitor, has impressive benefits for KRAS mutant lung cancer patients. Combination therapy of MEK inhibitors with either docetaxel, AKT inhibitors, or PI3K inhibitors also led to improved clinical responses in some KRAS mutant cancer patients. This review discusses knowledge gained from GEMMs, human cancer cells, and patient-related studies on RAS-mediated tumorigenesis and anti-RAS therapy. Emerging evidence demonstrates that RAS mutant cancers are heterogeneous because of the presence of different mutant alleles and/or co-mutations in other cancer driver genes. Effective subclassifications of RAS mutant cancers may be necessary to improve patients' outcomes through personalized precision medicine. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology

Genetic analysis of Ras genes in epidermal development and tumorigenesis

PubMed Central

Drosten, Matthias; Lechuga, Carmen G; Barbacid, Mariano

2013-01-01

Proliferation and differentiation of epidermal keratinocytes are tightly controlled to ensure proper development and homeostasis of the epidermis. The Ras family of small GTPases has emerged as a central node in the coordination of cell proliferation in the epidermis. Recent genetic evidence from mouse models has revealed that the intensity of Ras signaling modulates the proliferative capacity of epidermal keratinocytes. Interfering with Ras signaling either by combined elimination of the 3 Ras genes from the basal layer of the epidermis or by overexpression of dominant-negative Ras isoforms caused epidermal thinning due to hypoproliferation of keratinocytes. In contrast, overexpression of oncogenic Ras mutants in different epidermal cell layers led to hyperproliferative phenotypes including the development of papillomas and squamous cell carcinomas. Here, we discuss the value of loss- and gain-of-function studies in mouse models to assess the role of Ras signaling in the control of epidermal proliferation. PMID:24150175

Membrane fouling in a submerged membrane bioreactor: effect of pH and its implications.

PubMed

Zhang, Ye; Zhang, Meijia; Wang, Fangyuan; Hong, Huachang; Wang, Aijun; Wang, Juan; Weng, Xuexiang; Lin, Hongjun

2014-01-01

The effect of pH on membrane fouling in a submerged membrane bioreactor (MBR) was investigated in this study. It was found that, pH increase slightly increased the resistance of virgin membrane and fouled membrane. Pore clogging resistance was quite low, which was not apparently affected by the pH variation. Lower pH resulted in higher adherence of sludge flocs on membrane surface. Thermodynamic analysis showed that a repulsive energy barrier existed in the process of the foulants approaching to membrane surface. This energy barrier would decrease with pH decreased, suggesting the existence of a critical pH below which the repulsive energy barrier would disappear, which would facilitate attachment of the foulants. The resistance of the formed cake layer would significantly increase with the feed pH. This result could be explained by the osmotic pressure mechanism. The obtained findings also provided important implications for membrane fouling mitigation in MBRs. Copyright © 2013 Elsevier Ltd. All rights reserved.

End-anchored polymers in good solvents from the single chain limit to high anchoring densities.

PubMed

Whitmore, Mark D; Grest, Gary S; Douglas, Jack F; Kent, Michael S; Suo, Tongchuan

2016-11-07

An increasing number of applications utilize grafted polymer layers to alter the interfacial properties of solid substrates, motivating refinement in our theoretical understanding of such layers. To assess existing theoretical models of them, we have investigated end-anchored polymer layers over a wide range of grafting densities, σ, ranging from a single chain to high anchoring density limits, chain lengths ranging over two orders of magnitude, for very good and marginally good solvent conditions. We compare Monte Carlo and molecular dynamics simulations, numerical self-consistent field calculations, and experimental measurements of the average layer thickness, h, with renormalization group theory, the Alexander-de Gennes mushroom theory, and the classical brush theory. Our simulations clearly indicate that appreciable inter-chain interactions exist at all simulated areal anchoring densities so that there is no mushroom regime in which the layer thickness is independent of σ. Moreover, we find that there is no high coverage regime in which h follows the predicted scaling, h ∼ Nσ 1/3 , for classical polymer brushes either. Given that no completely adequate analytic theory seems to exist that spans wide ranges of N and σ, we applied scaling arguments for h as a function of a suitably defined reduced anchoring density, defined in terms of the solution radius of gyration of the polymer chains and N. We find that such a scaling approach enables a smooth, unified description of h in very good solvents over the full range of anchoring density and chain lengths, although this type of data reduction does not apply to marginal solvent quality conditions.

Endocytosis of glycosylphosphatidylinositol-anchored proteins

PubMed Central

2009-01-01

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) represent an interesting amalgamation of the three basic kinds of cellular macromolecules viz. proteins, carbohydrates and lipids. An unusually hybrid moiety, the GPI-anchor is expressed in a diverse range of organisms from parasites to mammalian cells and serves to anchor a large number of functionally diverse proteins and has been the center of attention in scientific debate for some time now. Membrane organization of GPI-APs into laterally-organized cholesterol-sphingolipid ordered membrane domains or "rafts" and endocytosis of GPI-APs has been intensely debated. Inclusion into or exclusion from these membrane domains seems to be the critical factor in determining the endocytic mechanisms and intracellular destinations of GPI-APs. The intracellular signaling as well as endocytic trafficking of GPI-APs is critically dependent upon the cell surface organization of GPI-APs, and the associations with these lipid rafts play a vital role during these processes. The mechanism of endocytosis for GPI-APs may differ from other cellular endocytic pathways, such as those mediated by clathrin-coated pits (caveolae), and is necessary for unique biological functions. Numerous intracellular factors are involved in and regulate the endocytosis of GPI-APs, and these may be variably dependent on cell-type. The central focus of this article is to describe the significance of the endocytosis of GPI-APs on a multitude of biological processes, ranging from nutrient-uptake to more complex immune responses. Ultimately, a thorough elucidation of GPI-AP mediated signaling pathways and their regulatory elements will enhance our understanding of essential biological processes and benefit as components of disease intervention strategies. PMID:19832981

Phloretin induces apoptosis in H-Ras MCF10A human breast tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase signaling.

PubMed

Kim, Mi-Sung; Kwon, Jung Yeon; Kang, Nam Joo; Lee, Ki Won; Lee, Hyong Joo

2009-08-01

Mutations in Ras play a critical role in the development of human cancers, including breast cancer. We investigated the possible antiproliferative effects of the naturally occurring dihydrochalcone phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on H-Ras-transformed MCF10A human breast epithelial (H-Ras MCF10A) cells. Phloretin suppressed H-Ras MCF10A cell proliferation in a dose-dependent manner and induced nuclear condensation in the cells, indicating that phloretin-induced cell death occurs mainly via the induction of apoptosis. Prominent upregulation of p53 and Bax and cleavage of poly (ADP)-ribose polymerase were also detected in the phloretin-treated cells. Finally, phloretin markedly increased caspase-3 activity as well as JNK and p38 mitogen-activated protein kinase signaling. Our findings suggest that the phloretin-induced apoptosis of breast tumor cells contributes to the chemopreventive potential of phloretin against breast cancer.

GPI-anchored protein organization and dynamics at the cell surface.

PubMed

Saha, Suvrajit; Anilkumar, Anupama Ambika; Mayor, Satyajit

2016-02-01

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or Förster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Ground anchors and anchored systems

DOT National Transportation Integrated Search

1999-06-01

This document presents state-of-the-practice information on the design and installation of cement-grouted ground anchors and anchored systems for highway applications. The anchored systems discussed include flexible anchored walls, slopes supported u...

Thermally induced anchoring of fullerene in copolymers with Si-bridging atom: Spectroscopic evidences

NASA Astrophysics Data System (ADS)

Marchiori, Cleber F. N.; Garcia-Basabe, Yunier; de A. Ribeiro, Fabio; Koehler, Marlus; Roman, Lucimara S.; Rocco, Maria Luiza M.

2017-01-01

We use X-ray photoelectron spectroscopy (XPS), Near-edge X-ray absorption fine structure (NEXAFS), resonant Auger spectroscopy (RAS), Attenuation Total Reflection Infrared (ATR-IR) and Atomic Force Microscopy (AFM) to study the blend between the copolymer poly[2,7-(9,9-bis(2-ethylhexyl)-dibenzosilole)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole] (PSiF-DBT) and the fullerene derivative PC71BM submitted to different annealing temperatures. Those measurements indicate that there is an incidental anchoring of a fullerene derivative to the Si-bridging atoms of a copolymer induced by thermal annealing of the film. Insights about the physical properties of one possible PSiF-DBT/PC71BM anchored structure are obtained using Density Functional Theory calculations. Since the performance of organic photovoltaic based on polymer-fullerene blends depends on the chemical structure of the blend components, the anchoring effect might affect the photovoltaic properties of those devices.

Live-cell confocal microscopy and quantitative 4D image analysis of anchor cell invasion through the basement membrane in C. elegans

PubMed Central

Kelley, Laura C.; Wang, Zheng; Hagedorn, Elliott J.; Wang, Lin; Shen, Wanqing; Lei, Shijun; Johnson, Sam A.; Sherwood, David R.

