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REGULATION OF VLDL TRAFFICKING BY ORP 10
Title
REGULATION OF VLDL TRAFFICKING BY ORP 10.
Creator
Wessels, Philip, Siddiqi, Shadab, University of Central Florida
Abstract / Description
Of the challenges facing the improvement of human health, none has taken the forefront quite like the endeavor to discover novel treatments for heart disease. As heart disease has now become the leading cause of death throughout the world , the medical community has made incredible strides in the mission to treat atherosclerosis which is the major contributor to heart disease. Very Low Density Lipoproteins (VLDL) are secreted by the liver and subsequently converted to Low Density Lipoproteins...
Show moreOf the challenges facing the improvement of human health, none has taken the forefront quite like the endeavor to discover novel treatments for heart disease. As heart disease has now become the leading cause of death throughout the world , the medical community has made incredible strides in the mission to treat atherosclerosis which is the major contributor to heart disease. Very Low Density Lipoproteins (VLDL) are secreted by the liver and subsequently converted to Low Density Lipoproteins (LDL). Many factors contribute to the narrowing of the arterial walls, however oxidized LDL is the main factor that leads to the deposition of plaque, leading to atherosclerosis pathologies. Recently, a main focus of research into atherosclerotic processes has been the synthesis and trafficking of VLDL in hepatocytes. The rate-limiting step for the secretion of VLDL from the liver has been determined to be the transport of VLDL from the endoplasmic reticulum (ER) to the Golgi apparatus. VLDL molecules are transported in a specialized transport vesicle the Very Low Density Lipoprotein Transport Vesicle (VTV) . VLDL's core protein, apolipoproteinB-100 (apoB100), is initially lipidated in the ER, and then subsequently delivered to the Golgi apparatus where the VLDL molecule undergoes maturation involving further lipidation and glycosylation of apoB100. Oxysterol Binding Proteins (OSBP) and the sub family OSBP Related Proteins (ORP) have been implicated in many different trafficking processes, mainly the trafficking of sterols, cholesterol, and lipids. Recently, ORP 10 was shown to be a negative regulator of apoB100 secretion in growth medium . Using co-immunoprecipitation, the current study shows that ORP 10 interacts with VLDL's core protein apoB100 directly. Employing an in vitro budding assay, we show that the blocking of ORP 10 with a specific antibody against ORP10 increases VTV formation from the ER. Given that the ER to Golgi pathway is the rate-limiting step in overall VLDL secretion, these findings support the conclusion that ORP 10 is a negative regulator of VLDL trafficking between the ER and Golgi, and that this process is mediated by the ORP 10 protein binding with apoB100.
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Date Issued
2015
Identifier
CFH0004866, ucf:45491
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004866
HEPATIC LIPASE REGULATES LIPOPROTEIN TRAFFICKING IN HEPATOCYTES
Title
HEPATIC LIPASE REGULATES LIPOPROTEIN TRAFFICKING IN HEPATOCYTES.
Creator
Thibeaux, Simeon, Siddiqi, Shadab, University of Central Florida
Abstract / Description
The production of very low density lipoprotein and high density lipoprotein particles by the liver is a tightly regulated process, which begins with synthesis and assembly of core protein components in the rough endoplasmic reticulum. Factors influencing the production and metabolism of these particles are of immediate medical relevance, as their malfunction or hyperactivity can lead to an assortment of disease states. Hepatic lipase is a secreted liver enzyme, with many previously described...
