Search results for "CELLULAR"
showing 10 items of 6449 documents
Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR
2005
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Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.
1998
The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar …
Curvature and Torsion of Protein Main Chain as Local Order Parameters of Protein Unfolding
2020
International audience; Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the protein main chain. Because chemical shift (CS) measured by NMR spectroscopy is extremely sensitive to the local atomic environment, CS has served as a local probe of thermal unfolding of proteins by varying the position of the atomic isotope along the amino-acid sequence. The variation of the CS of each C(alpha) atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We…
Curcumin Affects HSP60 Folding Activity and Levels in Neuroblastoma Cells.
2020
The fundamental challenge in fighting cancer is the development of protective agents able to interfere with the classical pathways of malignant transformation, such as extracellular matrix remodeling, epithelial−mesenchymal transition and, alteration of protein homeostasis. In the tumors of the brain, proteotoxic stress represents one of the main triggering agents for cell transformation. Curcumin is a natural compound with anti-inflammatory and anti-cancer properties with promising potential for the development of therapeutic drugs for the treatment of cancer as well as neurodegenerative diseases. Among the mediators of cancer development, HSP60 is a key factor for the maintenance of…
The production of 85 kDa N-terminal fragment of apolipoprotein B in mutant HepG2 cells generated by targeted modification of apoB gene occurs by ALLN…
2010
Abstract To study the mechanism of low levels of full length and truncated apoB in individuals heterozygous for apoB truncation, a non-sense mutation was introduced in one of the three alleles of apob gene of HepG2 cells by homologous recombination. Despite very low levels of apoB-82 (1–2%) in the media, a prominent N-terminal apoB protein of 85 kDa (apoB-15) was secreted that fractionated at d > 1.065 in density gradient ultracentrifugation. The mechanism of production of this short protein was studied by 35S-methionine pulse–chase experiment. Oleate prevented presecretory degradation of apoB-100 in the cell and resulted in increased secretion of newly synthesized apoB-100 with decreases i…
A Stevedore's protein knot.
2009
Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein Data Bank, we encountered the most complex and the smallest knots to date: A recently uncovered α-haloacid dehalogenase structure contains a knot with six crossings, a so-called Stevedore knot, in a projection onto a plane. The smallest protein knot is present in an as yet unclassified …
Paraoxonase-2 Reduces Oxidative Stress in Vascular Cells and Decreases Endoplasmic Reticulum Stress–Induced Caspase Activation
2007
Background— In the vascular system, elevated levels of reactive oxygen species (ROS) produce oxidative stress and predispose to the development of atherosclerosis. Therefore, it is important to understand the systems producing and those scavenging vascular ROS. Here, we analyzed the ROS-reducing capability of paraoxonase-2 (PON2) in different vascular cells and its involvement in the endoplasmic reticulum stress pathway known as the unfolded protein response. Methods and Results— Quantitative real-time polymerase chain reaction and Western blotting revealed that PON2 is equally expressed in vascular cells and appears in 2 distinct glycosylated isoforms. By determining intracellular ROS, we…
Calcium negatively regulates meprin β activity and attenuates substrate cleavage
2015
The meprin β metalloproteinase is an important enzyme in extracellular matrix turnover, inflammation, and neurodegeneration in humans and mice. Previous studies showed a diminished cleavage of certain meprin β substrates in the presence of calcium, although the mechanism was not clear. With the help of a specific fluorogenic peptide assay and the human amyloid precursor protein as substrate, we demonstrated that the influence of calcium is most likely a direct effect on human meprin β itself. Analyzing the crystal structures of pro- and mature meprin β helped to identify a cluster of negatively charged amino acids forming a potential calcium binding site. Mutation of 2 of these residues (D2…
Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing
2012
The vast majority of membrane proteins are anchored to biological membranes through hydrophobic alpha-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in t…
A Membrane-Bound Vertebrate Globin
2011
The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2)-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and m…