2018-01-01

Cell invasion through basement membrane (BM) barriers is crucial during development, leukocyte trafficking, and for the spread of cancer. Despite its importance in normal and diseased states, the mechanisms that direct invasion are poorly understood, in large part because of the inability to visualize dynamic cell-basement membrane interactions in vivo. This protocol describes multi-channel time-lapse confocal imaging of anchor cell invasion in live C. elegans. Methods presented include outline slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min), and quantitative analysis (variable timing). Images acquired enable direct measurement of invasive dynamics including invadopodia formation, cell membrane protrusions, and BM removal. This protocol can be combined with genetic analysis, molecular activity probes, and optogenetic approaches to uncover molecular mechanisms underlying cell invasion. These methods can also be readily adapted for real-time analysis of cell migration, basement membrane turnover, and cell membrane dynamics by any worm laboratory. PMID:28880279

Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation

PubMed Central

Šolman, Maja; Ligabue, Alessio; Blaževitš, Olga; Jaiswal, Alok; Zhou, Yong; Liang, Hong; Lectez, Benoit; Kopra, Kari; Guzmán, Camilo; Härmä, Harri; Hancock, John F; Aittokallio, Tero; Abankwa, Daniel

2015-01-01

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer. DOI: http://dx.doi.org/10.7554/eLife.08905.001 PMID:26274561

Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling

PubMed Central

Kinoshita, Taroh; Fujita, Morihisa

2016-01-01

Glycosylphosphatidylinositols (GPIs) act as membrane anchors of many eukaryotic cell surface proteins. GPIs in various organisms have a common backbone consisting of ethanolamine phosphate (EtNP), three mannoses (Mans), one non-N-acetylated glucosamine, and inositol phospholipid, whose structure is EtNP-6Manα-2Manα-6Manα-4GlNα-6myoinositol-P-lipid. The lipid part is either phosphatidylinositol of diacyl or 1-alkyl-2-acyl form, or inositol phosphoceramide. GPIs are attached to proteins via an amide bond between the C-terminal carboxyl group and an amino group of EtNP. Fatty chains of inositol phospholipids are inserted into the outer leaflet of the plasma membrane. More than 150 different human proteins are GPI anchored, whose functions include enzymes, adhesion molecules, receptors, protease inhibitors, transcytotic transporters, and complement regulators. GPI modification imparts proteins with unique characteristics, such as association with membrane microdomains or rafts, transient homodimerization, release from the membrane by cleavage in the GPI moiety, and apical sorting in polarized cells. GPI anchoring is essential for mammalian embryogenesis, development, neurogenesis, fertilization, and immune system. Mutations in genes involved in remodeling of the GPI lipid moiety cause human diseases characterized by neurological abnormalities. Yeast Saccharomyces cerevisiae has >60 GPI-anchored proteins (GPI-APs). GPI is essential for growth of yeast. In this review, we discuss biosynthesis of GPI-APs in mammalian cells and yeast with emphasis on the lipid moiety. PMID:26563290

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity*

PubMed Central

Peters, Diane E.; Szabo, Roman; Friis, Stine; Shylo, Natalia A.; Uzzun Sales, Katiuchia; Holmbeck, Kenn; Bugge, Thomas H.

2014-01-01

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8Cat−/Cat− mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8−/− and Prss8Cat−/Cat− mice born within the same litter. Furthermore, Prss8Cat−/Cat− mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease. PMID:24706745

The RAS Initiative

Cancer.gov

NCI established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to ultimately create effective, new therapies for RAS-related cancers.

GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface.

PubMed

Hiscox, Stephen; Hallett, Maurice B; Morgan, B Paul; van den Berg, Carmen W

2002-05-03

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent. Copyright 2002 Elsevier Science (USA).

Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS.

PubMed

Schneider, Falk; Waithe, Dominic; Clausen, Mathias P; Galiani, Silvia; Koller, Thomas; Ozhan, Gunes; Eggeling, Christian; Sezgin, Erdinc

2017-06-01

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signaling and are suggested to be strongly associated with the actin cytoskeleton. Here we use superresolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live-cell plasma membrane and in actin cytoskeleton-free, cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids is abolished in the GPMVs, whereas transient nanodomain incorporation of ganglioside lipid GM1 is apparent in both the live-cell membrane and GPMVs. For GPI-APs, we detect two molecular pools in living cells; one pool shows high mobility with transient incorporation into nanodomains, and the other pool forms immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules and highlight a powerful experimental approach to decipher specific influences on molecular plasma membrane dynamics. © 2017 Schneider et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Essential role for SUN5 in anchoring sperm head to the tail

PubMed Central

Wang, Lina; Ouyang, Ying-Chun; Dong, Ming-Zhe; Liu, Chao; Zhao, Haichao; Cui, Xiuhong; Ma, Dongyuan; Zhang, Zhiguo; Yang, Xiaoyu; Guo, Yueshuai; Liu, Feng; Yuan, Li

2017-01-01

SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5−/− mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility. PMID:28945193

Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application

PubMed Central

2018-01-01

Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell’s functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine–poly(ethylene glycol)–dibenzocyclooctyne (DSPE–PEG2000–DBCO) and cholesterol–PEG–dibenzocyclooctyne (CHOL–PEG2000–DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids. PMID:29503972

Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application.

PubMed

Vabbilisetty, Pratima; Boron, Mallorie; Nie, Huan; Ozhegov, Evgeny; Sun, Xue-Long

2018-02-28

Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell's functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine-poly(ethylene glycol)-dibenzocyclooctyne (DSPE-PEG 2000 -DBCO) and cholesterol-PEG-dibenzocyclooctyne (CHOL-PEG 2000 -DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids.

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.

PubMed

Lechuga, Carmen G; Simón-Carrasco, Lucía; Jacob, Harrys K C; Drosten, Matthias

2017-01-01

Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Ras lox (H-Ras -/- , N-Ras -/- , K-Ras lox/lox , RERT ert/ert ) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

PubMed

Chakravarti, D; Mailander, P C; Li, K M; Higginbotham, S; Zhang, H L; Gross, M L; Meza, J L; Cavalieri, E L; Rogan, E G

2001-11-29

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution

PubMed Central

Coyle, Scott M; Lim, Wendell A

2016-01-01

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras’s ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease. DOI: http://dx.doi.org/10.7554/eLife.12435.001 PMID:26765565

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-β-Glucanase Heterologously Expressed in Pichia pastoris1

PubMed Central

Mølhøj, Michael; Ulvskov, Peter; Dal Degan, Florence

2001-01-01

The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-β-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-β-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine1-lysine70), a hydrophobic transmembrane domain (isoleucine71-valine93), and a periplasmic catalytic core (lysine94-proline621). Here, we report the functional analysis of Δ1-90Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Δ1-90Cel16 in a pure form. The molecular mass of Δ1-90Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Δ1-90Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Δ1-90Cel16 had a pH optimum of 6.0. The activity of Δ1-90Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Δ1-90Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1→3),(1→4)-β-d-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Δ1-90Cel16-hydrolyzed carboxymethylcellulose showed that Δ1-90Cel16 is a true endo-acting glucanase. PMID:11598241

Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha.

PubMed

Okumura, Masaki; Inoue, Shin-ichiro; Takahashi, Koji; Ishizaki, Kimitsune; Kohchi, Takayuki; Kinoshita, Toshinori

2012-06-01

The plasma membrane H(+)-ATPase generates an electrochemical gradient of H(+) across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H(+)-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H(+)-ATPase in vascular plants. However, there is very little information on the plasma membrane H(+)-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H(+)-ATPase (pT H(+)-ATPase) and non-penultimate threonine-containing H(+)-ATPase (non-pT H(+)-ATPase) as in the green algae and that pT H(+)-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H(+)-ATPase genes, designated MpHA (for M. polymorpha H(+)-ATPase). Four isoforms are the pT H(+)-ATPase; the remaining isoforms are non-pT H(+)-ATPase. An apparent 95-kD protein was recognized by anti-H(+)-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H(+)-ATPase. Furthermore, we found that the pT H(+)-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H(+)-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H(+)-ATPase.

Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers*

PubMed Central

Öztürk, Yavuz; Lee, Dong-Woo; Mandaci, Sevnur; Osyczka, Artur; Prince, Roger C.; Daldal, Fevzi

2008-01-01

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt cy. In species like R. capsulatus, cyt cy functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt cy (S-cy), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt cy. This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-cy regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-cy variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-cy was about 340 mV, similar to that of cyts c2 or cy. Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-cy was distinct from those seen with the cyt c2 or cyt cy. The kinetics exhibited a pronounced slow phase, suggesting that cyt S-cy interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-cy revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt cy, whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers. PMID:18343817

Structure of the Dominant Negative S17N Mutant of Ras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nassar, N.; Singh, K; Garcia-Diaz, M

2010-01-01

The use of the dominant negative mutant of Ras has been crucial in elucidating the cellular signaling of Ras in response to the activation of various membrane-bound receptors. Although several point mutants of Ras exhibit a dominant negative effect, the asparagine to serine mutation at position 17 (S17N) remains the most popular and the most effective at inhibiting the activation of endogenous Ras. It is now widely accepted that the dominant negative effect is due to the ability of the mutant to sequester upstream activators and its inability to activate downstream effectors. Here, we present the crystal structure of RasS17Nmore » in the GDP-bound form. In the three molecules that populate the asymmetric unit, the Mg{sup 2+} ion that normally coordinates the {beta}-phosphate is absent because of steric hindrance from the Asn17 side chain. Instead, a Ca{sup 2+} ion is coordinating the {alpha}-phosphate. Also absent from one molecule is electron density for Phe28, a conserved residue that normally stabilizes the nucleotide's guanine base. Except for Phe28, the nucleotide makes conserved interactions with Ras. Combined, the inability of Phe28 to stabilize the guanine base and the absence of a Mg{sup 2+} ion to neutralize the negative charges on the phosphates explain the weaker affinity of GDP for Ras. Our data suggest that the absence of the Mg{sup 2+} should also dramatically affect GTP binding to Ras and the proper positioning of Thr35 necessary for the activation of switch 1 and the binding to downstream effectors, a prerequisite for the triggering of signaling pathways.« less

Centriolar satellite– and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly

PubMed Central

Hori, Akiko; Peddie, Christopher J.; Collinson, Lucy M.; Toda, Takashi

2015-01-01

Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction–induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring. PMID:25833712

Inhibition of SHP2-mediated dephosphorylation of Ras suppresses oncogenesis

PubMed Central

Bunda, Severa; Burrell, Kelly; Heir, Pardeep; Zeng, Lifan; Alamsahebpour, Amir; Kano, Yoshihito; Raught, Brian; Zhang, Zhong-Yin; Zadeh, Gelareh; Ohh, Michael

2015-01-01

Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously ‘undruggable' oncogenic or hyperactive Ras. PMID:26617336

Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

PubMed Central

Valentine, Cathleen D.; Haggie, Peter M.

2011-01-01

The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

Confinement of β(1)- and β(2)-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae.

PubMed

Valentine, Cathleen D; Haggie, Peter M

2011-08-15

The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β(1)- and β(2)AR, are structurally similar but mediate distinct signaling responses. Scaffold protein-mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β(1)- and β(2)AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)-domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β(2)AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β(2)AR confinement. For both β(1)- and β(2)AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β(1)- or β(2)AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a

A Raf-competitive K-Ras binder can fail to functionally antagonize signaling.

PubMed

Kauke, Monique J; Tisdale, Alison W; Kelly, Ryan L; Braun, Christian J; Hemann, Michael T; Wittrup, K Dane

2018-05-02

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically-encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacological efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Copyright ©2018, American Association for Cancer Research.

Nitrative and oxidative DNA damage caused by K-ras mutation in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohnishi, Shiho; Saito, Hiromitsu; Suzuki, Noboru

2011-09-23

Highlights: {yields} Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. {yields} The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. {yields} Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role. -- Abstract: Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-{kappa}B, IKK, MAPK, MEK,more » and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.« less

Structural and functional specificity of Influenza virus haemagglutinin and paramyxovirus fusion protein anchoring peptides.

PubMed

Kordyukova, Larisa

2017-01-02

Two enveloped virus families, Orthomyxoviridae and Paramyxoviridae, comprise a large number of dangerous pathogens that enter the host cell via fusion of their envelope with a target cell membrane at acidic or neutral pH. The Class I prototypic glycoproteins responsible for this reaction are the Influenza virus haemagglutinin (HA) protein or paramyxovirus fusion (F) protein. X-ray crystallography and cryoelectron microscopy data are available for the HA and F ectodomains in pre- and post-fusion conformations, revealing similar spiky architectures, albeit with clear differences in the details. In contrast, their anchoring segments, which possess a linker region, transmembrane domain and cytoplasmic tail that is specifically modified with long fatty acids (highly conserved in HA and occasional in F), are not resolved. Recent experimental, bioinformatics and molecular modelling data showing the primary, secondary and quaternary organization of the HA and F anchoring segments are summarized in this review. Some amino acid patterns that are crucial for protein thermal stability or lipid membrane order/cholesterol binding are addressed, and new achievements in vaccine practice using HA transmembrane domain chimaeras are discussed. The oligomerization properties of the transmembrane domains are considered in the context of Group-1 and Group-2 antigenic HA subtypes and various genera/subfamilies of paramyxoviruses. A specific focus is the late steps of fusion that are reportedly facilitated by (1) β-sheet-promoting β-branched amino acids (valine and isoleucine) that are enriched in the transmembrane domain of paramyxovirus F or (2) a post-translational modification of C-terminal cysteines with palmitate/stearate (differential S-acylation) that is highly conserved in Influenza virus HA. Copyright © 2016 Elsevier B.V. All rights reserved.

Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric

2014-08-22

Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellularmore » vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.« less

RotundRacGAP Functions with Ras during Spermatogenesis and Retinal Differentiation in Drosophila melanogaster

PubMed Central

Bergeret, Evelyne; Pignot-Paintrand, Isabelle; Guichard, Annabel; Raymond, Karine; Fauvarque, Marie-Odile; Cazemajor, Michel; Griffin-Shea, Ruth

2001-01-01

Our analysis of rotund (rn) null mutations in Drosophila melanogaster revealed that deletion of the rn locus affects both spermatid and retinal differentiation. In the male reproductive system, the absence of RnRacGAP induced small testes, empty seminal vesicles, short testicular cysts, reduced amounts of interspermatid membrane, the absence of individualization complexes, and incomplete mitochondrial condensation. Flagellar growth continued within the short rn null cysts to produce large bulbous terminations of intertwined mature flagella. Organization of the retina was also severely perturbed as evidenced by grossly misshapen ommatidia containing reduced numbers of photoreceptor and pigment cells. These morphological phenotypes were rescued by genomic rnRacGAP transgenes, demonstrating that RnRacGAP function is critical to spermatid and retinal differentiation. The testicular phenotypes were suppressed by heterozygous hypomorphic mutations in the Dras1 and drk genes, indicating cross talk between RacGAP-regulated signaling and that of the Ras pathway. The observed genetic interactions are consistent with a model in which Rac signaling is activated by Ras and negatively regulated by RnRacGAP during spermatid differentiation. RnRacGAP and Ras cross talk also operated during retinal differentiation; however, while the heterozygous hypomorphic drk mutation continued to act as a suppressor of the rn null mutation, the heterozygous hypomorphic Dras1 mutation induced novel retinal phenotypes. PMID:11509670

Carcinogenicity assessment of the Hedgehog pathway inhibitor, vismodegib in Tg.rasH2 mice and Sprague-Dawley rats.

PubMed

Li, Jinze; Morinello, Eric; Larsen, Thomas; Frost, Denzil; Caro, Ivor; Gould, Stephen; Wong, Lisa; Hendricks, Angela; Dybdal, Noel; Dambach, Donna; Schutten, Melissa

2018-02-01

Vismodegib (also known as GDC-0449) is a novel small molecule inhibitor of the Hedgehog (Hh) signaling pathway currently approved for the treatment of metastatic or locally advanced basal cell carcinoma (BCC) in humans. Its tumorigenic potential was assessed in dedicated carcinogenicity studies in rasH2 transgenic (Tg.rasH2) mice and Sprague Dawley (SD) rats. Tumorigenicity potential of vismodegib was identified in rats only and was limited to benign hair follicle tumors, including pilomatricomas and keratoacanthomas at exposures of ≥0.1-fold and ≥0.6-fold, respectively, of the steady-state exposure (AUC 0-24h ) of the recommended human dose. No malignant tumors were identified in either species. Overall, the totality of pharmacology and nonclinical safety data (lack of genotoxicity, in vitro secondary pharmacological binding, and immunoregulatory effects, and limited effects on the endocrine system) suggests that the development of the benign hair follicle tumors may be related to pharmacologically-mediated disruption of hair follicle morphogenesis, although the exact mechanism of tumorigenesis is unclear. Hair follicle tumors have not been reported in vismodegib-treated patients. The relevance of this finding in rats to patients is uncertain. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative Roles of Overexpressed and Mutated H- and K-ras in Mammary Carcinogenesis.

