1,721,028 research outputs found
Mithocondria defect and cisplatin resistance: relationships on ovarian cancer cells.
No full textThe Warburg effect, a phenomenon discovered by Otto Warburg in 1924, reflects a shift to an inefficient metabolism in cancer cells, in which an increase in the inefficient production of ATP via glycolysis leads to the secretion of lactate, even in the presence of oxygen (termed aerobic glycolysis) The underlying biochemical and molecular mechanism of the Warburg’s effect remain unclear, but it is likely a combination of mitochondrial malfunction (Wallace 2005) oncogenic alteration (Dang, Semenza 1999), as well as adaptive responses to the tumour microenvironment. We questioned the relevance of “metabolic reprogramming” in cisplatin-resistance, by studying mitochondrial function and metabolism in human ovary carcinoma cisplatin-resistant (C13) and -sensitive (2008) cell lines. C13 cells, in comparison 2008 cells, showed lower basal oxygen consumption (4.2±0.2 vs 6.5±0.7 fmol/cell/min, P<0.005) and lower basal transmembrane mitochondrial potential (ΔΨm) (18.7±1.5 vs 32.2±1 MFI P<0.001). Also C13 cells showed lower sensitivity to rotenone and oligomycin mitochondrial inhibitors. To further investigate the impact of mitochondria in the cisplatin- resistance, 2008 and C13 cells were depleted of their mitochondrial DNA (rho0-clones). Notably, the cytotoxicity of cisplatin was lower in 2008-rho0clones than in 2008 cells (IC50 3.56 μM and 0.72 μM) but similar between C13-rho0 and C13 cells (5.49 μM and 6.49 μM ). Next, the time-course of cell viability in glucose free-galactose medium, indicated that C13 cells were more strictly dependent on glucose, than 2008 cells. 1H-NMR spectroscopy evidenced higher basal content of intracellular GSH and mobile lipids (MLs) in C13 cells, as compared to 2008 cells, and Nile red staining confirmed the higher lipid accumulation, mainly within cytoplasmic droplets, in C13 cells. The findings allow to argue a “metabolic remodelling” of ovary carcinoma cells to a lipogenic phenotype, including alteration of mitochondrial function, as an advantageous mechanism to escape cisplatin-induced apoptosis. Investigation on how this metabolic change is achieved and how it influences resistance to platinum drugs in other neoplasms will be of relevance in chemotherapy and should be further evaluated to improve the clinical impact of platinum drugs.
Dang, Semenza. (1999). Trends Biochem Sci. 24:68-72.
Wallace. (2005) Annu Rev Genet. 39:359-407
Molecular Mapping of Functionalities in the Solution Structure of Reduced Grx4, a Monothiol Glutaredoxin from Escherichia coli
Full text linkThe ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems
Molecular architecture and the structural basis for anion interaction in prestin and SLC26 transporters
Full text linkPrestin (SLC26A5) is a member of the SLC26/SulP anion transporter family. Its unique quasi-piezoelectric mechanical activity generates fast cellular motility of cochlear outer hair cells, a key process underlying active amplification in the mammalian ear. Despite its established physiological role, it is essentially unknown how prestin can generate mechanical force, since structural information on SLC26/SulP proteins is lacking. Here we derive a structural model of prestin and related transporters by combining homology modelling, MD simulations and cysteine accessibility scanning. Prestin's transmembrane core region is organized in a 7+7 inverted repeat architecture. The model suggests a central cavity as the substrate-binding site located midway of the anion permeation pathway, which is supported by experimental solute accessibility and mutational analysis. Anion binding to this site also controls the electromotile activity of prestin. The combined structural and functional data provide a framework for understanding electromotility and anion transport by SLC26 transporters
A molecular dynamics strategy for CSαβ peptides disulfide-assisted model refinement
