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(1 - 10 of 10)
- Title
- Resistive Pulse study of Vesicles and Liposomes.
- Creator
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Lin, Yuqing, Chow, Lee, Schulte, Alfons, Tatulian, Suren, Yuan, Jiann-Shiun, University of Central Florida
- Abstract / Description
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In this work, the properties of the liposomes, the artificially created vesicles by various methods, are explored by a resistive pulse method using micropipettes. The fact that vesicles are fundamental in the wide range of functionalities they fulfill as organelles strengthen the desire of understanding the properties of them. The motivation of this work comes from the significant roles that liposomes play in the development of targeted drug delivery systems. Among other significant variables...
Show moreIn this work, the properties of the liposomes, the artificially created vesicles by various methods, are explored by a resistive pulse method using micropipettes. The fact that vesicles are fundamental in the wide range of functionalities they fulfill as organelles strengthen the desire of understanding the properties of them. The motivation of this work comes from the significant roles that liposomes play in the development of targeted drug delivery systems. Among other significant variables, the size of liposomes is found to be one of the dominating parameters in liposome based drug delivery, and the correlation between liposome size and delivery efficiency is discussed. To help improving the size evaluation ability, a few mainstream methods for liposome size detection and measurements are reviewed. As a reliable and accessible alternative method for liposomes detection, the resistive pulse method is introduced and the measurement on liposomes size change upon pH gradient was performed using this method. With our current liposome composition, we found the size increases as environmental pH increases. Further investigation is performed with vesicular pH=6, 7, and 8, respectively. Lastly, the stability of the small unilamellar vesicles (SUV) was studied via resistive pulse method, by monitoring the size change of 50nm liposomes as function of time. A significant size change in freshly prepared 50nm liposomes is recorded. This information will provide invaluable knowledge for targeting tumor with tight tissues, where small size liposomes are needed.
Show less - Date Issued
- 2015
- Identifier
- CFE0005826, ucf:50933
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005826
- Title
- UNDERSTANDING THE ROLE OF PLASMODIUM FALCIPARUM VAMP8 SNARE HOMOLOGUE.
- Creator
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Camacho Ferreira, Katherine, Chakrabarti, Debopam, University of Central Florida
- Abstract / Description
-
Malaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia . According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium,...
Show moreMalaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia . According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium, which has three distinct life cycles occurring in the midgut of the female Anopheles mosquito, the liver of the human host, and human erythrocytes. When the parasite infects the erythrocyte, some induced cell host modifications are made in order to accommodate growth. During its intra-erythrocytic life cycle, the malaria parasite traffics numerous proteins to a set of unique destinations within its own plasma membrane including the digestive vacuole, the apicoplast, rhoptries, and micronemes. Vesicular transport is an essential process in eukaryotic cells. This coordinated process is responsible for moving thousands of proteins between compartments within the cell. Essential to the targeting and fusion of protein transport vesicles in eukaryotes are SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), a family of fusogenic proteins that are localized to distinct intracellular compartments . Studies performed in our laboratory have identified 18 proteins putatively belonging to the PfSNARE family . To date the exact role of PfSNAREs in the unique trafficking pathways of malaria is undetermined. Of particular interest to our study is PfVAMP8. In model eukaryotic organisms, VAMP8 containing vesicles deliver cargo to lysosomes and are involved in endocytosis. The food vacuole of the parasite is very similar to that of lysosomes and is essential to parasite survival. The study aims to identify the organelle(s) to which PfVAMP8 is localized and characterize membrane-association properties of this parasite's R-SNARE protein. We believe that PfVAMP8 would localize to unique compartments in the parasite protein network flow. An in depth understanding of its mechanisms and localizations could be a key in developing novel anti-malarials. This study aims to identify the organelle(s) to which PfVAMP8 are localized, determine the trafficking determinants of this protein and determine this proteins' expression and membrane association during the intra-erythrocytic stages of Plasmodium falciparum. Our immunofluorescence studies with known biological markers reveals that, PfVAMP8 passes through the endoplasmic reticulum, Golgi, and localizes to the food vacuole during trophozoite and schizont stage. Further characterization of the membrane association properties of the protein in this study reveals that PfVAMP8 is a soluble integral membrane protein with amphipathic characteristics.
Show less - Date Issued
- 2013
- Identifier
- CFH0004525, ucf:45157
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004525
- Title
- VESICLE TARGETING IN PLASMODIUM FALCIPARUM: THE IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF PLASMODIUM FALCIPARUM FAMILY OF SNARE PROTEINS.
- Creator
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Ayong, Lawrence, Chakrabarti, Debopam, University of Central Florida
- Abstract / Description
-
Proteins of the SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) super-family have been characterized as playing an essential role in vesicle targeting and fusion in all eukaryotes. The intracellular malaria parasite Plasmodium falciparum exhibits an unusual endomembrane system that is characterized by an unstacked Golgi apparatus, a developmentally induced apical complex, and various organellar structures of parasite origin in the infected host cells. How malaria...
