Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutarylCoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ringmodified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.Cellular cholesterol requirements are met through de novo cholesterol synthesis and uptake of lipoprotein cholesterol. These homeostatic responses are regulated at multiple steps through a negative feedback loop that responds to elevations in cellular cholesterol. Central to this pathway is the sterol regulatory element-binding protein (SREBP) 3 family of transcription factors, which directly activates expression of genes involved in the synthesis and uptake of cholesterol and lipogenesis. In the endoplasmic reticulum (ER), cholesterol modulates SREBP processing by binding to the sterol-sensing domain of SREBP cleavage-activating protein (SCAP) and inducing conformational change in SCAP (1). Cholesterol also promotes interaction between SCAP and the ER retention proteins, Insig-1 and Insig-2, which in turn prevents movement of the SCAP-SREBP complex to the Golgi and maturation of the SREBP transcription factors (1, 2). Cholesterol homeostasis is governed not only by its end product, cholesterol, but also by oxygenated derivatives of cholesterol, known as oxysterols. Side chain oxysterols, such as 24-, 25-, and 27-hydroxycholesterol (HC), are generated enzymatically in vivo and are important physiological regulators of cholesterol homeostasis. These side chain oxysterols contribute to the maintenance of cellular cholesterol balance by serving as endogenous ligands for the liver X receptors (LXRs), which activate cholesterol elimination and efflux pathways (3), and through suppression of SREBP proteolysis. Unlike cholesterol, the side chain oxygenated sterols 25-HC and 27-HC do not induce conformational changes in SCAP and are unable to bind to the sterol-sensing domain of SCAP (1, 4). Rather, 25-HC and 27-HC have been shown to bind to the Insig proteins, promo...
The importance of BCL-2-family proteins in the control of cell death has been clearly established. One of the key members of this family, BAX, has soluble, membrane bound and membrane integrated forms that are central to the regulation of apoptosis. Using purified monomeric human BAX, defined liposomes and isolated human mitochondria we have characterized the soluble to membrane transition and pore formation by this protein. For the purified protein, activation but not oligomerization, is required for membrane binding. The transition to the membrane environment includes a binding step that is reversible and distinct from the membrane integration step. Oligomerization and pore activation occur after the membrane integration. In cells, BAX targets several intracellular membranes, but notably does not target the plasma membrane while initiating apoptosis. When cholesterol was added to either the liposome bilayer or mitochondrial membranes we observed increased binding but markedly reduced integration of BAX into both membranes. This cholesterol inhibition of membrane integration accounts for the reduction of BAX pore activation in liposomes and mitochondrial membranes. Our results indicate that the presence of cholesterol in membranes inhibits the pore forming activity of BAX by reducing the ability of BAX to transition from a membrane associated to a membrane integral protein.
Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very 'deep' mutagenesis at selected residues: Ͼ10 5 substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants. Leukemia (2001) 15, 1277-1285.
A deletion derivative of the cytokine human interleukin-3 (hIL-3(15-125), comprising amino acids 15-125 of the native protein) was produced as a fusion to the filamentous phage surface protein pIII. The cytokine was detected in association with phage particles by protein immunoblotting. Compared to an equivalent quantity of soluble-cytokine, phage-presented hIL-3(15-125) exhibited reduced biological activity in a hIL-3-dependent cell proliferation assay. The reduction in activity was attributable to presence of phage particles in the assay, rather than directly owing to physical incorporation of the cytokine into the phage particle. Owing to the position of the amber codon in the phagemid vector, the phagemid-produced free hIL-3(15-125) species (designated hIL-3(15-125) epsilon) had 20 amino acids appended to its C-terminus; hIL-3(15-125) epsilon did not exhibit reduced bioactivity. hIL-3(15-125)-presenting phage were affinity-selected with either a hIL-3-reactive polyclonal antibody or with cells expressing the heterodimeric hIL-3 receptor. These data are consistent with the use of phage-display technology for the affinity selection of hIL-3 variants with modified biological properties.
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