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Godfrey S Getz

Publications and source records attributed to Godfrey S Getz.

At least 19 recordsLinked to original sources
PubMed2006

Self-assembly of HEK cell-secreted ApoE particles resembles ApoE enrichment of lipoproteins as a ligand for the LDL receptor-related protein.

Recent studies have shown that the lipidation and assembly state of apolipoprotein E (apoE) determine receptor recognition and amyloid-beta peptide (Abeta) binding. We previously demonstrated that apoE secreted by HEK cells stably expressing apoE3 or apoE4 (HEK-apoE) binds Abeta and inhibits Abeta-induced neurotoxicity by an isoform-specific process that requires apoE receptors. Here we characterized the structure of HEK-apoE assemblies and determined their receptor binding specificity. By chromatography, HEK-apoE elutes in high molecular mass fractions and is the size of plasma HDL, consistent with a multiprotein assembly. No lipid was associated with these apoE assemblies. Several methods for analyzing receptor binding indicate that HEK-apoE is a ligand for low-density lipoprotein (LDL) receptor-related protein (LRP) but not the LDL receptor. This suggests that self-assembly of apoE may induce a functional conformation necessary for binding to LRP. Our results indicate that, in addition to lipid content, the assembly state of apoE influences Abeta binding and receptor recognition.

Animals↗
PubMed2005

Diet and murine atherosclerosis.

Lipid-enriched diets are often used to induce or accelerate the rate of atherosclerotic lesion development in murine models of atherosclerosis. It appears that the induction of persistent hypercholesterolemia to levels > or approximately to 300 mg/dL is required for the development of experimental atherosclerosis in the mouse. A variety of different diets have been used that vary in the level of cholesterol, the level and type of fatty acid, and the absence or presence of cholate. Each of these components as well as the protein source has been shown to influence lipoprotein level and/or atherosclerosis, with dietary cholesterol being the major proatherogenic component. In some instances the effects of these components on the expression of hepatic genes relevant to lipid homeostasis has been observed. An appreciation of the effect of the differences in diet composition on these processes is important to compare results from different atherosclerosis studies, so the composition of the diets used should always be reported or referenced. Cholate should not be used unless its effects are being specifically investigated.

Animal Feed↗
PubMed2005

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination.

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.

Animals↗
PubMed2005

Site-specific influence of polyunsaturated fatty acids on atherosclerosis in immune incompetent LDL receptor deficient mice.

Polyunsaturated fatty acids (PUFA) are thought to influence plasma lipid levels, atherosclerosis, and the immune system. In this study, we fed male LDL receptor deficient (LDLR(-/-)) mice and immune incompetent LDLR(-/-) RAG2(-/-) mice diets containing predominantly saturated fats (milk fat) or PUFA (safflower oil) to determine if the response to diet was influenced by immune status. Relative to milk fat diet, plasma lipid and VLDL levels in both the LDLR(-/-) and LDLR(-/-) RAG2(-/-) mice fed safflower oil diet were lower, suggesting that the primary effect of PUFA on plasma lipids was not due to its inhibition of the immune system. Neither diet nor immune status influenced hepatic triglyceride production and post-heparin lipase activity, suggesting that the differences in triglyceride levels are due to differences in rates of catabolism of triglyceride-rich lipoproteins. While both diets promoted atherogenesis, both aortic root and innominate artery atherosclerosis in LDLR(-/-) mice was less in safflower oil fed animals. In contrast, a site-specific effect of PUFA was observed in the immune incompetent LDLR(-/-) RAG2(-/-). In these mice, aortic root atherosclerosis, but not innominate artery atherosclerosis, was less in PUFA fed animal. These results suggest that PUFA and the immune system may influence innominate artery atherosclerosis by some overlapping mechanisms.

Animals↗
PubMed2005

Helix-turn-helix peptides that form alpha-helical fibrils: turn sequences drive fibril structure.

