Getting a handle on "good" cholesterol with the high-density lipoprotein receptor.
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Animals↗
Biomedical subjects
M Krieger
Publications and source records attributed to M Krieger.
Structure modelling and site-directed mutagenesis of the rat aromatic L-amino acid pyridoxal 5'-phosphate-dependent decarboxylase: a functional study.
The pyridoxal-5'-phosphate-dependent enzymes (B6 enzymes) are grouped into three main families named alpha, beta, and gamma. Proteins in the alpha and gamma families share the same fold and might be distantly related, while those in the beta family exhibit specific structural features. The rat aromatic L-amino acid decarboxylase (AADC; EC(4.1.1.28)) catalyzes the synthesis of two important neurotransmitters: dopamine and serotonin. It binds the cofactor pyridoxal-5'-phosphate and belongs to the alpha family. Despite the low level of sequence identity (approximately 10%) shared by the rat AADC and the sequences of the enzymes belonging to the B6 enzymes family, including the known three-dimensional structures, a multiple sequence alignment was deduced. A model was built using segments belonging to seven of the eleven known structures. By homology, and based on knowledge of the biochemistry of the aspartate aminotransferase, structurally and functionally important residues were identified in the rat AADC. Site-directed mutagenesis of the conserved residues D271, T246, and C311 was carried out in order to confirm our predictions and highlight their functional role. Mutation of D271A and D271N resulted in complete loss of enzyme activity, while the D271E mutant exhibited 2% of the wild-type activity. Substitution of T246A resulted in 5% of the wild-type activity while the C311A mutant conserved 42% of the wild-type activity. A functional model of the AADC is discussed in view of the structural model and the complementary mutagenesis and labelling studies.
Amino Acid Sequence↗
Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation.
The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by (1)H NMR as a 4-methyl-24, 25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.
Amino Acid Sequence↗
Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.
The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can influence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI/apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (i) plasma lipoprotein levels and/or structure, (ii) cholesterol flux into or out of peripheral tissues (ovary, aortic wall), and (iii) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI/apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.
Animals↗
Expression cloning of LDLB, a gene essential for normal Golgi function and assembly of the ldlCp complex.
The Chinese hamster ovary (CHO) cell mutants ldlC and ldlB, which exhibit almost identical phenotypes, define two genes required for multiple steps in the normal medial and trans Golgi-associated processing of glycoconjugates. The LDLC gene encodes ldlCp, an approximately 80-kDa protein, which in wild-type, but not ldlB, cells associates reversibly with the cytoplasmic surface of the Golgi apparatus. Here, we have used a retrovirus-based expression cloning system to clone a murine cDNA, LDLB, that corrects the pleiotropic mutant phenotypes of ldlB cells. The corresponding mRNA was not detected in ldlB mutants. LDLB encodes an approximately 110-kDa protein, ldlBp, which lacks homology to known proteins and contains no common structural motifs. Database searches identified short segments of homology to sequences from Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans, and the essentially full-length homologous human sequence (82% identity); however, as was the case for ldlCp, no homologue was identified in Saccharomyces cerevisiae. We have found that in wild-type cell cytosols, ldlCp is a component of an approximately 950-kDa "ldlCp complex," which is smaller, approximately 700 kDa, in ldlB cytosols. Normal assembly of this complex is ldlBp-dependent and may be required for Golgi association of ldlCp and for the normal activities of multiple luminal Golgi processes.
3T3 Cells↗
Influence of the HDL receptor SR-BI on atherosclerosis.
The scavenger receptor class B, type I (SR-BI) is an HDL receptor that mediates selective cholesterol uptake from HDL to cells. In rodents, SR-BI has a critical influence on plasma HDL-cholesterol concentration and structure, the delivery of cholesterol to steroidogenic tissues, female fertility, and biliary cholesterol concentration. SR-BI can also serve as a receptor for non-HDL lipoproteins and appears to play an important role in reverse cholesterol transport. Recent studies involving the manipulation of SR-BI expression in mice, either using adenovirus-mediated or transgenic hepatic overexpression or using homologous recombination for complete functional ablation, indicate that the expression of SR-BI protects against atherosclerosis. If SR-BI has a similar activity in humans, it may become an attractive target for therapeutic intervention.
Animals↗
Charting the fate of the "good cholesterol": identification and characterization of the high-density lipoprotein receptor SR-BI.
Risk for cardiovascular disease due to atherosclerosis increases with increasing concentrations of low-density lipoprotein (LDL) cholesterol and is inversely proportional to the levels of high-density lipoprotein (HDL) cholesterol. The receptor-mediated control of plasma LDL levels has been well understood for over two decades and has been a focus for the pharmacologic treatment of hypercholesterolemia. In contrast, the first identification and characterization of a receptor that mediates cellular metabolism of HDL was only recently reported. This receptor, called scavenger receptor class B type I (SR-BI), is a fatty acylated glycoprotein that can cluster in caveolae-like domains on the surfaces of cultured cells. SR-BI mediates selective lipid uptake from HDL to cells. The mechanism of selective lipid uptake is fundamentally different from that of classic receptor-mediated endocytic uptake via coated pits and vesicles (e.g. the LDL receptor pathway) in that it involves efficient receptor-mediated transfer of the lipids, but not the outer shell proteins, from HDL to cells. In mice, SR-BI plays a key role in determining the levels of plasma HDL cholesterol and in mediating the regulated, selective delivery of HDL-cholesterol to steroidogenic tissues and the liver. Significant alterations in SR-BI expression can result in cardiovascular and reproductive disorders. SR-BI may play a similar role in humans; thus, modulation of its activity may provide the basis of future approaches to the treatment and prevention of atherosclerotic disease.