DTIC Science & Technology

1996-08-01

transgene of these tumors. 14. SUBJECT TERMS 15. NUMBER OF PAGES Breast Cancer , mammary carcinogenesis, oncogenes, ras genes, 44 replication defective...27 Appendix 5 29 Appendix 6 31 Appendix 7 33 Appendix 8 35 Appendix 9 37 Appendix 10 39 Introduction Breast cancer development involves multiple poorly...understood steps (25). Currently, several genes that may participate in breast cancer development are under investigation. The ras family of genes

Membrane protein properties revealed through data-rich electrostatics calculations

PubMed Central

Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

2015-01-01

SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

The Bcr Kinase Downregulates Ras Signaling by Phosphorylating AF-6 and Binding to Its PDZ Domain

PubMed Central

Radziwill, G.; Erdmann, R. A.; Margelisch, U.; Moelling, K.

2003-01-01

The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state. PMID:12808105

[Expressions of Ras and Sos1 in epithelial ovarian cancer tissues and their clinical significance].

PubMed

Xiao, Zheng-Hua; Linghu, Hua; Liu, Qian-Fen

2016-11-20

To detect the expressions of Ras and Sos1 proteins in human epithelial ovarian cancer (EOC) tissues and explore their correlation with the clinicopathological features of the patients. The expressions of Ras and Sos1 proteins were detected immunohistochemically in 62 EOC tissues, 5 borderline ovarian cancer tissues, 15 benign epithelial ovarian neoplasm tissues, and 18 normal ovarian tissues. The EOC tissues showed significantly higher expression levels of both Ras and Sos1 than the other tissues tested (P<0.05). In EOC tissues, Ras and Sos1 proteins were expressed mostly on the cell membrane and in the cytoplasm. The expression level of Ras was correlated with pathological types of the tumor (P<0.05) and was the highest in serous cystadenomcarcinoma; Sos1 expression did not show significant correlation with the clinicopathological indexes of the patients. High expressions of both Ras and Sos1 proteins were associated with shorter progression-free survival of the patients, but this association was not statistically significant. Ras and Sos1 protein may participate in in the occurrence and development of EOC. The tissue-specific variation of Ras expression can lend support to a specific diagnosis of ovarian serous adenocarcinoma. The association of Ras and Sos1 protein expression with the tumor-free survival time of the patients awaits further investigation with a larger sample size.

Cell Activation Mediated by Glycosylphosphatidylinositol-Anchored or Transmembrane Forms of CD14†

PubMed Central

Pugin, J.; Kravchenko, V. V.; Lee, J.-D.; Kline, L.; Ulevitch, R. J.; Tobias, P. S.

1998-01-01

CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-κB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking. PMID:9488411

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.

PubMed

Jia, Hao-Ran; Wang, Hong-Yin; Yu, Zhi-Wu; Chen, Zhan; Wu, Fu-Gen

2016-03-16

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.

Density control of dodecamanganese clusters anchored on silicon(100).

PubMed

Condorelli, Guglielmo G; Motta, Alessandro; Favazza, Maria; Nativo, Paola; Fragalà, Ignazio L; Gatteschi, Dante

2006-04-24

A synthetic strategy to control the density of Mn12 clusters anchored on silicon(100) was investigated. Diluted monolayers suitable for Mn12 anchoring were prepared by Si-grafting mixtures of the methyl 10-undecylenoate precursor ligand with 1-decene spectator spacers. Different ratios of these mixtures were tested. The grafted surfaces were hydrolyzed to reveal the carboxylic groups available for the subsequent exchange with the [Mn12O12(OAc)16(H2O)4]4 H2O2 AcOH cluster. Modified surfaces were analyzed by attenuated total reflection (ATR)-FTIR spectroscopy, X-ray photoemission spectroscopy (XPS), and AFM imaging. Results of XPS and ATR-FTIR spectroscopy show that the surface mole ratio between grafted ester and decene is higher than in the source solution. The surface density of the Mn12 cluster is, in turn, strictly proportional to the ester mole fraction. Well-resolved and isolated clusters were observed by AFM, using a diluted ester/decene 1:1 solution.

RAS Insight

Cancer.gov

David Heimbrook, now CEO of the Frederick National Laboratory for Cancer Research, played a major role in a large pharma as it tried to develop an anti-RAS drug. Lessons from that failure inform the RAS Initiative today.

Structural and mechanical heterogeneity of the erythrocyte membrane reveals hallmarks of membrane stability.

PubMed

Picas, Laura; Rico, Félix; Deforet, Maxime; Scheuring, Simon

2013-02-26

The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosphorylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme.

PubMed

Moriau, L; Michelet, B; Bogaerts, P; Lambert, L; Michel, A; Oufattole, M; Boutry, M

1999-07-01

The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility.

PubMed

Kabbani, Abir Maarouf; Woodward, Xinxin; Kelly, Christopher V

2017-10-18

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility

PubMed Central

Kabbani, Abir Maarouf; Woodward, Xinxin

2017-01-01

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity. PMID:29057801

ACB-PCR measurement of H-ras codon 61 CAA→CTA mutation provides an early indication of aristolochic acid I carcinogenic effect in tumor target tissues.

PubMed

Wang, Yiying; Arlt, Volker M; Roufosse, Candice A; McKim, Karen L; Myers, Meagan B; Phillips, David H; Parsons, Barbara L

2012-08-01

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen, which induces forestomach and kidney tumors in mice and is associated with development of urothelial cancer in humans. This study sought to gain mechanistic insight into AAI-induced carcinogenesis through analysis of a tumor-relevant endpoint. Female Hupki mice were treated daily with 5 mg AAI/kg body weight by gavage for 3, 12, or 21 days. Histopathology and DNA adduct analysis confirmed kidney and forestomach as target tissues for AAI-induced toxicity. H-ras codon 61 CAA→CTA mutations were measured in mouse kidney and forestomach, as well as liver and glandular stomach (nontarget organs) by allele-specific competitive blocker-PCR (ACB-PCR), because A→T transversion is the predominant mutation induced by AA and this particular mutation was found previously in AA-induced rodent forestomach tumors. Treatment-related differences were observed, with the H-ras mutant fraction (MF) of mouse kidney and forestomach exposed to 5 mg AAI/kg body weight for 21 days significantly higher than that of vehicle-treated controls (Fisher's exact test, P < 0.05). Statistically significant correlations between dA-AAI adduct levels (measured previously in the same animals) and induced H-ras MFs were evident in forestomach of mice treated for 21 days (linear regression, P < 0.05). The significant increase in H-ras MF in kidney and forestomach, along with the correlation between DNA adducts, histopathology, and oncogene mutation, provide definitive evidence that AA induces tumors through a directly mutagenic mode of action. Thus, measurement of tumor-associated mutations is a useful tool for elucidating the mechanisms underlying the tissue specificity of carcinogenesis. Copyright © 2012 Wiley Periodicals, Inc.

Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

PubMed

Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

2015-08-04

The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

PubMed

Branco, Patrícia; Albergaria, Helena; Arneborg, Nils; Prista, Catarina

2018-05-01

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling

DOE PAGES

Erickson, Keesha E.; Rukhlenko, Oleksii S.; Posner, Richard G.; ...

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisitionmore » of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers.« less

New insights into RAS biology reinvigorate interest in mathematical modeling of RAS signaling.

PubMed

Erickson, Keesha E; Rukhlenko, Oleksii S; Posner, Richard G; Hlavacek, William S; Kholodenko, Boris N

2018-03-05

RAS is the most frequently mutated gene across human cancers, but developing inhibitors of mutant RAS has proven to be challenging. Given the difficulties of targeting RAS directly, drugs that impact the other components of pathways where mutant RAS operates may potentially be effective. However, the system-level features, including different localizations of RAS isoforms, competition between downstream effectors, and interlocking feedback and feed-forward loops, must be understood to fully grasp the opportunities and limitations of inhibiting specific targets. Mathematical modeling can help us discern the system-level impacts of these features in normal and cancer cells. New technologies enable the acquisition of experimental data that will facilitate development of realistic models of oncogenic RAS behavior. In light of the wealth of empirical data accumulated over decades of study and the advancement of experimental methods for gathering new data, modelers now have the opportunity to advance progress toward realization of targeted treatment for mutant RAS-driven cancers. Copyright © 2018 Elsevier Ltd. All rights reserved.

"Twin copper source" growth of metal-organic framework membrane: Cu(3)(BTC)(2) with high permeability and selectivity for recycling H(2).

PubMed

Guo, Hailing; Zhu, Guangshan; Hewitt, Ian J; Qiu, Shilun

2009-02-11

In this communication, the copper net supported Cu(3)(BTC)(2) membranes have been successfully synthesized by means of a "twin copper source" technique. Separation studies on gaseous mixtures (H(2)/CO(2), H(2)/CH(4), and H(2)/N(2)) using the membrane revealed that the membrane possesses high permeability and selectivity for H(2) over CO(2), N(2), and CH(4). Compared with the conventional zeolite membranes, the copper net supported Cu(3)(BTC)(2) membrane exhibited high permeation flux in gas separation. Such highly efficient copper net supported Cu(3)(BTC)(2) membranes could be used to separate, recycle, and reuse H(2) exhausted from steam reforming natural gas.