Models of apolipoprotein A-I (apo A-I), the main protein of high-density lipoprotein, predict that it contains 10 amphiphilic, alpha-helical segments connected by turns. We synthesized four peptides with two identical 18-residue, amphiphilic, alpha-helical segments (Anantharamaiah, G. M., et al. (1985) J. Biol. Chem. 260, 10248-10255) connected by putative turn sequences from apo A-I: (1) Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2, (2) Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2, (3) Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2, and (4) Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2. Surprisingly, peptides 1-3 formed fibrils after incubation (37 degrees C, 10 mM sodium phosphate, pH 7.60), but in contrast to beta-sheet amyloid fibrils, these did not bind thioflavin T and they induced a blue shift in the spectrum of Congo red. CD (peptides 1-3) and FTIR (peptides 1 and 2) of the fibrils showed significant alpha-helical character. Synchrotron X-ray fiber diffraction on a magnetically aligned sample of 1 confirmed the alpha-helical character in the fibrils and indicated that the helical axes are oriented perpendicular to the fibril axis. In contrast, peptide 4, containing two Gly residues but no Pro in the turn, formed only a small amount of nonfibrillar precipitate after prolonged incubation. Peptide 4P (peptide 4 with a Pro in place of the central Ala) and peptide 5, containing a PEG block in lieu of the central turn, were similar to peptide 4 in not forming fibrils, possibly because the region linking the helices was unstructured. These studies indicate that varying turn sequences between longer amphiphilic alpha-helical segments can drive the structure of fibrils.

Amino Acid Sequence↗
PubMed2005

Chemical colloids versus biological colloids: a comparative study for the elucidation of the mechanism of protein fiber formation.

Fiber formation from murine serum amyloid A1 (SAA) was compared to the linear aggregation and fiber formation of colloidal gold particles. Here we report the similarities of these processes. Upon incubation with acetic acid, SAA misfolds and adopts a new conformation, which we termed saa. saa apparently is less soluble than SAA in aqueous solution; it aggregates and forms nucleation units and then fibers. The fibers appear as a string of the nucleation units. Additionally, an external electric field promotes saa fiber formation. These properties of saa are reminiscent of colloidal gold formation from gold ions and one-dimensional aggregation of the gold colloids. Colloidal gold particles were also found to be capable of aggregating one-dimensionally under an electric field or in the presence of polylysine. These gold fibers resembled in structure that of saa fibers. In summary, protein aggregation and formation of fibers appear to follow the generalized principles derived in colloidal science for the aggregation of atoms and molecules, including polymers such as polypeptides. The analysis of colloidal gold formation and of one-dimensional aggregation provides a simple model system for the elucidation of some aspects of protein fiber formation.

Animals↗
PubMed2004

Thematic review series: the immune system and atherogenesis. Immune function in atherogenesis.

In this overview to a new thematic series on the immune system and atherogenesis, I provide a very brief summary of current conceptions of atherogenesis, of the innate and adaptive immune systems, and of the participation of the latter in atherogenesis, with particular emphasis on studies of the involvement of the immune system in atherosclerosis reported in the last 2 years. This is followed by a short outline of the eight reviews that will make up this thematic series. The overview is concluded with some caveats that should be considered in the analysis of atherosclerosis in experimental animals.

Animals↗
PubMed2004

Requirement of different mitochondrial targeting sequences of the yeast mitochondrial transcription factor Mtf1p when synthesized in alternative translation systems.

