Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors

The NR1I subfamily of nuclear receptors contains a phylogenetically diverse array of receptors related to the mammalian pregnane X receptor (PXR) (NR1I2) and constitutive androstane receptor (CAR) (NR1I3). We have carried out an extensive comparative analysis of this subgroup with representatives from fish, birds, amphibians, and mammals. Four novel receptors were isolated from fish, dog, pig, and monkey for this study and combined with a previously reported set of related receptors including human PXR, rabbit PXR, mouse PXR, chicken CXR, frog benzoate X receptors (BXRalpha, BXRbeta), and human and mouse CAR. A broad range of xenobiotics, steroids, and bile acids were tested for their ability to activate the ligand binding domain of each receptor. Three distinct groups of receptors were identified based on their pharmacological profiles: 1) the PXRs were activated by a broad range of xenobiotics and, along with the mammalian PXRs, included the chicken and fish receptors; 2) the CARs were less promiscuous, had high basal activities, and were generally repressed rather than activated by those compounds that modulated their activity; and 3) the BXRs were selectively activated by a subset of benzoate analogs and are likely to be specialized receptors for this chemical class of ligands. The PXRs are differentiated from the other NR1I receptors by a stretch of amino acids between helices 1 and 3, which we designate the H1-3 insert. This insert was present in the mammalian, chicken, and fish PXRs but absent in the CARs and BXRs. Modeling studies suggest that the H1-3 insert contributes to the promiscuity of the PXRs by facilitating the unwinding of helices-6 and -7, thereby expanding the ligand binding pocket.     

Structure and energy of fusion stalks: the role of membrane edges

Fusion of lipid bilayers proceeds via a sequence of distinct structural transformations. Its early stage involves a localized, hemifused intermediate in which the proximal but not yet the distal monolayers are connected. Whereas the so-called stalk model most successfully accounts for the properties of the hemifused intermediate, there is still uncertainty about its microscopic structure and energy. We reanalyze fusion stalks using the theory of membrane elasticity. In our calculations, a short (cylindrical micelle-like) tether connects the two proximal monolayers of the hemifused membranes. The shape of the stalk and the length of the tether are calculated such as to minimize the overall free energy and to avoid the formation of voids within the hydrocarbon core. Our free energy expression is based on three internal degrees of freedom of a perturbed lipid layer: thickness, splay, and tilt deformations. Based on exactly the same model, we compare fusion stalks with and without the ability included to form sharp edges at the interfacial region between the hydrocarbon core and the polar environment. Requiring the interface to be smooth everywhere, our detailed calculations recover previous results: the stalk energies are far too high to account for the experimental observation of fusion intermediates. However, if we allow the interface to be nonsmooth, we find a remarkable reduction of the stalk free energy down to more realistic values. The corresponding structure of a nonsmooth stalk exhibits sharp edges at the transition regions between the bilayer and tether parts. In addition to that, a corner is formed at each of the two distal monolayers. We discuss the mechanism how membrane edges reduce the energy of fusion stalks.     

Cholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking

A variety of signal transduction pathways including PI turnover, MAP kinase activation, and PI 3-kinase activation have been shown to be affected by changes in cellular cholesterol content. However, no information is available regarding the locus (or loci) in the pathways that are susceptible to modulation by cholesterol. We report here that depletion of cholesterol with methyl-beta-cyclodextrin increases cell surface (125)I-EGF binding by approximately 40% via a mechanism that does not involve externalization of receptors from an internal pool. Cholesterol depletion also enhances in vivo EGF receptor autophosphorylation 2-5-fold without altering the rate of receptor dephosphorylation. In vitro kinase assays, which are done under conditions where phosphotyrosine phosphatases are inhibited and receptor trafficking cannot occur, demonstrate that treatment with methyl-beta-cyclodextrin leads to an increase in intrinsic EGF receptor tyrosine kinase activity. EGF receptors are localized in cholesterol-enriched lipid rafts but are released from this compartment upon treatment with methyl-beta-cyclodextrin. These data are consistent with the interpretation that localization to lipid rafts partially suppresses the binding and kinase functions of the EGF receptor and that depletion of cholesterol releases the receptor from lipid rafts, relieving the functional inhibition of the receptor. Cholesterol depletion also inhibits EGF internalization and down-regulation of the EGF receptor, and this likely contributes to the enhanced ability of EGF to stimulate downstream signaling pathways such as the activation of MAP kinase.     

The effects of lysosomotropic agents on normal and INCL cells provide further evidence for the lysosomal nature of palmitoyl-protein thioesterase function

Fatty acylation of proteins on cysteine residues is a common post-translational modification that plays roles in protein-membrane and protein-protein interactions. Recently, we described a lysosomal palmitoyl-protein thioesterase that removes long-chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in palmitoyl-protein thioesterase results in a human lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis (INCL), which primarily affects the central nervous system. The pathological hallmark of the disorder is the accumulation of granular osmiophilic deposits (GROD) that resemble lipofuscin, or aging pigment. In previous work, we have shown that [35S]cysteine-labeled lipid thioesters derived from fatty acylated proteins accumulate in cultured cells derived from palmitoyl-protein thioesterase-deficient patients. In the present work, we show that the lipid cysteine thioesters accumulate in the lysosomal fraction, and we further show that the appearance of these compounds in the organic phase is blocked by inhibitors of lysosomal proteolysis, demonstrating through biochemical means the lysosomal nature of the site of palmitoyl-protein thioesterase action. Furthermore, substrates for palmitoyl-protein thioesterase accumulate even in normal cells after leupeptin or chloroquine treatment. This was demonstrated by subjecting extracts of treated cells to exhaustive proteolysis to release protein-bound cysteine lipid for analysis. Cysteamine, a lysosomotropic drug recently proposed for the treatment of INCL, was found to have effects similar to leupeptin and chloroquine, suggesting that its mechanism of action may be more complex than previously understood.     

Functional analysis of mouse hepatocytes differing in DNA content: volume,receptor expression,and effect of IFNgamma

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule‐1 (ICAM‐1, CD54) or IFNγR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNγ)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM‐1 increases with increasing DNA content and IFNγR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNγ‐induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro. J. Cell. Physiol. 191: 138–144, 2002. © 2002 Wiley‐Liss, Inc.     

The kinesin-related protein MCAK is a microtubule depolymerase that forms an ATP-hydrolyzing complex at microtubule ends

MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.     

Combinatorial discovery of tumor targeting peptides using phage display

Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.     

Function,diversity, and evolution of signal transduction in prokaryotes

Major areas covered at the Bacterial Locomotion and Signal Transduction (BLAST) meeting included the clustering of chemoreceptors and its significance to signal amplification, organelle biogenesis, motility, developmental responses mediated by "chemotaxis" operons, and advances in two-component signaling mechanisms.