bMany plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.
Melanomas display poor response rates to adjuvant therapies because of their intrinsic resistance to proapoptotic stimuli. This study indicates that such resistance can be overcome, at least partly, through the targeting of eEF1A elongation factor with narciclasine, an Amaryllidaceae isocarbostyril controlling plant growth. Narciclasine displays IC(50) growth inhibitory values between 30-100 nM in melanoma cell lines, irrespective of their levels of resistance to proapoptotic stimuli. Normal noncancerous cell lines are much less affected. At nontoxic doses, narciclasine also significantly improves (P=0.004) the survival of mice bearing metastatic apoptosis-resistant melanoma xenografts in their brain. The eEF1A targeting with narciclasine (50 nM) leads to 1) marked actin cytoskeleton disorganization, resulting in cytokinesis impairment, and 2) protein synthesis impairment (elongation and initiation steps), whereas apoptosis is induced at higher doses only (≥200 nM). In addition to molecular docking validation and identification of potential binding sites, we biochemically confirmed that narciclasine directly binds to human recombinant and yeast-purified eEF1A in a nanomolar range, but not to actin or elongation factor 2, and that 5 nM narciclasine is sufficient to impair eEF1A-related actin bundling activity. eEF1A is thus a potential target to combat melanomas regardless of their apoptosis-sensitivity, and this finding reconciles the pleiotropic cytostatic of narciclasine. -
Molecular dynamics simulations have been used to compute the difference in the unfolding free energy between wild-type barnase and the mutant in which Ile-96 is replaced by alanine. The simulations yield results (-3.42 and -5.21 kcal/mol) that compare favorably with experimental values (-3.3 and -4.0 kcal/mol). The major contributions to the free energy difference arise from bonding terms involving degrees of freedom of the mutated side chain and from nonbonded interactions of that side chain with its environment in the folded protein. By comparison with simulations of an extended peptide in the absence of solvent, used as a reference state, hydration effects are shown to play a minor role in the overall free energy balance for the Ile -* Ala transformation. The implications of these results for our understanding of the hydrophobic effect and its contribution to protein stability are discussed.Hydrophobic interactions are believed to make a major contribution to stabilizing the native structure of proteins in an aqueous environment (1-4). According to the classical picture (1) and more recent theoretical analyses (4), the hydrophobic effect leads to structures in which many, but not all, of the nonpolar side chains are packed together in the protein interior where they avoid contact with water. Measurements of solvation effects have shown that the hydrophobic effect is largely entropic in origin at room temperature (5). The unfavorable entropy of solvation is ascribed to the entropy decrease of water surrounding the nonpolar groups (6, 7); this is in accord with simulation studies of hydrophobic solutes (8, 9) and peptides in water (10).Although the general role of the hydrophobic effect in protein folding is understood, a more detailed quantitative description of its contributions to protein stability is essential. Site-directed mutagenesis experiments combined with thermal and spectroscopic stability measurements are now being used to dissect the contributions of individual amino acids (11). Substitutions of buried or partly buried nonpolar residues in several proteins (12-15) have shown that the change in thermodynamic stability between the wild type and mutant can be related to the free energies of transfer (16)(17)(18) and/or the accessible surface areas (19) of the individual substituted residues. Although there is a good overall correlation when a wide range of substitutions are considered (12), the variation seen among the hydrophobic aliphatic side chains does not show any simple relation with transfer free energies. Moreover, the interpretation is somewhat confused by the use of transfer free energy values from different solvents or from the gas phase to water for analyzing the experimental data (12,14). There are also more basic questions concerning the effect of hydrophobic solvation of individual nonpolar groups and its relationship to their bulk properties in the protein interior (20). Whether the protein interior corresponds to a nonpolar or a slightly polar liquid (e.g., alcohol-like) ...
Oxidized low-density lipoproteins (LDLs) accumulate in the vascular wall and promote local inflammation, which contributes to the progression of the atheromatous plaque. The key role of myeloperoxidase (MPO) in this process is related to its ability to modify APO B-100 in the intima and at the surface of endothelial cells. A series of 3-(aminoalkyl)-5-fluoroindole analogues was designed and synthesized by exploiting the structure-based docking of 5-fluorotryptamine, a known MPO inhibitor. In vitro assays were used to study the effects of these compounds on the inhibition of MPO-mediated taurine chlorination and oxidation of LDLs. The kinetics of the interaction between the MPO redox intermediates, Compounds I and II, and these inhibitors was also investigated. The most potent molecules possessed a 4- or 5-carbon aminoalkyl side chain and no substituent on the amino group. The mode of binding of these analogues and the mechanism of inhibition is discussed with respect to the structure of MPO and its halogenation and peroxidase cycles.
Background: Can1 is a yeast plasma membrane permease catalyzing specific uptake of arginine. Results: Two residues in the binding pocket of Can1 affect its selectivity. Conclusion: Subtle amino acid changes can convert Can1 to a lysine-specific permease. Significance: Understanding, at the molecular level, the translocation mechanism(s) of yeast amino acid transporters has bearings on our knowledge of other transporters featuring the same fold.
The voltage-dependent anion-selective channel (VDAC) is the most abundant protein in the mitochondrial outer membrane and the major transport pathway for a large variety of compounds ranging from ions to large polymeric molecules such as DNA and tRNA. Plant VDACs feature a secondary structure content and electrophysiological properties akin to those of VDACs from other organisms. They however undergo a specific regulation. The general importance of VDAC in plant physiology has only recently emerged. Besides their role in metabolite transport, plant VDACs are also involved in the programmed cell death triggered in response to biotic and abiotic stresses. Moreover, their colocalization in non-mitochondrial membranes suggests a diversity of function. This review summarizes our current understanding of the structure and function of plant VDACs. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
The interactions between HIV-1 protease and its bound inhibitors have been investigated by molecular mechanics calculations and by analysis of crystal structures of the complexes in order to determine general rules for inhibitor and substrate binding to the protease. Fifteen crystal structures of HIV-1 protease with different peptidomimetic inhibitors showed conservation of hydrogen bond interactions between the main chain C = O and NH groups of the inhibitors and the C = O and NH groups of the protease extending from P3 C = O to P3' NH. The mean length of the hydrogen bonds between the inhibitor and the flexible flaps and the conserved water molecule (2.9 A) is slightly shorter than the mean length of hydrogen bonds between the inhibitor and the more rigid active site region (3.1 A) of the protease. The two hydrogen bonds between the conserved water and P2 and P1' carbonyl oxygen atoms of the inhibitor are the shortest and are predicted to be important for the tight binding of inhibitors. Molecular mechanics analysis of three crystal structures of HIV-1 protease with different inhibitors with independent calculations using the programs Discover and Brugel gave an estimate of 56-68% for the contribution of all the inhibitor main chain atoms to the total calculated protease-inhibitor interaction energy. The contribution of individual inhibitor residues to the interaction energy was calculated using Brugel. The main chain atoms of residue P2 had a consistently large favorable contribution to the total interaction energy, probably due to the presence of the two short hydrogen bonds to the flexible flap.(ABSTRACT TRUNCATED AT 250 WORDS)
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