No full textMany cysteine-stabilized antimicrobial peptides from a variety of living organisms could be good candidates for the development of anti-infective agents. In the absence of experimentally obtained structural data, peptide modeling is an essential tool for understanding structure–activity relationships and for optimizing the bioactive moieties. Focusing on cysteine-rich peptide structures, we reproduced the case of structure predictions in the so-called midnight zone. We developed our protocol on a training set derived by clustering the available cysteine-stabilized αβ (CSαβ) structures in nine different representative families and tested it on peptides randomly selected from each family. Starting from draft models, we tested a structure-based disulfide predictor and we used cysteine distances as constraints during molecular dynamics. Finally, we proposed an analysis for final structure selection. Accordingly, we obtained a mean root mean square deviation improvement of 21% for the test set. Our findings demonstrate that it is possible to predict the network of disulfide bridges in cysteine-stabilized peptides and to use this result to improve the accuracy of structural predictions. Finally, we applied the methods to predict the structure of royalisin, a cysteine-rich peptide with unknown structure
Direct observation of intramolecular hydrogen bonds in peptide 3(10) helices by (3h)J(N,C ') scalar couplings
No full textDifferentiating helices: The direct observation of hydrogen bonds by (3h)J(N,C′) scalar couplings is not only possible for alpha-helix and beta-sheet peptides and proteins, but for 3(10)-helical peptides as well. The method also provides information on terminal fraying of helices and allows the discrimination between alpha and 3(10) helices
Structure of the Cytosolic Portion of the Motor Protein Prestin and Functional Role of the STAS Domain in SLC26/SulP Anion Transporters
No full textPrestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases. SLC26 and related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and protein function. This C-terminal portion is mainly composed of a STAS (sulfate transporters and anti- sigma factor antagonist) domain, whose name is due to a remote but significant sequence similarity with bacterial ASA (anti- sigma factor antagonist) proteins. Here we present the crystal structure at 1.57 Å resolution of the cytosolic portion of prestin, the first structure of a SulP transporter STAS domain, and its characterization in solution by heteronuclear multidimensional NMR spectroscopy. Prestin STAS significantly deviates from the related bacterial ASA proteins, especially in the N-terminal region, which-although previously considered merely as a generic linker between the domain and the last transmembrane helix-is indeed fully part of the domain. Hence, unexpectedly, our data reveal that the STAS domain starts immediately after the last transmembrane segment and lies beneath the lipid bilayer. A structure-function analysis suggests that this model can be a general template for most SLC26 and SulP anion transporters and supports the notion that STAS domains are involved in functionally important intramolecular and intermolecular interactions. Mapping of disease-associated or functionally harmful mutations on STAS structure indicates that they can be divided into two categories: those causing significant misfolding of the domain and those altering its interaction properties
1H-NMR Spectroscopy and cytotoxicity of natural phenolic compounds on human wild and cis-platin resistant cancer cell
No full textsito web: http://farmacologiasif.unito.it/cong32/final/sifcong32_prog_scie_def.pd
Conformational study of an Aib-rich peptide in DMSO by NMR
No full textThe strong propensity of 2-amino-2-methyl propanoic acid (Aib)-rich peptides to form stable helical structures is well documented. NMR analysis of the short peptide Z-(Aib)5-L-Leu-(Aib)2-OMe indicates the presence of a well-characterized 3(10)-helix even in dimethylsulfoxide (DMSO), a solvent known to disrupt helical structures. The structure remains stable at least up to 348 K. Stereospecific assignment of the diastereotopic methyls of Aib was achieved, with the assumption of a specific helical screw sense. The methyl more eclipsed with respect to the CO vector resonates at a higher field in the carbon dimension. Molecular dynamics simulations successfully predict the 3J(CHNH) coupling constant of Leu6 and most of the H-bonding pattern. Discrepancies were found for Aib3 and Aib7 amide protons which can be explained by a higher sensitivity of the simulations to the helix fraying at the end of the peptide and by the presence of extended conformations for Leu6 during most of the simulations
Effects of natural semisynthetic and sinthetic phenolics and nuclear magnetic resonance on human ovarian cancer cell lines
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