Lack of HXK2 induces localization of active Ras in mitochondria and triggers apoptosis in the yeast Saccharomyces cerevisiae.

PubMed

Amigoni, Loredana; Martegani, Enzo; Colombo, Sonia

2013-01-01

We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

A cross-sectional study examining the expression of splice variants K-RAS4A and K-RAS4B in advanced non-small-cell lung cancer patients.

PubMed

Aran, Veronica; Masson Domingues, Pedro; Carvalho de Macedo, Fabiane; Moreira de Sousa, Carlos Augusto; Caldas Montella, Tatiane; de Souza Accioly, Maria Theresa; Ferreira, Carlos Gil

2018-02-01

Mammalian cells differently express 4 RAS isoforms: H-RAS, N-RAS, K-RAS4A and K-RAS4B, which are important in promoting oncogenic processes when mutated. In lung cancer, the K-RAS isoform is the most frequently altered RAS protein, being also a difficult therapeutic target. Interestingly, there are two K-RAS splice variants (K-RAS4A and K-RAS4B) and little is known about the role of K-RAS4A. Most studies targeting K-RAS, or analysing it as a prognostic factor, have not taken into account the two isoforms. Consequently, the in-depth investigation of them is needed. The present study analysed 98 specimens from advanced non-small cell lung cancer (NSCLC) adenocarcinoma patients originated from Brazil. The alterations present in K-RAS at the DNA level (Sanger sequencing) as well as the expression of the splicing isoforms at the RNA (qRT-PCR) and protein levels (immunohistochemistry analysis), were evaluated. Possible associations between clinicopathological features and the molecular findings were also investigated. Our results showed that in the non-smoking population, the cancer incidence was higher among women. In contrast, in smokers and former smokers, the incidence was higher among men. Regarding sequencing results, 10.5% of valid samples presented mutations in exon 2, being all wild-type for exon 3, and the most frequently occurring base change was the transversion G → T. Our qRT-PCR and immunohistochemical analysis showed that both, K-RAS4A and K-RAS4B, were differently expressed in NSCLC tumour samples. For example, tumour specimens showed higher K-RAS4A mRNA expression in relation to commercial normal lung control than did K-RAS4B. In addition, K-RAS4B protein expression was frequently stronger than K-RAS4A in the patients analysed. Our results highlight the differential expression of K-RAS4A and K-RAS4B in advanced adenocarcinoma NSCLC patients and underline the need to further clarify the enigma behind their biological significance in various cancer

pH-Dependence of Binding Constants and Desorption Rates of Phosphonate- and Hydroxamate-Anchored [Ru(bpy)3]2+ on TiO2 and WO3.

PubMed

Esarey, Samuel L; Bartlett, Bart M

2018-04-17

The binding constants and rate constants for desorption of the modified molecular dye [Ru(bpy) 3 ] 2+ anchored by either phosphonate or hydroxamate on the bipyridine ligand to anatase TiO 2 and WO 3 have been measured. In aqueous media at pH 1-10, repulsive electrostatic interactions between the negatively charged anchor and the negatively charged surface govern phosphonate desorption under neutral and basic conditions for TiO 2 anatase due to the high acidity of phosphonic acid (p K a,4 = 5.1). In contrast, the lower acidity of hydroxamate (p K a,1 = 6.5, p K a,2 = 9.1) leads to little change in adsorption/desorption properties as a function of pH from 1 to 7. The binding constant for hydroxamate is 10 3 in water, independent of pH in this range. These results are true for WO 3 as well, but are not reported at pH > 4 due to its Arrhenius acidity. Kinetics for desorption as a function of pH are reported, with a proposed mechanism for phosphonate desorption at high pH being the electrostatic repulsion of negative charges between the surface and the anionic anchor. Further, the hydroxamic acid anchor itself is likely the site of quasi-reversible redox activity in [Ru(bpy) 2 (2,2'-bpy-4,4'-(C(O)N(OH)) 2 )] 2+ , which does not lead to any measurable deterioration of the complex within 2 h of dark cyclic voltammogram scans in aqueous media. These results posit phosphonate as the preferred anchoring group under acidic conditions and hydroxamate for neutral/basic conditions.

Structural and Biophysical Analysis of BST-2/Tetherin Ectodomains Reveals an Evolutionary Conserved Design to Inhibit Virus Release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swiecki, M.; Allaire, M.; Scheaffer, S.

2011-01-28

BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-{angstrom} crystal structure of the complete mouse BST-2 ectodomain reveals an {approx}145-{angstrom} parallel dimer in an extended {alpha}-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are requiredmore » for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 {angstrom} for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.« less

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

PubMed

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-05-22

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

RAS - Target Identification - Informatics

Cancer.gov

The RAS Informatics lab group develops tools to track and analyze “big data” from the RAS Initiative, as well as analyzes data from external projects. By integrating internal and external data, this group helps improve understanding of RAS-driven cancers.

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

PubMed

Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

Phosphatidylcholine Membrane Fusion Is pH-Dependent.

PubMed

Akimov, Sergey A; Polynkin, Michael A; Jiménez-Munguía, Irene; Pavlov, Konstantin V; Batishchev, Oleg V

2018-05-03

Membrane fusion mediates multiple vital processes in cell life. Specialized proteins mediate the fusion process, and a substantial part of their energy is used for topological rearrangement of the membrane lipid matrix. Therefore, the elastic parameters of lipid bilayers are of crucial importance for fusion processes and for determination of the energy barriers that have to be crossed for the process to take place. In the case of fusion of enveloped viruses (e.g., influenza) with endosomal membrane, the interacting membranes are in an acidic environment, which can affect the membrane's mechanical properties. This factor is often neglected in the analysis of virus-induced membrane fusion. In the present work, we demonstrate that even for membranes composed of zwitterionic lipids, changes of the environmental pH in the physiologically relevant range of 4.0 to 7.5 can affect the rate of the membrane fusion notably. Using a continual model, we demonstrated that the key factor defining the height of the energy barrier is the spontaneous curvature of the lipid monolayer. Changes of this parameter are likely to be caused by rearrangements of the polar part of lipid molecules in response to changes of the pH of the aqueous solution bathing the membrane.

BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax.

PubMed

Wilfling, F; Weber, A; Potthoff, S; Vögtle, F-N; Meisinger, C; Paschen, S A; Häcker, G

2012-08-01

During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

PubMed

Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

PubMed

Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

2008-01-04

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

Functional Anchoring Lipids for Drug Delivery Carrier Fabrication and Cell Surface Re-Engineering Applications

NASA Astrophysics Data System (ADS)

Vabbilisetty, Pratima

For decades, lipid vesicular bodies such as liposomes have been widely used and explored as biomimetic models of cell membranes and as drug/gene delivery carrier systems. Similarly, micellar iron oxide nanoparticles have also been investigated as potential MRI agents as well as drug delivery carrier systems. Cell surface carbohydrate-protein interactions allow them to serve as markers for recognition of many molecular and cellular activities thereby, are exploited as attractive molecules for surface modification of nanocarrier systems with purpose for tissues specific targeting and biocompatibility. In addition, the cell lipid membrane serves as an important platform for occurrence of many biological processes that are governed and guided by cell surface receptors. Introduction of chemoselective functional groups, via bio-orthogonal conjugation strategies, at the cell surface facilitates many cellular modifications and paves path for novel and potential biomedical applications. Anchoring lipids are needed for liposome surface functionalization with ligands of interest and play important roles in ligand grafting density, liposomes stability and biological activity. On the other hand, anchoring lipids are also needed for cell surface re-engineering by lipid fusion approach and have high impact for ligand insertion efficiency and biological activity. Overall, in this dissertation study, functional anchoring lipids for glyco-functionalized carrier systems and for efficient cell surface re-engineering applications were systematically investigated, respectively. Firstly, investigation of the synthesis of glyco-functionalized liposome systems based on phosphatidylethonalamine (PE) and cholesterol (Chol) anchoring lipids, prepared by post chemically selective functionalization via Staudinger ligation were carried out. The effect of anchor lipids on the stability, encapsulation and releasing capacity of the glycosylated liposomes were investigated by dynamic light

Activated release of membrane-anchored TGF-alpha in the absence of cytosol

PubMed Central

1993-01-01

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA- sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol. PMID:8314849

RAP/RAS workshop.

DOT National Transportation Integrated Search

2013-01-01

: RAP & RAS increases mix stiffness : : Most WMA additives decrease stiffness : : Tear-Off shingles are stiffer than Man-waste shingles : : Using multiple recycled bins improves consistency : : Finer RAS material improves consiste...

RAS Initiative - Events

Cancer.gov

The NCI RAS Initiative has organized multiple events with outside experts to discuss how the latest scientific and technological breakthroughs can be applied to discover vulnerabilities in RAS-driven cancers.

Deconstructing Ras Signaling in the Thymus

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Pinski, John M.; Alexander, Clayton P.; Merrill, Robert K.; Li, Wenmei; Love, Paul E.