Show moreProteins of the SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) super-family have been characterized as playing an essential role in vesicle targeting and fusion in all eukaryotes. The intracellular malaria parasite Plasmodium falciparum exhibits an unusual endomembrane system that is characterized by an unstacked Golgi apparatus, a developmentally induced apical complex, and various organellar structures of parasite origin in the infected host cells. How malaria parasites target nuclear-encoded proteins to these novel compartments is a central question in Plasmodium cell biology. Ultrastructural studies elsewhere have implicated the participation of specialized vesicular elements in transport of virulence proteins, including various cytoadherance and host cell remodeling factors, into the infected erythrocyte cytoplasm. However, little is known about the machineries that define the directionality of vesicle trafficking in malaria parasites. We hypothesized that the P. falciparum SNARE proteins would exhibit novel features required for vesicle targeting to the parasite-specific compartments. We then identified for the first time and confirmed the expression of eighteen SNARE genes in P. falciparum. Members of the PfSNAREs exhibit atypical structural features (Ayong et al., 2007, Molecular & Biochemical Parasitology, 152(2), 113-122). Among the atypical PfSNAREs, PfSec22 contains an unusual insertion of the Plasmodium export element (PEXEL) within its profilin-like longin domain, preceded by an N-terminal hydrophobic segment. Localization analyses suggest that PfSec22 is predominantly a vesicle-associated SNARE of the ER/Golgi interface, but which associates partially with mobile extraparasitic vesicles in P. falciparum-infected erythrocytes at trophozoite stages. We showed that PfSec22 export into host cells occurs via a two-step model that involves extraparasitic vesicle budding from the parasite plasma membrane and fusion with the parasitophorous vacuolar membrane. Export of PfSec22 was independent of its membrane-insertion suggesting that this protein might cross the vacuolar space as a single-pass type IV membrane protein. We demonstrated that the atypical longin domain dictates the steady-state localization of PfSec22, regulating its ER/Golgi trafficking and export into host cells. Our study provides the first experimental evidence for SNARE protein export in P. falciparum, and suggests a role of PfSec22 in vesicle trafficking within the infected host cell (Ayong et al, Eukaryotic Cell, Epub Jul 17, 2009). Next, to define the physiological function of the PfSec22 protein in Plasmodium parasites, we investigated its cognate partners. Using purified recombinant proteins we showed that PfSec22 forms direct binding interactions with six other PfSNAREs in vitro. These included the PfSyn5, PfBet1, PfGS27, PfSyn6, PfSyn16 and PfSyn18 PfSNAREs. By generating GFP-expressing parasites, we successfully localized the SNARE proteins PfSyn5, PfBet1 and PfGS27 to the parasite cis-Golgi compartment. We confirmed the association of PfSec22 with PfSyn5, PfBet1 and PfGS27 in vivo by immunoprecipitation analyses. Our data indicate a conserved ER-to-Golgi SNARE assembly in P. falciparum, and suggest that the malaria Sec22 protein might form novel SNARE complexes required for vesicle traffic within P. falciparum-infected erythrocytes
Show less - Date Issued
- 2009
- Identifier
- CFE0002852, ucf:48053
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002852
- Title
- IDENTIFYING THE EFFECTS OF A HUMAN DYNEIN MUTATION ON GFP-RAB7 AXONAL TRANSPORT IN EMBRYONIC MOUSE NEURONS.
- Creator
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Wilson, Natalie E, King, Stephen, University of Central Florida
- Abstract / Description
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The first dynein mutation found in humans that caused disease was a cytoplasmic dynein 1 heavy chain (DYNC1H1 in humans) p.His306Arg mutation, first described by Weedon et al. in 2011. This mutation caused Charcot-Marie-Tooth (CMT) subtype 2O. CMT has a prevalence of approximately 1 in 2500 people, making it the most common hereditary neuromuscular disorder. Cytoplasmic dynein 1 is used by eukaryotic cells for minus-end directed microtubule-based transport of cargo. One such cargo is Rab7, a...