Mitochondrial (mt) translocation of the nuclearly encoded mt transcription factor Mtf1p appears to occur independent of a cleavable presequence, mt receptor, mt membrane potential or ATP [Biswas and Getz (2002) J. Biol. Chem. 277, 45704-45714]. To understand further the import strategy of Mtf1p, we investigated the import of the wild-type and N-terminal-truncated Mtf1p mutants synthesized in two different in vitro translation systems. These Mtf1p derivatives were generated either in the RRL (rabbit reticulocyte lysate) or in the WGE (wheat germ extract) translation system. Under the in vitro import conditions, the RRL-synthesized full-length Mtf1p but not the N-terminal-truncated Mtf1p product was efficiently imported into mitochondria, suggesting that the N-terminal sequence is important for its import. On the other hand, when these Mtf1p products were generated in the WGE system, surprisingly, the N-terminal-truncated products, but not the full-length protein, were effectively translocated into mitochondria. Despite these differences between the translation systems, in both cases, import occurs at a low temperature and has no requirement for a trypsin-sensitive mt receptor, mt membrane potential or ATP hydrolysis. Together, these observations suggest that, in the presence of certain cytoplasmic factors (derived from either RRL or WGE), Mtf1p is capable of using alternative import signals present in different regions of the protein. This appears to be the first example of usage of different targeting sequences for the transport of a single mt protein into the mt matrix.

Animals↗
PubMed2004

Autoantibodies to OxLDL fail to alter the clearance of injected OxLDL in apolipoprotein E-deficient mice.

This study tests the hypothesis that autoantibodies to oxidation epitopes on oxidized LDL (OxLDL) promote the clearance of OxLDL from the plasma. Human LDL (hLDL) was injected into immune-competent apolipoprotein E-deficient (apoE(-/-)) mice and immune-deficient apoE(-/-)/recombination-activating gene-deficient mice that lack mature T and B cells and thus antibodies. There was a progressive decrease in human apoB-100 in the plasma in all mice, but the rate of clearance was not greater in the immune-competent mice than in the immune-deficient mice. Interestingly, oxidized phospholipid (OxPL) epitopes as detected by the EO6 antibody on the hLDL increased over time, suggesting de novo oxidation of the LDL or transfer of OxPL to the particles. Because the native LDL was not extensively modified, we also examined the clearance of copper OxLDL. Although the extensively OxLDL was cleared faster than the native LDL, there was no difference in the rate of clearance as a function of immune status. There appeared to be some transfer of OxPL to the endogenous murine LDL. Together, these results suggest that oxidation-specific antibodies do not participate to any great extent in the clearance of OxLDL from plasma. However, it is possible that such antibodies may bind to oxidation epitopes and modulate lesion formation within the vessel wall.

Animals↗
PubMed2004

Paraoxonase, a cardioprotective enzyme: continuing issues.

PURPOSE OF REVIEW: The paraoxonase family consists of three members (PON1, PON2 and PON3) that share structural properties and enzymatic activities, among which is the ability to hydrolyze oxidized lipids in LDL. The exact function of the different family members is not clear although the conservation among the individual family members across species suggests a strong evolutionary pressure to preserve these functional differences. The purpose of this review is to highlight several problems with respect to the mechanism of action of paraoxonase and differences between the family members that merit further study. RECENT FINDINGS: PON1 transgenic mice are at lower risk for atherosclerosis, which is consistent with PON1 gene knockout studies in mice and human genetic polymorphism studies. The exact mechanism by which paraoxonase is cardioprotective is not clear, although it is likely to be related to its antioxidant properties especially on LDL. PON1 levels are influenced by a variety of environmental factors, including statins and cytokines. The preferential association of PON1 with HDL is mediated in part by its signal peptide and by desorption from the plasma membrane of expressing cells by HDL or phospholipid. Apolipoprotein A-I is not necessary for PON1 association with HDL, but its activity is stabilized in the presence of the apolipoprotein. Only in the absence of both lecithin cholesterol acyltransferase and apolipoprotein E is paraoxonase associated with non-HDL lipoproteins. The displacement of paraoxonase by serum amyloid A may explain in part the proinflammatory nature of HDL in the acute phase. The mechanism by which PON3 associates with HDL has not been studied. In addition to the ability to hydrolyze oxidized lipids in LDL, paraoxonase also alters the oxidative state of macrophages. Exogenous PON1 is able to reverse the oxidative stress in macrophages in aged apolipoprotein E deficient and PON1 deficient mice. The increase in oxidative stress in macrophages from PON1 deficient mice occurs despite the expression of PON2 and PON3 in macrophages. PON1 has recently been shown to contain phospholipase A2 activity, with the subsequent release of lysophosphatidylcholine that influences macrophage cholesterol biosynthesis. SUMMARY: PON1 mass and activity in the plasma significantly influence the risk of developing cardiovascular disease. This is likely mediated by its antioxidation properties on LDL and/or macrophages. The precise mechanism by which this HDL associated protein prevents or attenuates oxidation of LDL and the oxidative stress of macrophages remains to be clarified. The role of PON2 and PON3 in atherosclerosis and their antioxidant properties with respect to LDL and macrophages also merit further investigation.