2012-01-01

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance. PMID:22586275

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

PubMed

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

Carcinogenicity assessment of baricitinib in Tg.rasH2 mice and Sprague-Dawley (Crl:CD) rats.

PubMed

Carfagna, Mark; Cannady, Ellen; Ryan, Thomas; Herman, Jay; Truex, Lew; Narwani, Kanchan; Sullivan, John

2018-02-01

Baricitinib is a potent and selective Janus kinase (JAK)1 and JAK2 inhibitor, and is approved for the treatment of moderately to severely active RA in adults in Europe, Japan, and other countries. This study evaluated the carcinogenic potential of baricitinib in Tg. rasH2 mice and Sprague-Dawley (Crl:CD) rats. Baricitinib was administered daily by oral gavage to Crl:CD rats for up to 94 weeks (dose levels of 0, 1, 3, or 8 mg/kg for males and 0, 3, 8, or 25 mg/kg for females) and to Tg. rasH2 mice for 26 weeks (dose levels of 0, 15, 40, or 300 mg/kg for males and 0, 10, 30, or 150 mg/kg for females). Baricitinib was well tolerated with no incidence of compound-related neoplasms at any dose levels in rats and mice. In mice, non-neoplastic events observed were bone marrow hypocellularity and increased adipocytes. In rats, baricitinib administration was associated with a dose-dependent increase in survival, with a decreased incidence of neoplasm (hematopoietic and mammary), potentially secondary to drug-related decreased weight gain. The incidence of proliferative changes such as neoplastic and hyperplastic lesions in the mammary glands of females and in the livers of males and females also decreased. In conclusion, baricitinib is not considered to be carcinogenic. Copyright © 2018 Elsevier Inc. All rights reserved.

Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

PubMed

Stanhill, A; Levin, V; Hendel, A; Shachar, I; Kazanov, D; Arber, N; Kaminski, N; Engelberg, D

2006-03-09

Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

Defined spatiotemporal features of RAS-ERK signals dictate cell fate in MCF-7 mammary epithelial cells.

PubMed

Herrero, Ana; Casar, Berta; Colón-Bolea, Paula; Agudo-Ibáñez, Lorena; Crespo, Piero

2016-06-15

Signals conveyed through the RAS-ERK pathway are essential for the determination of cell fate. It is well established that signal variability is achieved in the different microenvironments in which signals unfold. It is also known that signal duration is critical for decisions concerning cell commitment. However, it is unclear how RAS-ERK signals integrate time and space in order to elicit a given biological response. To investigate this, we used MCF-7 cells, in which EGF-induced transient ERK activation triggers proliferation, whereas sustained ERK activation in response to heregulin leads to adipocytic differentiation. We found that both proliferative and differentiating signals emanate exclusively from plasma membrane-disordered microdomains. Of interest, the EGF signal can be transformed into a differentiating stimulus by HRAS overexpression, which prolongs ERK activation, but only if HRAS localizes at disordered membrane. On the other hand, HRAS signals emanating from the Golgi complex induce apoptosis and can prevent heregulin-induced differentiation. Our results indicate that within the same cellular context, RAS can exert different, even antagonistic, effects, depending on its sublocalization. Thus cell destiny is defined by the ability of a stimulus to activate RAS at the appropriate sublocalization for an adequate period while avoiding switching on opposing RAS signals. © 2016 Herrero et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Thermo-and pH-sensitive hydrogel membranes composed of poly(N-isopropylacrylamide)-hyaluronan for biomedical applications: Influence of hyaluronan incorporation on the membrane properties.

PubMed

Kamoun, Elbadawy A; Fahmy, Alaa; Taha, Tarek H; El-Fakharany, Esmail M; Makram, Mohamed; Soliman, Hesham M A; Shehata, Hassan

2018-01-01

Interpenetrating hydrogel membranes consisting of pH-sensitive hyaluronan (HA) and thermo-sensitive poly(N-isopropylacrylamide) (PNIPAAM) were synthesized using redox polymerization, followed by N,N-methylenebisacrylamide (BIS) and epichlorohydrin (EPI) were added as chemical crosslinkers. The interaction between membrane compositions has been characterized by FTIR spectroscopy and discussed intensively. The result indicates that HA incorporation in membranes increase the gel fraction, swelling uptake, and the flexibility/elasticity of crosslinked membranes, however it reduced oppositely the mechanical elongation of membranes. PNIPAAm-HA hydrogels responded to both temperature and pH changes and the stimuli-responsiveness was reversible. However, in vitro bioevaluation results revealed that the released ampicillin during the burst release time was sharply influenced and increased with increasing HA contents in membranes; afterwards it became sustainable. Whereas, high HA contents in hydrogels unexpectedly impacted negatively on the cells viability, owing to the viscosity of cell culture media changed. A big resistance was observed against microbial growth of Staphylococcus aureus, Salmonella typhi, and Candida albicans in case of pure PNIPAAm hydrogel membranes without HA or ampicillin. However, HA incorporation or the loaded ampicillin in membranes showed unexpected easily microbial growth. The fast release performance with dual pH-thermo-sensitive hydrogels were suggested as promising materials for quick drug carrier in the biomedical field. Copyright © 2017 Elsevier B.V. All rights reserved.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors.

PubMed

Loboda, Andrey; Nebozhyn, Michael; Klinghoffer, Rich; Frazier, Jason; Chastain, Michael; Arthur, William; Roberts, Brian; Zhang, Theresa; Chenard, Melissa; Haines, Brian; Andersen, Jannik; Nagashima, Kumiko; Paweletz, Cloud; Lynch, Bethany; Feldman, Igor; Dai, Hongyue; Huang, Pearl; Watters, James

2010-06-30

Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.

A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors

PubMed Central

2010-01-01

Background Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. Methods We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. Results The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. Conclusions These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

PubMed Central

Lehto, M T; Sharom, F J

1998-01-01

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion

Functional characterization of the essential tail anchor of the herpes simplex virus type 1 nuclear egress protein pUL34.

PubMed

Ott, Melanie; Tascher, Georg; Hassdenteufel, Sarah; Zimmermann, Richard; Haas, Jürgen; Bailer, Susanne M

2011-12-01

Release of herpes simplex virus type 1 (HSV-1) nucleocapsids from the host nucleus relies on the nuclear egress complex consisting of the two essential proteins pUL34 and pUL31. The cytoplasmically exposed N-terminal region of pUL34 interacts with pUL31, while a hydrophobic region followed by a short luminal part mediates membrane association. Based on its domain organization, pUL34 was postulated to be a tail-anchor (TA) protein. We performed a coupled in vitro transcription/translation assay to show that membrane insertion of pUL34 occurs post-translationally. Transient transfection and localization experiments in mammalian cells were combined with HSV-1 bacterial artificial chromosome mutagenesis to reveal the functional properties of the essential pUL34 TA. Our data show that a minimal tail length of 15 residues is sufficient for nuclear envelope targeting and pUL34 function. Permutations of the pUL34 TA with orthologous regions of human cytomegalovirus pUL50 or Epstein-Barr virus pBFRF1 as well as the heterologous HSV-1 TA proteins pUL56 or pUS9 or the cellular TA proteins Bcl-2 and Vamp2 revealed that nuclear egress tolerates TAs varying in sequence and hydrophobicity, while a non-α-helical membrane anchor failed to complement the pUL34 function. In conclusion, this study provides the first mechanistic insights into the particular role of the TA of pUL34 in membrane curving and capsid egress from the host nucleus.

Relationship of body weight parameters with the incidence of common spontaneous tumors in Tg.rasH2 mice.

PubMed

Paranjpe, Madhav G; Denton, Melissa D; Vidmar, Tom J; Elbekai, Reem H

2014-10-01

The mechanistic relationship between increased food consumption, increased body weights, and increased incidence of tumors has been well established in 2-year rodent models. Body weight parameters such as initial body weights, terminal body weights, food consumption, and the body weight gains in grams and percentages were analyzed to determine whether such relationship exists between these parameters with the incidence of common spontaneous tumors in Tg.rasH2 mice. None of these body weight parameters had any statistically significant relationship with the incidence of common spontaneous tumors in Tg.rasH2 males, namely lung tumors, splenic hemangiosarcomas, nonsplenic hemangiosarcomas, combined incidence of all hemangiosarcomas, and Harderian gland tumors. These parameters also did not have any statistically significant relationship with the incidence of lung and Harderian gland tumors in females. However, in females, increased initial body weights did have a statistically significant relationship with the nonsplenic hemangiosarcomas, and increased terminal body weights did have a statistically significant relationship with the incidence of splenic hemangiosarcomas, nonsplenic hemangiosarcomas, and the combined incidence of all hemangiosarcomas. In addition, increased body weight gains in grams and percentages had a statistically significant relationship with the combined incidence of all hemangiosarcomas in females, but not separately with splenic and nonsplenic hemangiosarcomas. © 2013 by The Author(s).