Show moreThe production of very low density lipoprotein and high density lipoprotein particles by the liver is a tightly regulated process, which begins with synthesis and assembly of core protein components in the rough endoplasmic reticulum. Factors influencing the production and metabolism of these particles are of immediate medical relevance, as their malfunction or hyperactivity can lead to an assortment of disease states. Hepatic lipase is a secreted liver enzyme, with many previously described roles in the metabolism and clearance of both high and low density lipoproteins. Increased production and assembly of this enzyme is an indicator of metabolic dysfunction, while its absence or insufficiency leads to pre-mature atherosclerosis and death. The present study shows that this enzyme's role in lipoprotein metabolism is not confined to the degradation and clearance of these particles after they have been secreted. Experiments using co-immunoprecipitation targeted at hepatic lipase demonstrate that this protein interacts with ApoA1 and ApoB100, the core protein components of HDL and VLDL respectively, at the ER level in hepatocytes, as part of an enormous multi-subunit protein complex. This interaction with ApoA1 leads to decreased competence of hepatocytes to secrete HDL, which confers a pro-atherogenic phenotype. Analysis of ER to Golgi VLDL transport vesicles, produced with a cell-free in vitro budding assay, has revealed that hepatic lipase is co-secreted between these compartments with immature VLDL particles. Further analysis of cytosol isolated from hepatocytes demonstrates an interaction between hepatic lipase and the LDL-receptor related protein in a post-Golgi vesicle; the significance of which will be investigated in future studies.
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Date Issued
2015
Identifier
CFH0004736, ucf:45366
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004736
Differential Expression Of Proteins Involved In VLDL Trafficking Causes Reduced VLDL Secretion In Male Ames Dwarf Mice
Title
Differential Expression Of Proteins Involved In VLDL Trafficking Causes Reduced VLDL Secretion In Male Ames Dwarf Mice.
Creator
Ahmed Moinuddin, Faisal, Siddiqi, Shadab, Masternak, Michal, Naser, Saleh, University of Central Florida
Abstract / Description
Cardiovascular diseases (CVDs) have been recorded as the number one cause of death worldwide, accounting for 32% of total deaths annually. More than two-thirds of all CVD cases are associated with atherosclerosis, which is the accumulation of fats and other substances causing plaque formation in the interior walls of major arteries. This leads to narrowing of the lumen and hardening of the arteries, ultimately resulting in angina, heart attack and/or stroke. Studies have shown that the...
Show moreCardiovascular diseases (CVDs) have been recorded as the number one cause of death worldwide, accounting for 32% of total deaths annually. More than two-thirds of all CVD cases are associated with atherosclerosis, which is the accumulation of fats and other substances causing plaque formation in the interior walls of major arteries. This leads to narrowing of the lumen and hardening of the arteries, ultimately resulting in angina, heart attack and/or stroke. Studies have shown that the pathogenesis of atherosclerosis and associated CVDs is strongly linked to elevated secretion of liver-specific lipoproteins called very-low-density-lipoprotein (VLDL). VLDLs are crucial lipoproteins responsible for transportation of triacylglycerides (TAGs), chemically inert particles that are physiologically significant for their energy storing capacity, from the liver to peripheral tissues. These VLDL particles are synthesized in the lumen of the endoplasmic reticulum (ER) of hepatocytes, transported from the ER to the cis-Golgi in special transport vesicles called VLDL-transport-vesicles (VTVs) and secreted into plasma through a highly regulated secretory pathway. Previous studies from our laboratory have shown that VTV-mediated ER-to-Golgi VLDL trafficking is the rate-limiting step in overall VLDL secretion from hepatocytes into plasma. In this project, we investigated intracellular VLDL trafficking and VLDL secretion in Ames dwarf (Prop1df, df/df) mice, a mutant mouse model homozygous for a recessive mutation at Prop1 gene locus (Prop1df) having deficiency of growth hormone (GH), thyroid stimulating hormone (TSH) and prolactin (PRL). This model is characteristic of prolonged longevity (~50% longer) and improved insulin sensitivity in comparison to their wild-type (N) counterparts. Ames dwarf (df/df) mice have recently been shown to have highly reduced plasma TAG levels, associating them with reduced susceptibility to atherosclerosis and associated CVDs. The underlying mechanism responsible for reduced VLDL secretion in Ames dwarf mice is yet to be characterized. We hypothesize that VTV-mediated trafficking of VLDL is reduced in Ames dwarf mice because of reduced expression of proteins regulating VLDL and VTV formation. To test our hypothesis, we first performed VTV-budding assay using cellular fractions isolated separately from Ames dwarf (df/df) and wild-type (N) mice livers. Our results show a significant (45%) reduction in VTV-budding process in Ames dwarf (df/df) mice compared to wild-type (N). Next we performed 2-dimensional differential gel electrophoresis (2-DIGE) on VTV and whole cell lysate (WCL) samples in order to examine the differences in protein expression and to have highly specific protein separation. ExPASy database was used to analyze protein spots that allowed us in identifying proteins specifically expressed in each of the mouse groups. Employing western blotting, samples (ER, cytosol, VTV and WCL) from both sets of mice were tested for expression levels of VLDL and VTV associated proteins (ApoB100, Sec22b, CideB, MTP, Apo-A1 and Apo-AIV) with ?-actin as the loading control. Significant differences in expression level of these proteins were observed which strongly suggest that the formation of VTV from ER in male Ames dwarf (df/df) mice is reduced compared to wild-type (N). Overall, we conclude that the differential expression of proteins required for VLDL transport causes reduced VLDL secretion in male Ames dwarf (df/df) mice.
