Structural origins of the functional divergence of human insulin-like growth factor-I and insulin

Human insulin-like growth factors I and II (hIGF-I, hIGF-II) are potent stimulators of cell and growth processes. They display high sequence similarity to both the A and B chains of insulin but contain an additional connecting C-domain, which reflects their secretion without specific packaging or precursor conversion. IGFs also have an extension at the C-terminus known as the D-domain. This paper describes four homologous hIGF-1 structures, obtained from crystals grown in the presence of the detergent SB12, which reveal additional detail in the C- and D-domains. Two different detergent binding modes observed in the crystals may reflect different hIGF-I biological properties such as the interaction with IGF binding proteins and self-aggregation. While the helical core of hIGF-I is very similar to that in insulin, there are distinct differences in the region of hIGF-I corresponding to the insulin B chain C-terminus, residues B25-B30. In hIGF-I, these residues (24-29) and the following C-domain form an extensive loop protruding 20 A from the core, which results in a substantially different conformation for the receptor binding epitope in hIGF-I compared to insulin. One notable feature of the structures presented here is demonstration of peptide-bond cleavage between Ser35 and Arg36 resulting in an apparent gap between residues 35 and 39. The equivalent region of proinsulin is involved in hormone processing demanding a reassessment of the structural integrity of hIGF-I in relation to its biological function.     

Glycated high-density lipoprotein induces apoptosis of endothelial cells via a mitochondrial dysfunction

Glycation of plasma proteins may contribute to an excess risk of developing atherosclerosis in patients with diabetes mellitus. Although it is believed that high-density lipoprotein (HDL) is nonenzymatically glycosylated at an increased level in diabetic individuals, little is known about a possible linkage between glycated HDL and endothelium dysfunction in diabetes. This study set out to clarify whether glucose-modified HDL affects the function of endothelial cells by examining the apoptosis of cultured human aortic endothelial cells (HAECs) exposed to a glycated-oxidized HDL (gly-ox-HDL) prepared in vitro. Incubation of HAECs with 100 microg/ml of gly-ox-HDL for 48 h showed apoptotic features, such as cell shrinkage, membrane blebbing, and concentration and fragmentation of the nucleus, and the degree of apoptosis was dose-dependent on the glucose used in the preparation of gly-ox-HDL. Stimulation of HAECs with gly-ox-HDL elicited a marked increase in caspase 3 activity and the expressions of active caspase 3 and caspase 9, whereas concomitant treatment with a caspase 3 inhibitor significantly blocked gly-ox-HDL-induced apoptosis of HAECs. The release of cytochrome c into cytosols markedly increased in HAECs during the treatment with gly-ox-HDL. The increased expressions of Bax and Bad were detected in HAECs incubated for 24 h with gly-ox-HDL, but gly-ox-HDL failed to interfere with the expression of Bcl-2 and Bcl-x. Moreover, in vitro experiments with HDL (gly-HDL) glycated in the presence of 2 mM EDTA and Cu(2+)-oxidized HDL suggested that the apoptotic effect of gly-ox-HDL on endothelial cells might be due to an additional oxidative modification of gly-HDL. Taken altogether, additional oxidation of HDL under hyperglycemic conditions may induce endothelial apoptosis through a mitochondrial dysfunction, following the deterioration of vascular function.     

Carcinogen-Induced, Free Radical-Mediated Reduction in Microsomal Membrane Fluidity: Reversal by Indole-3-propionic Acid