Animals↗
PubMed2003

Site specificity of atherosclerosis: site-selective responses to atherosclerotic modulators.

Atherosclerosis is a complex disease process that affects very specific sites of the vasculature. It is recognized that hemodynamic forces are largely responsible for dictating which vascular sites are either susceptible or resistant to developing atherosclerosis. In addition, a number of systemic and local factors also modulate the pathogenesis of the disease. By studying the development of atherosclerosis in mice, investigators have gained insights into the molecular mechanisms of this disease, although studies have largely focused on a single vascular site. Here, we review those recent studies in which vascular site-specific effects on atherosclerosis were reported when more than 1 site was examined. We assess the hypothesis that regional differences in the hemodynamic profile prime the endothelial phenotype to respond distinctly to such systemic risk factors as hypercholesterolemia, genetics, immune status, gender, and oxidative stress. Because a given treatment may differentially affect the development of atherosclerotic lesions throughout the vasculature, the sites chosen for study are critically important. By accounting for the complex interplay of factors that may operate at these different sites, a more complete understanding of the overriding mechanisms that control the initiation and progression of the atherosclerotic lesion may be realized.

Adolescent↗
PubMed2003

Influence of apoA-I and apoE on the formation of serum amyloid A-containing lipoproteins in vivo and in vitro.

Serum amyloid A (SAA) circulates bound to HDL3 during the acute-phase response (APR), and recent evidence suggests that elevated levels of SAA may be a risk factor for cardiovascular disease. In this study, SAA-HDL was produced in vivo during the APR and without the APR by injection of an adenoviral vector expressing human SAA-1. SAA-HDL was also produced in vitro by incubating mouse HDL with recombinant mouse SAA and by SAA-expressing cultured hepatoma cells. Whether produced in vivo or in vitro, SAA-HDL floated at a density corresponding to that of human HDL3 (d 1.12 g/ml) separate from other apolipoproteins, including apolipoprotein A-I (apoA-I; d 1.10 g/ml) when either apoA-I or apolipoprotein E (apoE) was present. In the absence of both apoA-I and apoE, SAA was found in VLDL and LDL, with low levels in the HDL and the lipid-poor fractions suggesting that other HDL apolipoproteins are incapable of facilitating the formation of SAA-HDL. We conclude that SAA does not exist in plasma as a lipid-free protein. In the presence of HDL-associated apoA-I or apoE, SAA circulates as SAA-HDL with a density corresponding to that of human HDL3. In the absence of both apoA-I and apoE, SAA-HDL is not formed and SAA associates with any available lipoprotein.

Acute-Phase Reaction↗
PubMed2003

Lysine-phosphatidylcholine adducts in kringle V impart unique immunological and potential pro-inflammatory properties to human apolipoprotein(a).

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

Amino Acid Sequence↗
PubMed2003

Genetic background selectively influences innominate artery atherosclerosis: immune system deficiency as a probe.

