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Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616–8741 and a Cardiovascular Research Institute and b Department of Anatomy, University of California, San Francisco, CA 94143–0130
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INTRODUCTION |
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 TOP INTRODUCTION REFERENCES |
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Dr. Stephen Young and associates discuss genetic engineering experiments with the human apolipoprotein B (apoB)4gene in mice. Initially driven by questions about atherogenesis, their efforts rapidly revealed new structure-function relationships for apoB-containing lipoprotein metabolism. Perhaps their most surprising discovery was that the human heart, traditionally recognized as a target for apoB-lipoproteins, assembles and secretes its own apoB-containing lipoproteins, albeit mostly of LDL size.
Dr. Gregory Shelness and co-authors describe exciting new connections between apoB protein turnover and lipoprotein assembly processes. Experiments designed to dissect very early events of VLDL assembly in the rough endoplasmic reticulum define the interaction of microsomal triacylglycerol transfer protein with the globular amino terminal domain of apoB. This protein-protein interaction appears to be necessary for the early proper folding of apoB before its recruitment of core-lipids. Fundamental questions concerning the topography of apoB in the rough endoplasmic reticulum, of whether or not transmembrane apoB exists in native lipoprotein secreting cells, and if and how cytosolic proteosome-mediated degradation of apoB occurs, remain technically difficult to investigate and hence, controversial.
Dr. Jan Borén and colleagues discuss evidence, derived from pulse-chase studies of apoB isoforms in cultured hepatoma cells (human and rat), that supports the hypothesis that both apoB48 and apoB100 undergo two steps of core-lipidation during VLDL assembly. The molecular mechanisms of the core-lipidation steps of apoB in hepatic VLDL assembly remain an important but unresolved issue. Another important unresolved issue is the mechanism of action of Brefeldin-A. This compound has proved to be a key metabolic reagent because when used in low concentrations, it selectively dissociates the second step of VLDL assembly from first-step assembly processes. Suggestions by Dr. Borén et al. that novel protein chaperons are involved in second-step assembly events hold promise for resolution of these issues.
Dr. Rosemary Walzem and collaborators discussed the unique apoB100 biology of birds wherein estrogen stimulates VLDLy (yolk-targeted) secretion approximately fivefold by the liver of the laying hen. Intriguingly, these nascent VLDLy particles are reduced in diameter by one half in spite of surplus liver triacylglycerol. Specific changes in the physical properties of VLDLy are shown to be essential for optimal yolk formation. Ultrastructural studies of avian kidney revealed nascent generic VLDL assembly and secretion by the proximal tubule epithelium. These new studies provoked the hypothesis that these particles may ensure adequate delivery of triacylglycerol fatty acids and perhaps other nutrients to somatic tissues during egg laying.
Drs. Joachim Herz and Robert Farese present their new research that provides genetic and molecular explanations for old observations related to teratogenic effects of cholesterol deficiencies. Common new threads in developmental biology link cholesterol availability to proper embryonic growth and brain development. Yolk sac VLDL secretion and members of the LDL-receptor gene family are pivotal participants, and each is required for normal embryonic development, particularly the maturation of the central nervous system.
As is characteristic of virtually all innovative advances in science, the new concepts brought to light by each speaker's discussion raise more questions than answers. The primary objective for this symposium was to highlight our expanding understanding of the role of apoB-containing lipoproteins in normal tissue functions. What has emerged is a wealth of new information regarding relationships among apoB protein structure, lipoprotein particle assembly events, and subsequent lipoprotein particle metabolism. Some information was obtained through application of the powerful techniques of genetic engineering. These approaches, in conjunction with detailed biochemical and ultrastructural analyses, are providing unprecedented new insights into apoB-containing lipoprotein assembly events and particle metabolism. It is becoming more apparent that apoB-containing lipoproteins can serve as vehicles for specialized lipid transport. It is notable that the apoB-containing lipoprotein ligands and their attendant receptor systems play fundamental roles in nutrient delivery in reproductive biology. Finally, cholesterol has become an unexpectedly important nutrient in early embryonic development, with particular importance for central nervous system development.
Several presentations described how changes in the primary structure of
apoB changed quantitative or qualitative aspects of subsequent
lipoprotein particle assembly. Together with apoB, microsomal
triglyceride transfer protein (MTP) is only the second protein with a
documented role that is essential for apoB-containing lipoprotein
assembly. This may not be the case by the time these proceedings are
published. Dr. Borén and his colleagues mention a number of
partially sequenced putative chaperones whose functions in VLDL
assembly are being studied. Dr. Borén was unexpectedly prevented
from speaking at our symposium, but graciously reviewed the work that
he and his colleagues would have presented. In his absence, Dr. Robert
V. Farese, Jr. presented very recent work from his laboratory regarding
the cloning of two hepatic acyl-cholesterol acyl transferases (ACAT)
and one diacylglycerol acyl transferase (DGAT), enzymes that may be
directly involved in VLDL particle assembly (Cases, et al. 1998a and 1998b
).
Finally, we have chosen to try to articulate important or intriguing research question(s), elicited from each of the speakers' discussions, in the order presented in the symposium.