Animals↗
QEEG and traumatic brain injury: rebuttal of the American Academy of Neurology 1997 report by the EEG and Clinical Neuroscience Society.
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Brain Injuries↗
The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.
The class B type I scavenger receptor, (SR-BI), is a member of the CD36 superfamily of proteins and is a physiologically relevant, high affinity cell surface high density lipoprotein (HDL) receptor that mediates selective lipid uptake. The mechanism of selective lipid uptake is fundamentally different from that of classic receptor-mediated uptake via coated pits and vesicles (e.g. the low density lipoprotein receptor pathway) in that it involves efficient transfer of the lipids, but not the outer shell proteins, from HDL to cells. The abilities of SR-BI and CD36, both of which are class B scavenger receptors, to bind HDL and mediate cellular uptake of HDL-associated lipid when transiently expressed in COS cells were examined. For these experiments, the binding of HDL to cells was assessed using either 125I- or Alexa (a fluorescent dye)-HDL in which the apolipoproteins on the surface of the HDL particles were covalently modified. Lipid transfer was measured using HDL noncovalently labeled by the fluorescent lipid 1,1'-dioctadecyl-3,3, 3',3'-tetramethylindocarbocyanine perchlorate. Although both mSR-BI and human CD36 (hCD36) could mediate the binding of HDL in a punctate pattern across the surfaces of cells, only mSR-BI efficiently mediated the transfer of lipid to the cells. Analysis of point mutants established that the major sites of fatty acylation of mSR-BI are Cys462 and Cys470 and that fatty acylation is not required for receptor clustering, HDL binding, or efficient lipid transfer. Generation of mSR-BI/hCD36 domain swap chimeras showed that the differences in lipid uptake activities between mSR-BI and hCD36 were not due to differences between their two sets of transmembrane and cytoplasmic domains but rather result from differences in their large extracellular loop domains. These results show that high affinity binding to a cell surface receptor is not sufficient to ensure efficient cellular lipid uptake from HDL. Thus, SR-BI-mediated binding combined with SR-BI-dependent facilitated transfer of lipid from the HDL particle to the cell appears to be the most likely mechanism for the bulk of the selective uptake of cholesteryl esters from HDL to the liver and steroidogenic tissues.
Amino Acid Sequence↗
The "best" of cholesterols, the "worst" of cholesterols: a tale of two receptors.
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Animals↗
Immunolocalization of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, dopamine, and serotonin in the forebrain of Ambystoma mexicanum.
To improve basic knowledge about the neurochemical organization of the urodele brain, and to study discrepancies in the localization of monoaminergic markers, we immunohistochemically charted the distribution of four such markers (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine, and serotonin) in the axolotl (Ambystoma mexicanum) forebrain. Catecholaminergic and serotoninergic systems were found in similar locations to those seen in other Urodela. As seen in other vertebrates, the localization of the different monoaminergic markers reveals some inconsistencies. Cells that are exclusively tyrosine hydroxylase-immunoreactive are observed in the olfactory bulb, anterior olfactory nucleus/nucleus accumbens region, the epichiasmatic portion of the preoptic nucleus, and in the pars intercalaris thalami, whereas cells that are only labelled by aromatic L-amino acid decarboxylase are seen in the anterior olfactory nucleus/nucleus accumbens region, the bed nuclei of the anterior commissure, the posterior portion of the preoptic nucleus, the ventral hypothalamus, and the pars intercalaris thalami. The presence of cells solely serotonin (5-HT)-immunoreactive is suggested for the nucleus infundibularis dorsalis. Conversely, there were no areas that appeared to be exclusively immunoreactive for dopamine. Double-labelling for aromatic L-amino acid decarboxylase/tyrosine hydroxylase and aromatic L-amino acid decarboxylase/serotonin, together with cell counting, confirmed the existence of neurons that express only one monoaminergic marker in amphibian, supporting the hypothesis that these cells are universally present in the central nervous system of vertebrates.
Ambystoma↗
Temporal and spatial pattern of expression of the HDL receptor SR-BI during murine embryogenesis.