OBJECTIVE: We sought to examine whether there is a site-specific effect on atherosclerosis of the absence of mature T and B cells caused by a recombination activating-gene deficiency in LDL receptor-deficient mice and whether this effect is influence by the extent of backcrossing to C57BL/6 mice. METHODS AND RESULTS: Male mice were fed atherogenic diets for 3 months. In strain 1 mice, in which approximately 93% of the genes were from C57BL/6 mice, the absence of mature T and B cells led to a significant reduction in atherosclerosis in both the aortic sinus and the innominate artery. In strain 2 mice, in which approximately 99+% of the genes were from C57BL/6 mice, immune system deficiency led to a site-specific effect on atherosclerosis, with a reduction in atherosclerosis in the aortic sinus but not in the innominate artery, similar to previous results obtained with apolipoprotein E-/- mice. All of the immune system-incompetent mice had lower plasma total and VLDL cholesterol levels regardless of strain or diet, indicating that differences in lipid levels were unlikely to be responsible for these site-specific effects of immune system deficiency. CONCLUSIONS: These results suggest that immune system deficiency has a site-specific effect on atherosclerosis that is sensitive to the genetic background of the mice.

Animals↗
PubMed2003

Serum paraoxonase: effect of the apolipoprotein composition of HDL and the acute phase response.

Genetic variations of paraoxonase (PON) correlate with HDL cholesterol and apolipoprotein A-I (apoA-I), suggesting antiatherogenic properties. Atherosclerosis occurs naturally in humans and rabbits but not in mice. We compared variations of PON arylesterase activity (PON AEase, phenylacetate substrate) in humans, rabbits, and mice. In humans and rabbits, >95% of PON AEase is HDL associated. In mice, about 30% of PON AEase is lipid poor. In the absence of apoA-I in mice, total PON AEase is reduced and >60% is lipid poor. PON AEase level and distribution is restored in apoA-I-/- mice injected with adenoviruses encoding human apoA-I and in transgenic mice expressing human apoA-I at a steady-state level. Thus, while apoA-I is not required for the HDL association of PON AEase, induced variations in apoA-I correlate with changes in HDL-associated, but not lipid-poor, PON AEase. PON AEase associates only with apoA-I- or apoE-containing HDL but not VLDL. In the absence of both apoA-I and apoE, PON AEase is all-lipid-poor. PON AEase is displaced from HDL by ultracentrifugation and following incubation with serum amyloid A. Variations in the PON distribution between HDL and lipid-poor fractions may have important consequences in its antioxidant activity and in atherogenesis.

Acute-Phase Reaction↗
PubMed2003

Lipoproteins produced by ApoE-/- astrocytes infected with adenovirus expressing human ApoE.

We have developed an astrocyte cell culture system that is attractive for the study of apoE structure and its impact on astrocyte lipoproteins and neuronal function. Primary astrocytes from apoE-/- mice were infected with adenovirus expressing apoE3 or apoE4 and the nascent lipoproteins secreted were characterized. The nascent apoE-containing astrocyte particles were predominantly the size of plasma high density lipoprotein (HDL). ApoE4, in contrast to apoE3, appeared to be distributed in two distinct lipoprotein peaks and the apoE4-containing lipoproteins contained significantly more radiolabeled triglyceride. On electron micrographs the astrocyte particles were both discoidal and spherical in shape with a prevalence of stacked discs in apoE3 particles, but single discs and larger spheres in apoE4 particles. The apoE4 discs were significantly wider than apoE3 discs. These properties of the astrocyte lipoproteins are similar to those obtained from apoE isoform transgenic mice. Astrocyte lipoproteins containing apoE3, but not apoE4, stimulated neurite outgrowth in Neuro-2a cells. These studies suggest that the isoform-specific effects of apoE lipoproteins may involve differences in particle size and composition. Finally we demonstrate the usefulness of this system by expressing a truncated apoE3 (delta202-299) mutant and show preliminary data indicating that a liver X receptor agonist promotes HDL output by the astrocytes without an increase in apoE in the media. This cell culture system is more flexible and allows for more rapid expression of apoE mutants.

Adenoviridae↗