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Date Issued
2015
Identifier
CFE0005916, ucf:50829
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005916
Molecular Regulators of Post-Golgi VLDL Transport Vesicle (PG-VTV) Biogenesis
Title
Molecular Regulators of Post-Golgi VLDL Transport Vesicle (PG-VTV) Biogenesis.
Creator
Riad, Aladdin, Siddiqi, Shadab, Jewett, Travis, Naser, Saleh, University of Central Florida
Abstract / Description
Amongst its numerous functions, the liver is responsible for the synthesis and secretion of very low-density lipoprotein (VLDL). VLDL particles play the important role of facilitating the transport of lipids within the aqueous environment of the plasma; yet high plasma concentrations of these particles result in the pathogenesis of atherosclerosis, while low VLDL secretion from the liver results in hepatic steatosis. VLDL synthesis in the hepatocyte is completed in the Golgi apparatus, which...
Show moreAmongst its numerous functions, the liver is responsible for the synthesis and secretion of very low-density lipoprotein (VLDL). VLDL particles play the important role of facilitating the transport of lipids within the aqueous environment of the plasma; yet high plasma concentrations of these particles result in the pathogenesis of atherosclerosis, while low VLDL secretion from the liver results in hepatic steatosis. VLDL synthesis in the hepatocyte is completed in the Golgi apparatus, which serves as the final site of VLDL maturation prior to its secretion to the bloodstream. The mechanism by which VLDL's targeted transport to the plasma membrane is facilitated has yet to be identified. Our lab has identified this entity. Our findings suggest that upon maturation, VLDL is directed to the plasma membrane through a novel trafficking vesicle, the Post-Golgi VLDL Transport Vesicle (PG-VTV). PG-VTVs containing [3H] radiolabeled VLDL were generated in a cell-free in vitro budding assay for study. First, the fusogenic capabilities of PG-VTVs were established. Vesicles were capable of fusing with the plasma membrane and delivering the VLDL cargo for secretion in a vectorial manner. The next goal of our study is to characterize key regulatory molecular entities necessary for PG-VTV biosynthesis. A detailed analysis was undertaken to determine the PG-VTV proteome via western blot and two-dimensional difference in gel electrophoresis. The identification of key molecular regulators will potentially offer therapeutic targets to control VLDL secretion to the bloodstream.
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Date Issued
2013
Identifier
CFE0005236, ucf:50602
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005236
Validation of a novel hypothesis of generating foam cells by its use to study reverse cholesterol transport
Title
Validation of a novel hypothesis of generating foam cells by its use to study reverse cholesterol transport.
Creator
Sengupta, Bhaswati, Parthasarathy, Sampath, Singla, Dinender, Jewett, Mollie, Rohde, Kyle, University of Central Florida
Abstract / Description
Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho...