Overexpression of K-p21Ras play a prominent role in lung cancer

NASA Astrophysics Data System (ADS)

Zhang, Peng-bo; Zhou, Xin-liang; Yang, Ju-lun

2018-06-01

The proto-oncogene ras product, p21Ras, has been found overexpression in many human tumors. However, the subtypes of overexpressed p21Ras still remain unclear. The purpose of this study was to investigate overexpressed isoforms of p21Ras and their roles in the progress of lung cancer. Method: The expression of total p21Ras in normal lung tissues and lung cancers was determined by immunohistochemically staining with monoclonal antibody (Mab) KGHR-1 which could recognize and broad spectrum reaction with the (K/H/N) ras protein. Then, the isoforms of p21Ras was examined by specific Mab for each p21Ras subtypes. Results: Low expression of total p21Ras was found in 26.67% (8/30) of normal lung tissues, and 81.31% (87/107) of adenocarcinoma harbored overexpressed total p21Ras. Besides, 70.00% (35/50) of squamous cell carcinoma were detected overexpressed total p21Ras. In addition, 122 lung cancer tissues from overexpression of total p21Ras protein were selected to detect the expression of each subtype. And all the 122 lung cancer tissues were K-p21Ras overexpression. Moreover, there was a statistical significance difference between the expression level of total p21Ras and differentiation, and the same results were observed between the expression level of total p21Ras and lymph node metastasis (P<0.05). However, there was no correlation between the expression level of total p21Ras and gender, age, tumor size (P>0.05). Conclusions: Overexpression of K-p21Ras plays a prominent role in the progress of lung cancer and it is suggested that the p21Ras could serve as a promising treatment target in lung cancer.

A Novel Soluble Peptide with pH-Responsive Membrane Insertion.

PubMed

Nguyen, Vanessa P; Alves, Daiane S; Scott, Haden L; Davis, Forrest L; Barrera, Francisco N

2015-11-03

Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH-dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane α-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments.

Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

PubMed

Wasylyk, Christine; Zheng, Hong; Castell, Christelle; Debussche, Laurent; Multon, Marie-Christine; Wasylyk, Bohdan

2008-03-01

Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

RB mutation and RAS overexpression induce resistance to NK cell-mediated cytotoxicity in glioma cells.

PubMed

Orozco-Morales, Mario; Sánchez-García, Francisco Javier; Golán-Cancela, Irene; Hernández-Pedro, Norma; Costoya, Jose A; de la Cruz, Verónica Pérez; Moreno-Jiménez, Sergio; Sotelo, Julio; Pineda, Benjamín

2015-01-01

Several theories aim to explain the malignant transformation of cells, including the mutation of tumor suppressors and proto-oncogenes. Deletion of Rb (a tumor suppressor), overexpression of mutated Ras (a proto-oncogene), or both, are sufficient for in vitro gliomagenesis, and these genetic traits are associated with their proliferative capacity. An emerging hallmark of cancer is the ability of tumor cells to evade the immune system. Whether specific mutations are related with this, remains to be analyzed. To address this issue, three transformed glioma cell lines were obtained (Rb(-/-), Ras(V12), and Rb(-/-)/Ras(V12)) by in vitro retroviral transformation of astrocytes, as previously reported. In addition, Ras(V12) and Rb(-/-)/Ras(V12) transformed cells were injected into SCID mice and after tumor growth two stable glioma cell lines were derived. All these cells were characterized in terms of Rb and Ras gene expression, morphology, proliferative capacity, expression of MHC I, Rae1δ, and Rae1αβγδε, mult1, H60a, H60b, H60c, as ligands for NK cell receptors, and their susceptibility to NK cell-mediated cytotoxicity. Our results show that transformation of astrocytes (Rb loss, Ras overexpression, or both) induced phenotypical and functional changes associated with resistance to NK cell-mediated cytotoxicity. Moreover, the transfer of cell lines of transformed astrocytes into SCID mice increased resistance to NK cell-mediated cytotoxicity, thus suggesting that specific changes in a tumor suppressor (Rb) and a proto-oncogene (Ras) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells and therefore provide some insight into the ability of tumor cells to evade immune responses.

The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting.

PubMed

Jonckheere, Nicolas; Vasseur, Romain; Van Seuningen, Isabelle

2017-03-01

RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives. Copyright © 2017 Elsevier B.V. All rights reserved.

Evidence from simultaneous intracellular- and surface-pH transients that carbonic anhydrase IV enhances CO2 fluxes across Xenopus oocyte plasma membranes

PubMed Central

Occhipinti, Rossana; Boron, Walter F.

2014-01-01

Human carbonic anhydrase IV (CA IV) is GPI-anchored to the outer membrane surface, catalyzing CO2/HCO3− hydration-dehydration. We examined effects of heterologously expressed CA IV on intracellular-pH (pHi) and surface-pH (pHS) transients caused by exposing oocytes to CO2/HCO3−/pH 7.50. CO2 influx causes a sustained pHi fall and a transient pHS rise; CO2 efflux does the opposite. Both during CO2 addition and removal, CA IV increases magnitudes of maximal rate of pHi change (dpHi/dt)max, and maximal pHS change (ΔpHS) and decreases time constants for pHi changes (τpHi) and pHS relaxations (τpHS). Decreases in time constants indicate that CA IV enhances CO2 fluxes. Extracellular acetazolamide blocks all CA IV effects, but not those of injected CA II. Injected acetazolamide partially reduces CA IV effects. Thus, extracellular CA is required for, and the equivalent of cytosol-accessible CA augments, the effects of CA IV. Increasing the concentration of the extracellular non-CO2/HCO3− buffer (i.e., HEPES), in the presence of extracellular CA or at high [CO2], accelerates CO2 influx. Simultaneous measurements with two pHS electrodes, one on the oocyte meridian perpendicular to the axis of flow and one downstream from the direction of extracellular-solution flow, reveal that the downstream electrode has a larger (i.e., slower) τpHS, indicating [CO2] asymmetry over the oocyte surface. A reaction-diffusion mathematical model (third paper in series) accounts for the above general features, and supports the conclusion that extracellular CA, which replenishes entering CO2 or consumes exiting CO2 at the extracellular surface, enhances the gradient driving CO2 influx across the cell membrane. PMID:24965590

Dragging ras back in the ring.

PubMed

Stephen, Andrew G; Esposito, Dominic; Bagni, Rachel K; McCormick, Frank

2014-03-17

Ras proteins play a major role in human cancers but have not yielded to therapeutic attack. Ras-driven cancers are among the most difficult to treat and often excluded from therapies. The Ras proteins have been termed "undruggable," based on failures from an era in which understanding of signaling transduction, feedback loops, redundancy, tumor heterogeneity, and Ras' oncogenic role was poor. Structures of Ras oncoproteins bound to their effectors or regulators are unsolved, and it is unknown precisely how Ras proteins activate their downstream targets. These knowledge gaps have impaired development of therapeutic strategies. A better understanding of Ras biology and biochemistry, coupled with new ways of targeting undruggable proteins, is likely to lead to new ways of defeating Ras-driven cancers. Copyright © 2014 Elsevier Inc. All rights reserved.

Anchoring of development workings in a zone of influence of mining in case of the level anchoring system

NASA Astrophysics Data System (ADS)

Demin, V. F.; Fofanov, O. B.; Demina, T. V.; Yavorskiy, V. V.

2017-02-01

Regularities of the change of the stress-strain state of coal containing rock masses, depending on mining-geological factors, were revealed. These factors allow establishing rational parameters of anchoring of wall rocks to enhance the stability of development workings. Specific conditions of the deflected mode, displays of rock pressure, terms of maintenance depending on technological parameters are investigated. Researches allowed determining the degree of their development influence on the efficiency of application of the anchoring of the hollow making and will allow a reasonable application of anchoring certificates, provide stability of the rocks mining and reduce expenses on its realization and maintenance.

RAS - Screens & Assays

Cancer.gov

A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology

DOE PAGES

Bolla, Jani Reddy; Su, Chih-Chia; Delmar, Jared A.; ...

2015-04-20

The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. However, many pathogens have developed resistance to these antibiotics, prompting a re-evaluation of potential drug targets within the pathway. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. Here we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. YdaH is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm tomore » halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins that suggest a plausible pathway for substrate transport. Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs.« less

Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology

NASA Astrophysics Data System (ADS)

Bolla, Jani Reddy; Su, Chih-Chia; Delmar, Jared A.; Radhakrishnan, Abhijith; Kumar, Nitin; Chou, Tsung-Han; Long, Feng; Rajashankar, Kanagalaghatta R.; Yu, Edward W.

2015-04-01

The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. However, many pathogens have developed resistance to these antibiotics, prompting a re-evaluation of potential drug targets within the pathway. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. Here we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. YdaH is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins that suggest a plausible pathway for substrate transport. Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs.