During rodent fetal development, maternal lipoproteins can be sources of cholesterol for the membrane synthesis required for tissue growth in the developing embryo and for steroid hormone production in the extraembryonic tissues. Although the mechanisms underlying the maternal-fetal lipoprotein cholesterol transport system are not well defined, the placenta and yolk sac seem to play major roles in this process, serving as functionally active interfaces between maternal circulation and the embryo. In rodents, the principal cholesterol transporter in the plasma is HDL, and the HDL receptor SR-BI is a physiologically important mediator of cholesterol uptake in adult liver and steroidogenic tissues. To begin to investigate SR-BI's role in maternal cholesterol uptake by the fetus, we used immunofluorescence microscopy to determine the pattern of SR-BI expression during murine embryogenesis. At day E7.5 in gestation, there was significant SR-BI expression in endothelial cells of the decidua, but little in intraembryonic and extraembryonic tissues. By day E8.5, there was a dramatic increase in SR-BI expression in the trophoblast cells which surround the developing embryo. Beginning at day E10, SR-BI was expressed in both the placenta and yolk sac. The expression in these extraembryonic tissues was correlated with significant uptake of fluorescent dye by the yolk sac visceral endodermal cells from DiI-labeled HDL injected into pregnant mice. Within the embryo proper, SR-BI expression appeared by day E14.5 at high levels in the adrenal gland. SR-BI expression was not detected in the embryonic liver through day E17.5 of gestation; however, it could be observed in neonatal livers. These findings suggest that SR-BI may play a role in the rodent maternal-fetal lipoprotein cholesterol transport system, supplying HDL cholesterol for either membrane or steroid hormone synthesis, or both.
Animals↗
Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells.
The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.
Adrenal Cortex↗
A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.
Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by approximately 31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.
Animals↗
Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels.
The risk of atherosclerosis, a leading cause of cardiovascular disease and death, is inversely related to plasma levels of high-density lipoprotein (HDL) cholesterol, although the mechanism of this protective effect is unclear. The class B scavenger receptor, SR-BI, is the first HDL receptor to be well defined at a molecular level and is a mediator of selective cholesterol uptake in vitro. It is expressed most abundantly in steroidogenic tissues, where it is coordinately regulated with steroidogenesis by adrenocorticotropic hormone (ACTH), human chorionic gonadotropin (hCG) and oestrogen, and in the liver, where its expression in rats is suppressed by oestrogen. Here we show that adenovirus-mediated, hepatic overexpression of SR-BI in mice on both sinusoidal and canalicular surfaces of hepatocytes results in the virtual disappearance of plasma HDL and a substantial increase in biliary cholesterol. SR-BI may directly mediate these effects by increasing hepatic HDL cholesterol uptake or by increasing cholesterol secretion into bile, or both. These results indicate that SR-BI may be important in hepatic HDL metabolism, in determining plasma HDL concentrations, and in controlling cholesterol concentrations in bile, and thus may influence the development and progression of atherosclerosis and gallstone disease.
Adenoviridae↗
Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.
The class B, type I scavenger receptor, SR-BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor-mediated endocytosis via clathrin-coated pits and vesicles. SR-BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR-BI (mSR-BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells. mSR-BI, an approximately 82-kDa glycoprotein, was initially synthesized with multiple high mannose N-linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [3H]palmitate and [3H]myristate demonstrated that mSR-BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol- and glycolipid-rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR-BI copurified with caveolin-1, a constituent of caveolae; and immunofluorescence microscopy demonstrated that mSR-BI colocalized with caveolin-1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR-BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR-BI-mediated transfer of lipids between lipoproteins and cells.
Acylation↗
Expression, purification, and characterization of rat aromatic L-amino acid decarboxylase in Escherichia coli.
A cDNA encoding rat aromatic L-amino acid decarboxylase (AADC) was successfully expressed in Escherichia coli using a T7 RNA polymerase expression system. Two types of expression vectors were tested and revealed to be equivalent to produce AADC. The enzyme was purified in both cases. The ratio of recovery of the pure active recombinant protein was better when the purification of the protein was made easier by addition of a short His-Tag at the C-terminal moiety of AADC, as achieved in the case of pET-20b+ vector expression. Spectral characteristics of the bound pyridoxal-5'-phosphate were essentially identical to the spectral properties of rat AADC. Kinetic constants Km and Vmax of recombinant AADC for the natural substrates L-dihydroxyphenylalanine and 5-hydroxytryptamine were 0.14 mM and 8444 U/mg, and 0.066 mM and 1813 U/mg, respectively. These values were in good agreement with previously reported values for AADC of the rat and other mammalian species.
5-Hydroxytryptophan↗
Active rat aromatic-L-amino acid decarboxylase as a fusion protein in Escherichia coli.
The DNA sequence encoding rat aromatic-L-amino acid decarboxylase (AADC) was inserted into the Escherichia coli (E. coli) expression vector pMAL-c2. This clone produced a fusion protein able to catalyze the conversion of L-DOPA to dopamine. After purification and treatment of the fusion protein by factor Xa (FXa), an enzymatically active form of the enzyme resistant to FXa was isolated. It showed kinetic constants, Vmax, K(m) and enzymatic properties very similar to those obtained previously for the mammalian enzyme. This method for obtaining active AADC appears to be useful for initiating the study of the catalytic activity of this protein because it permitted the rapid isolation and the stabilization of an active form of the enzyme.
Animals↗