1) If apoB-containing lipoprotein secretion is of physiologic relevance for the optimal function of the human heart, would defective or absent apoB expression in cardiac myocytes cause heart pathophysiologies, perhaps some that may underlie unusual forms of human congenital idiopathic cardiomyopathies?
2) Data were presented to show that early protein-protein disulfide interactions between MTP and the globular amino terminal region of apoB are critical for apoB assembly into VLDL. Do either MTP or apoB interact physically and functionally with recently cloned ACAT and DGAT enzymes? The putative catalytic domains of some of these acyltransferases have been predicted to lie within the lumen of the endoplasmic reticulum, a result that suggests to some researchers that these proteins have specific roles in VLDL assembly. If so, do they core-lipidate ß-sheet domains of both isoforms of apoB?
3) Several entirely different experimental settings support the hypotheses that both apoB48 and apoB100 undergo two separable steps for full core-lipidation. Burning questions for future research are as follows: Do both apoB isoforms undergo the same second-step core-lipidation process? What are the identities of putative second-step particle assembly proteins, and are there specific chaperones that facilitate the second-step mechanism?
4) By what biochemical mechanism does estrogen reduce nascent VLDLy diameters by one half in laying hen hepatocytes in the presence of surplus liver triglycerides? Does the resistance of VLDLy to lipoprotein lipase relate to the unique expression of apoB by the kidney of birds and that organ's secretion of generic VLDL instead of VLDLy in the presence of estrogen? Do renal VLDL assembly and secretion comprise a general mechanism to conserve urinary filtrate fatty acids, and what is the metabolic fate of kidney-derived generic VLDL nutrients?
5) Many experimental roads now lead to the conclusion that
cholesterol availability is crucial for normal embryonic development,
particularly that of the central nervous system. However, it is not
known what specific cellular events or processes are sensitive to the
delivery of this sterol to the developing brain. Some insight was
gained when it was demonstrated that hedgehog proteins undergo an
intramolecular autoprocessing reaction that requires the covalent
addition of cholesterol to generate the mature hedgehog signaling
molecule (Porter et al. 1996
). Since the time of this symposium, this
same group presented evidence for a second site of cholesterol
sensitivity within target tissues (Cooper et al. 1998
), possibly acting
through the sterol sensing domain within the Patched protein regulator
of Sonic hedgehog response. But why do defects in apoB-mediated
cholesterol delivery affect one region of the brain, whereas defects in
de novo cholesterol synthesis affect another? Where does vitamin E,
whose deficiency leads to similar early neurodevelopmental defects, fit
within this complex scenario of cholesterol?
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FOOTNOTES |
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1 Presented at the symposium "Assembly and
Physiology of Apolipoprotein B-Containing Lipoproteins It's Not Just
for Heart Disease Anymore!" as part of Experimental Biology 98, April
18–22, 1998, San Francisco, CA. The symposium was sponsored by the
Energy and Macronutrient Research Interest Section of the American
Society for Nutritional Sciences, the Egg Nutrition Center, the
American Heart Association-Western States Affiliate, Merck Research
Laboratories, Bristol-Meyers Squibb Pharmaceutical Research Institute
and Parke-Davis Pharmaceutical Research. Published as a supplement to
The Journal of Nutrition. Guest editors for this supplement
were Rosemary L. Walzem, University of California, Davis, and Robert L.
Hamilton, University of California, San Francisco, CA.
2 Supported by American Heart
Association-Grant-in-Aid #97–218 (R.L.H. and R.L.W.), U.S. Department
of Agriculture Formula Funds provided through the School of Veterinary
Medicine (R.L.W.) and UCSF-ASFRG (R.L.H).
3 Abbreviations used: ACAT, acyl-cholesterol acyl
transferase; apo, apolipoprotein; DCAT, diacylglycerol acyl
transferase; MTP, microsomal transfer protein; VLDLy, yolk-targeted
VLDL.
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REFERENCES |
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TOPINTRODUCTIONREFERENCES |
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1.
Cases S. C., Novak S., Zheng Y.-W., Myers H. M., Lear S. R., Sande E., Welch C. B., Lusis A. J., Spencer T. A., Krause B. R., Erickson S. E., Farese R. V., Jr. ACAT-2, a second mammalian acyl CoA:cholesterol acyltransferaseits cloning, expression, and characterization. J. Biol. Chem. 1998;273:26755-26764.
2.
Cases S., Smith S. J., Zheng Y-W., Myers H. M., Lear S. R., Sande E., Novak S., Collins C., Welch C. B., Lusis A. L., Erickson S. E., Farese R. V.. Identification of a gene encoding an acyl synthesis. Proc. Natl. Acad. Sci, USA 1998;95:13018-13023.
3.
Cooper M. K., Porter J. A., Young K. E., Beachy P.A.. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science (Washington, DC) 1998;280:1603-1607.
4.
Porter J. A., Young K. E., Beachy P. A.. Cholesterol modification of hedgehog signaling proteins in animal development. Science (Washington, DC) 1996;274:255-259.
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