Na+/H+ exchange activity in the plasma membrane of Arabidopsis.

PubMed

Qiu, Quan-Sheng; Barkla, Bronwyn J; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S

2003-06-01

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation

PubMed Central

Bettoun, Audrey; Surdez, Didier; Vallerand, David; Gundogdu, Ramazan; Sharif, Ahmad A.D.; Gomez, Marta; Cascone, Ilaria; Meunier, Brigitte; White, Michael A.; Codogno, Patrice; Parrini, Maria Carla; Camonis, Jacques H.; Hergovich, Alexander

2016-01-01

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells. PMID:27283898

Expression of membrane anchored cytokines and B7-1 alters tumor microenvironment and induces protective antitumor immunity in a murine breast cancer model.

PubMed

Bozeman, Erica N; Cimino-Mathews, Ashley; Machiah, Deepa K; Patel, Jaina M; Krishnamoorthy, Arun; Tien, Linda; Shashidharamurthy, Rangaiah; Selvaraj, Periasamy

2013-05-07

Many studies have shown that the systemic administration of cytokines or vaccination with cytokine-secreting tumors augments an antitumor immune response that can result in eradication of tumors. However, these approaches are hampered by the risk of systemic toxicity induced by soluble cytokines. In this study, we have evaluated the efficacy of 4TO7, a highly tumorigenic murine mammary tumor cell line, expressing glycosyl phosphatidylinositol (GPI)-anchored form of cytokine molecules alone or in combination with the costimulatory molecule B7-1 as a model for potential cell or membrane-based breast cancer vaccines. We observed that the GPI-anchored cytokines expressed on the surface of tumor cells greatly reduced the overall tumorigenicity of the 4TO7 tumor cells following direct live cell challenge as evidenced by transient tumor growth and complete regression within 30 days post challenge. Tumors co-expressing B7-1 and GPI-IL-12 grew the least and for the shortest duration, suggesting that this combination of immunostimulatory molecules is most potent. Protective immune responses were also observed following secondary tumor challenge. Further, the 4TO7-B7-1/GPI-IL-2 and 4TO7-B7-1/GPI-IL-12 transfectants were capable of inducing regression of a wild-type tumor growing at a distant site in a concomitant tumor challenge model, suggesting the tumor immunity elicited by the transfectants can act systemically and inhibit the tumor growth at a distant site. Additionally, when used as irradiated whole cell vaccines, 4TO7-B7-1/GPI-IL-12 led to a significant inhibition in tumor growth of day 7 established tumors. Lastly, we observed a significant decrease in the prevalence of myeloid-derived suppressor cells and regulatory T-cells in the tumor microenvironment on day 7 post challenge with 4TO7-B7-1/GPI-IL-12 cells, which provides mechanistic insight into antitumor efficacy of the tumor-cell membrane expressed IL-12. These studies have implications in designing membrane

Proteomic analysis of Herbaspirillum seropedicae reveals ammonium-induced AmtB-dependent membrane sequestration of PII proteins.

PubMed

Huergo, Luciano F; Noindorf, Lilian; Gimenes, Camila; Lemgruber, Renato S P; Cordellini, Daniela F; Falarz, Lucas J; Cruz, Leonardo M; Monteiro, Rose A; Pedrosa, Fábio O; Chubatsu, Leda S; Souza, Emanuel M; Steffens, Maria B R

2010-07-01

This study was aimed at describing the spectrum and dynamics of proteins associated with the membrane in the nitrogen-fixing bacterium Herbaspirillum seropedicae according to the availability of fixed nitrogen. Using two-dimensional electrophoresis we identified 79 protein spots representing 45 different proteins in the membrane fraction of H. seropedicae. Quantitative analysis of gel images of membrane extracts indicated two spots with increased levels when cells were grown under nitrogen limitation in comparison with nitrogen sufficiency; these spots were identified as the GlnK protein and as a conserved noncytoplasmic protein of unknown function which was encoded in an operon together with GlnK and AmtB. Comparison of gel images of membrane extracts from cells grown under nitrogen limitation or under the same regime but collected after an ammonium shock revealed two proteins, GlnB and GlnK, with increased levels after the shock. The P(II) proteins were not present in the membrane fraction of an amtB mutant. The results reported here suggest that changes in the cellular localization of P(II) might play a role in the control of nitrogen metabolism in H. seropedicae.

Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice

PubMed Central

Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

2014-01-01

Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR–Ras–Raf–MEK–ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [3H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras–MAPK activity could be important in its anticancer activity. PMID:24853419

Rabex-5 ubiquitin ligase activity restricts Ras signaling to establish pathway homeostasis in Drosophila.

PubMed

Yan, Hua; Jahanshahi, Maryam; Horvath, Elizabeth A; Liu, Hsiu-Yu; Pfleger, Cathie M

2010-08-10

The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid.

PubMed Central

Kinsella, B T; Erdman, R A; Maltese, W A

1991-01-01

ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Ali-Ali-Leu motif. We changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by [3H]geranylgeranyl instead of [3H]farnesyl in an in vitro assay. Gel-permeation chromatography of [3H]mevalonate-labeled hydrocarbons released from immunoprecipitated ras proteins overexpressed in COS cells indicated that Ha-ras p21(Leu-189) was also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21(Leu-189) with [3H]palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesyl- or geranylgeranyltransferases. Images PMID:1924354

Tea polyphenols can restrict benzo[a]pyrene-induced lung carcinogenesis by altered expression of p53-associated genes and H-ras, c-myc and cyclin D1.

PubMed

Manna, Sugata; Mukherjee, Sudeshna; Roy, Anup; Das, Sukta; Panda, Chinmay Kr

2009-05-01

The modulatory influence of tea polyphenols (epigallocatechin gallate, epicatechin gallate and theaflavin) on benzo[a]pyrene (B[a]P)-induced lung carcinogenesis in mice was analyzed using histopathological and molecular parameters. Progression of lung lesions was restricted at the hyperplastic stage by tea polyphenols. A significant reduction in cellular proliferative index and an increase in apoptotic index were noted in the restricted lung lesions. High expression of H-ras, c-myc, cyclin D1 and p53 genes was seen at the inflammatory stage (9th week) and in subsequent premalignant lesions, but down-regulation of H-ras at the hyperplastic stage (17th week). Expression of bcl-2 was high in hyperplastic lesions, whereas the expression of mdm2 and bcl-xl increased only at the moderately dysplastic stage (36th week). The tea polyphenols inhibited inflammatory response in the lung lesions on the 9th week, when decreased expression of H-ras and c-myc and increased expression of bax were noted. Prolonged treatment (>9th week) with tea polyphenols resulted in changes in the expression of some additional genes, such as reduced expression of cyclin D1 (from the 17th week), bcl-2 (from the 26th week; mild dysplasia) and p21 (on the 36th week), and high expression of p53 (from the 17th week) and p27 (on the 36th week). These observations indicate that the tea polyphenols can restrict B[a]P-induced lung carcinogenesis by differential modulation of the expression of p53 and its associated genes such as bax, bcl-2, mdm2, p21 and p27, along with H-ras, c-myc and cyclin D1, at different time points.

Ionic complexation as a non-covalent approach for the design of folate anchored rifampicin Gantrez nanoparticles.

PubMed

Date, Praveen V; Patel, Mitesh D; Majee, Sharmila B; Samad, Abdul; Devarajan, Padma V

2013-05-01

The present study discloses the design of folate anchored Rifampicin-Poly methylvinylether maleic anhydride copolymer (Gantrez AN-119, Gantrez) nanoparticles (RFMGzFa) by ionic complexation. Folic acid was anchored to the preformed drug loaded nanoparticles. Folic acid was anchored in different concentration by simply varying the amount of folic acid added during preparation. RFMGzFa nanoparticles were prepared by emulsion solvent diffusion method. Gantrez AN-119 rapidly hydrolyzes in aqueous medium releasing carboxylic acid groups, to create an acidic environment. This facilitates protonation and subsequent ionic complexation of folic acid with the carboxylic groups, to enable anchoring. FTIR spectra confirmed this interaction. Infrared imaging revealed distribution of folic acid across the nanoparticle surface. Nanoparticles were obtained in the size range 350-450 nm with RFM loading of 12-14% w/w. Zeta potential confirmed colloidal stability. TEM/SEM revealed spherical morphology. RFMGzFa nanoparticles exhibited sustained release of RFM and folic acid. Folic acid showed sustained release upto 12 h, which was ion exchange mediated. A 480% enhancement in RFM uptake with RFMGzFa nanoparticles compared to 300% with RFMGz nanoparticles in-vitro, in human macrophage cell line U-937, suggested the role of folic acid in folate receptor mediated uptake. Ionic complexation represents a simple non-covalent approach for anchoring folic acid on polymeric nanoparticles of Gantrez.

Anchored phosphatases modulate glucose homeostasis

PubMed Central