Methods for Site-Selective Chemical Pretein Immobilization

Els bioxips de proteïnes (micro arrays) requereixen, per a una implementació eficient, d'un desenvolupament de les tècniques d'immobilització de proteïnes.Es comenten diverses aproximacions químiques per tal d’assolir la immobilització de proteïnes d’una manera específica amb enllaços covalents. El desenvolupament en aquesta àrea condueix a millores tecnològiques per al desenvolupament de bioxips de proteïnes

Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions

The Autodepalmitoylating activity of APT maintains the spatial organization of Palmitoylated membrane proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.Fil: Vartak, Nachiket. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Papke, Bjoern. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Grecco, Hernan Edgardo. Institut Max Planck Fur Molekulare Physiologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossmannek, Lisaweta. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Waldmann, Herbert. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Hedberg, Christian. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Bastiaens, Philippe I. H.. Institut Max Planck Fur Molekulare Physiologie; Alemani

Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation

AbstractThe lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data

Stereoselective synthesis of a natural product inspired tetrahydroindolo[2,3-a]-quinolizine compound library

AbstractA natural product-inspired synthesis of a compound collection embodying the tetrahydroindolo[2,3-a]quinolizine scaffold was established with a five step synthesis route. An imino-Diels–Alder reaction between Danishefsky’s diene and the iminoesters derived from tryptamines was used as a key reaction. Reductive amination of the ketone function and amide synthesis with the carboxylic acid derived from the ethyl ester, were used to decorate the core scaffold. Thus a compound library of 530 tetrahydroindolo[2,3-a]quinolizines was generated and submitted to European lead factory consortium for various biological screenings

Methylation of H2AR29 is a novel repressive PRMT6 target

<p>Abstract</p> <p>Background</p> <p>Covalent histone modifications are central to all DNA-dependent processes. Modifications of histones H3 and H4 are becoming well characterised, but knowledge of how H2A modifications regulate chromatin dynamics and gene expression is still very limited.</p> <p>Results</p> <p>To understand the function of H2A modifications, we performed a systematic analysis of the histone H2A methylation status. We identified and functionally characterised two new methylation sites in H2A: R11 (H2AR11) and R29 (H2AR29). Using an unbiased biochemical approach in combination with candidate assays we showed that protein arginine methyltransferase (PRMT) 1 and PRMT6 are unique in their ability to catalyse these modifications. Importantly we found that H2AR29me2 is specifically enriched at genes repressed by PRMT6, implicating H2AR29me2 in transcriptional repression.</p> <p>Conclusions</p> <p>Our data establishes R11 and R29 as new arginine methylation sites in H2A. We identified the specific modifying enzymes involved, and uncovered a novel functional role of H2AR29me2 in gene silencing <it>in vivo</it>. Thus this work reveals novel insights into the function of H2A methylation and in the mechanisms of PRMT6-mediated transcriptional repression.</p

Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI

Small GTPases of the Rho family regulate cytoskeleton remodeling, cell polarity, and transcription, as well as the cell cycle, in eukaryotic cells. Membrane delivery and recycling of the Rho GTPases is mediated by Rho GDP dissociation inhibitor (RhoGDI), which forms a stable complex with prenylated Rho GTPases. We analyzed the interaction of RhoGDI with the active and inactive forms of prenylated and unprenylated RhoA. We demonstrate that RhoGDI binds the prenylated form of RhoA center dot GDP with unexpectedly high affinity (K-d = 5 pM). The very long half-life of the complex is reduced 25-fold on RhoA activation, with a concomitant reduction in affinity (K-d = 3 nM). The 2.8-angstrom structure of the RhoA center dot guanosine 5'-[beta,gamma-imido] triphosphate (GMPPNP)center dot RhoGDI complex demonstrated that complex formation forces the activated RhoA into a GDP-bound conformation in the absence of nucleotide hydrolysis. We demonstrate that membrane extraction of Rho GTPase by RhoGDI is a thermodynamically favored passive process that operates through a series of progressively tighter intermediates, much like the one that is mediated by RabGDI