Chromium (Cr) is a well established carcinogen, with Cr(III) accounting for much of the intracellular oxidative damage that this transition metal induces. Indole-3-propionic acid (IPA), a melatonin-related molecule, is a reported antioxidant and free radical scavenger. Concentration (1, 10, 100, 500, or 1000 M) and time (15, 30, 45, 60, or 90 min)-dependent effects of Cr(III) in the presence of H₂O₂ (0.5 mM), as well as the protective effect of IPA on Cr(III)-induced alterations in membrane fluidity (the inverse of membrane rigidity), as an index of membrane damage, were estimated by fluorescence spectroscopy. Cr(III), in a concentration- and a time-dependent manner, decreased membrane fluidity, with marked effects at a concentration of 500 M and 60 min of incubation. IPA (5, 3, or 1 mM) prevented the Cr(III)-induced decrease in membrane fluidity. It is concluded that the carcinogen Cr(III), in the presence of H₂O₂, generates free radicals, which decrease membrane fluidity in rat microsomal membranes. Membrane alterations are pharmacologically prevented by the antioxidant IPA.     

The Influence of Sequence Microvariation Among HLA-DR3 Alleles on their Structure and Complexes Formation with Presentation Peptides

The three-dimensional structure of human leukocyte antigens HLA-DR*0301 and HLA-DR*0302 have been calculated using the homology modeling approach. General structural features of our models are similar to those of related HLA molecules. The typical layout of segments of the secondary structure is well preserved. However polypeptide chains are less tightly bound, which causes slightly broader opening of the binding groove. It also results in the modified layout of pockets in the binding groove. Amino acids defining the restricted sequence diversity of studied proteins are easily available for interactions with ligands.A set of docking simulations was performed using modeled structures of both HLA molecules and various specific peptide ligands. The control docking of influenza hemagglutinin peptide into HLA-DR*0101 molecule gives the complex structure which is in good agreement with that from crystallographic studies. The extensive analysis of the structure of modeled complexes of HLA-DR*0301 and HLA-DR*0302 with various ligands indicates that sequence microvariation of both alleles is not directly controlling the binding specificity. Preferences for binding of specific ligands, as evaluated from interactions in modeled complexes, agree qualitatively with experimental observations. Thus the computer aided docking simulations can be successfully used to calculate the three-dimensional structure of HLA-ligand complexes. However detailed explanation of binding specificity can not be achieved using presently available modeling procedures.Electronic Supplementary Material available.     

Topological origins of chromosomal territories

Using freely jointed polymer model we compare equilibrium properties of crowded polymer chains whose segments are either permeable or not permeable for other segments to pass through. In particular, we addressed the question whether non-permeability of long chain molecules, in the absence of excluded volume effect, is sufficient to compartmentalize highly crowded polymer chains, similarly to what happens during formation of chromosomal territories in interphase nuclei. Our results indicate that even polymers without excluded volume compartmentalize and show strongly reduced intermingling when they are mutually non-permeable. Judging from the known fact that chromatin fibres originating from different chromosomes show very limited intermingling in interphase nuclei, we propose that regular chromatin fibres during chromosome decondensation can hardly serve as a substrate of cellular type II DNA topoisomerases.     

Regulation of S-amino acids biosynthesis in Saccharomycopsis lipolytica

Molecular Genetics and Genomics - ATP-sulfurylase, cysteine synthase, homocysteine synthase, arylsulfatase and β-cystathionase in Saccharomycopsis lipolytica are repressed on the addition of...     

Calponin-Like Chd64 Is Partly Disordered

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function.     

Laser interferometric investigation of solute transport through membrane-concentration boundary layer system

We investigate diffusive transport in a membrane system with a horizontally mounted membrane under concentration polarization conditions performed by a laser interferometry method. The data obtained from two different theoretical models are compared to the experimental results of the substance flux. In the first model, the membrane is considered as infinitely thin, while in the second one as a wall of finite thickness. The theoretical calculations show sufficient correspondence with the experimental results. On the basis of interferometric measurements, the relative permeability coefficient (ζ_s) for the system, consisting of the membrane and concentration boundary layers, was also obtained. This coefficient reflects the concentration polarization of the membrane system. The obtained results indicate that the coefficient ζ_s of the membrane-concentration boundary layer system decreases in time and seems to be independent of the initial concentration of the solute.