Mon. Mar 4th, 2024

The Tumor Procurement Core (TPC) within the Department of Pathology for
The Tumor Procurement Core (TPC) in the Division of Pathology for supplying the HNSCC tumor samples. The authors also thank Dr. Sushma Shivaswamy and Mr. John Simard for kindly giving the human neutralizing IL-1 antibody for use in our in vivo research. Lastly, we thank Nicholas Borcherding and Drs. Weizhou Zhang, Fayyaz Sutterwala and Hasem Habelhah for their useful recommendations and discussions relating to this function. Grant Assistance This operate was supported by grants NIH R01DE024550, NIH K01CA134941 and IRG-77-004-34 from the American Cancer Society, administered via the Holden Comprehensive Cancer Center at the University of Iowa.
H-Ras types dimers on membrane surfaces by means of a protein rotein interfaceWan-Chen Lina,b,1, Lars Iversena,b,1,2, Hsiung-Lin Tua,b, Christopher Rhodesa,b, Sune M. Christensena,b, Jeffrey S. Iwiga,c, Scott D. Hansena,b, William Y. C. Huanga,b, and Jay T. Grovesa,b,d,a Howard Hughes Health-related Institute and Departments of bChemistry and cMolecular and Cell Biology, University of California, Berkeley, CA 94720; and dPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CAEdited by Michael K. Rosen, University of Texas Southwestern Medical Center, Dallas, TX, and accepted by the Editorial Board January 15, 2014 (received for overview November 15, 2013)The lipid-anchored small GTPase Ras is an significant signaling node in mammalian cells. Several observations suggest that Ras is laterally organized within the cell membrane, and this may possibly play a regulatory function in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to become accountable for guiding protein organization in membranes. Here, we report that H-Ras types a dimer on membrane surfaces through a protein rotein binding interface. A Y64A point mutation within the switch II area, identified to stop Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, close to the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers by means of a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not call for lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes CXCR3 MedChemExpress applying a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization Kd is measured to become 1 103 moleculesm2, and no higher-order oligomers have been observed. Dimerization only happens around the membrane surface; H-Ras is strictly monomeric at comparable densities in resolution. Evaluation of several H-Ras constructs, which includes key adjustments to the lipidation pattern on the hypervariable area, recommend that dimerization can be a general home of native H-Ras on membrane surfaces.Ras signaling| Ras LTC4 Biological Activity assayIn addition to biochemical evidence for communication involving the C-terminal membrane binding area along with the nucleotide binding pocket, NMR and IR spectroscopic observations suggest that the HVR and lipid anchor membrane insertion impacts Ras structure and orientation (157). Molecular dynamics (MD) modeling of bilayer-induced H-Ras conformations has identified two nucleotide-dependent states, which differ in HVR conformation, membrane contacts, and G-domain orientation (18). In vivo FRET meas.