Show moreGeneration of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4(&)deg;C and retained the solubilized cholesterol during one month storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled or radiolabeled) / Lyso PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by thin layer chromatography (TLC). Such foam cells unloaded cholesterol when incubated with high density lipoprotein (HDL) and not with oxidized HDL (Ox-HDL). We propose that stable cholesterol or CE / Lyso PtdCho micelles would offer advantages over existing methods.Oxidative stress is associated with heart failure (HF). Previously our research group observed that the patients with low left-ventricular ejection fraction showed accumulation of high level of oxidized LDL (Ox-LDL) when compared with the heart failure patients with normal range of ejection fraction (EF). HDL is known to be atheroprotective and one of its important antioxidative functions is to protect LDL from oxidative modifications. However, HDL itself undergoes oxidation and Ox-HDL becomes functionally poor. It is expected to have a diminished ability to promote reverse cholesterol transport. Therefore, it was hypothesized that the quality of HDL present in the patients with EF would more compromised than those present in the patients with normal EF. Functionality of HDL was evaluated by measuring its cholesterol efflux capacity from foam cells generated in vitro. Functionality of HDL, which is strongly related to the oxidative modifications of HDL was further estimated by measuring paraoxonase 1 (PON1) enzyme activity associated with HDL. Higher the PON1 activity and RCT ability, better is the functionality of HDL.
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Date Issued
2014
Identifier
CFE0005250, ucf:50596
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005250
Alpha-Tocopherol Reduces VLDL Secretion Through Modulation of the VLDL Transport Vesicle
Title
Alpha-Tocopherol Reduces VLDL Secretion Through Modulation of the VLDL Transport Vesicle.
Creator
Clay, Ryan, Siddiqi, Shadab, Altomare, Deborah, Masternak, Michal, University of Central Florida
Abstract / Description
The liver distributes serum triacylglycerol (TAG) via the very low-density lipoprotein (VLDL), and an increase in VLDL production may result in hyperlipidemia. VLDL synthesis consists of lipidation of Apolipoprotein B100 (ApoB) as it is co- translationally translocated across the endoplasmic reticulum (ER) membrane, and this nascent VLDL particle must undergo subsequent maturation and post-translational modification in the Golgi. The ER-to-Golgi trafficking of VLDL represents the rate...
Show moreThe liver distributes serum triacylglycerol (TAG) via the very low-density lipoprotein (VLDL), and an increase in VLDL production may result in hyperlipidemia. VLDL synthesis consists of lipidation of Apolipoprotein B100 (ApoB) as it is co- translationally translocated across the endoplasmic reticulum (ER) membrane, and this nascent VLDL particle must undergo subsequent maturation and post-translational modification in the Golgi. The ER-to-Golgi trafficking of VLDL represents the rate-limiting step in VLDL secretion and is mediated by the VLDL Transport Vesicle (VTV). Many in vivo studies have indicated that vitamin E (alpha-tocopherol) supplementation protects against atherosclerosis and can reduce hepatic steatosis in nonalcoholic fatty liver disease (NAFLD), but its effects at the molecular level on hepatic lipid metabolism are poorly understood. To investigate the effects of alpha-tocopherol on hepatic VLDL secretion and cellular lipid retention, we performed several experiments in HepG2 (human) and McARH- 7777 (rat) hepatoma cell lines including pulse-chase experiments using 3H-oleic acid (3H- OA), confocal microscopy with BODIPY lipid droplet staining, and an in vitro VTV budding assay. Our results demonstrate a significant reduction of 3H-TAG secretion and ApoB media expression in response to 100 uM alpha-tocopherol, with a corresponding decrease in markers of VTV biogenesis in western blots of whole cell lysates (WCL) and retention of ApoB within the cell, indicating disruption of an early step in VLDL biogenesis. Further evidence indicates an increase in size and lipidation of the VTV and VLDL particle. BODIPY staining as well as 3H-TAG retention in WCLs was also sharply reduced. Overall, these results indicate that alpha-tocopherol reduces VLDL secretion, partially disrupts hepatic VLDL synthesis and VTV biogenesis, increases the lipidation of remaining VLDL particles, and diminishes overall cellular lipid droplet retention.
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Date Issued
2019
Identifier
CFE0007617, ucf:52538
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007617