Show moreThe first dynein mutation found in humans that caused disease was a cytoplasmic dynein 1 heavy chain (DYNC1H1 in humans) p.His306Arg mutation, first described by Weedon et al. in 2011. This mutation caused Charcot-Marie-Tooth (CMT) subtype 2O. CMT has a prevalence of approximately 1 in 2500 people, making it the most common hereditary neuromuscular disorder. Cytoplasmic dynein 1 is used by eukaryotic cells for minus-end directed microtubule-based transport of cargo. One such cargo is Rab7, a late endosomal marker. The purpose of this study is to identify the effects of this mutation on the transport of GFP-tagged Rab7 cargo in neurons from wild type (HH), heterozygous mutant (HR), and homozygous mutant (RR) mice harboring a DYNC1HI His306Arg mutation. Mouse embryos were euthanized, dissected to collect the hippocampal and cortical brain tissues, and these tissues were digested to isolate neurons. Nucleofection was used to introduce the exogenous GFP-Rab7 gene construct. These neurons were plated and imaged at 10 days in vitro using wide-field epifluorescence microscopy to generate image stacks of fluorescent GFP-Rab7 vesicles. Kymograph analysis was performed on the image stacks using MetaMorph software to measure several characteristics of movement. Statistical analysis of the data from each of the three genotypes shows there is no significant difference in Rab-7 transport between the three genotypes.
Show less - Date Issued
- 2017
- Identifier
- CFH2000265, ucf:45979
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000265
- Title
- REGULATION OF VLDL TRAFFICKING BY ORP 10.
- Creator
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Wessels, Philip, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2015
- Identifier
- CFH0004866, ucf:45491
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004866
- Title
- Differential Expression Of Proteins Involved In VLDL Trafficking Causes Reduced VLDL Secretion In Male Ames Dwarf Mice.
- Creator
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Ahmed Moinuddin, Faisal, Siddiqi, Shadab, Masternak, Michal, Naser, Saleh, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2015
- Identifier
- CFE0005916, ucf:50829
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005916
- Title
- Identification of proteins regulating VLDL sorting into the VLDL Transport Vesicle (VTV) and involved in the biogenesis of the VTV.
- Creator
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Tiwari, Samata, Siddiqi, Shadab, Zervos, Antonis, Singla, Dinender, Naser, Saleh, University of Central Florida
- Abstract / Description
-
Increased secretion of very low-density lipoprotein (VLDL), a triglyceride-rich lipoprotein, by the liver causes hypertriglyceridemia, which is a major risk factor for the development of atherosclerosis. The rate of VLDL-secretion from the liver is determined by its controlled transport from the endoplasmic reticulum (ER) to the Golgi. The ER-to-Golgi transport of newly synthesized VLDL is a complex multi-step process and is mediated by the VLDL transport vesicle (VTV). Once a nascent VLDL...
Show moreIncreased secretion of very low-density lipoprotein (VLDL), a triglyceride-rich lipoprotein, by the liver causes hypertriglyceridemia, which is a major risk factor for the development of atherosclerosis. The rate of VLDL-secretion from the liver is determined by its controlled transport from the endoplasmic reticulum (ER) to the Golgi. The ER-to-Golgi transport of newly synthesized VLDL is a complex multi-step process and is mediated by the VLDL transport vesicle (VTV). Once a nascent VLDL particle is synthesized in the lumen of the ER, it triggers the process of VTV-biogenesis and this process requires coat complex II (COPII) proteins that mediate the formation of classical protein transport vesicles (PTV). Even though, both VTV and PTV bud off the same ER at the same time and require the same COPII proteins, their cargos and sizes are different. The VTV specifically exports VLDL to the Golgi and excludes hepatic secretory proteins such as albumin and the size of the VTV is larger (~ 100 -120 nm) than PTV to accommodate VLDL-sized particles. These observations indicate (i) the existence of a sorting mechanism at the level of the ER; and (ii) the involvement of proteins in addition to COPII components. This doctoral thesis is focused on identification of proteins regulating VLDL sorting into the VTV and involved in the biogenesis of the VTV. In order to identify proteins present exclusively in VTV, we have characterized the proteome of VTV, which suggest CideB (cell death-inducing DFF45-like effector b) and SVIP (small VCP/P97 interacting protein) as candidates, present in VTV but excluded from PTV. We further confirmed the finding by performing co-immunoprecipitation studies and confocal microscopy studies. CideB, a 26-kDa protein was found to interact with apolipoprotein B100 (apoB 100), the structural protein of VLDL. Moreover, CideB interacts with two of the COPII components, Sar1 and Sec24. VTV generation was examined after blocking CideB by specific antibodies and by silencing CideB in rat primary hepatocytes. Knockdown of CideB in primary hepatocytes showed significant reduction in VTV generation, however, CideB was concentrated in VTV as compared with the ER suggesting its functional role in the sorting of VLDL into the VTV. SVIP, a small (~ 9-kDa) protein was found to interact with Sar1, a COPII component that initiates the budding of vesicles from ER membrane. SVIP has sites for myristoylation and we found increased recruitment of SVIP on ER membrane upon myristic acid (MA) treatment. Sar1 that lacks sites for myristoylation also is recruited more on ER upon myristoylation indicating that SVIP promotes Sar1 recruitment on ER. Additionally, our data suggest that Sar1 interacts with SVIP and forms a multimer that facilitates the biogenesis of VTV. Interestingly, silencing of SVIP reduced the VTV generation significantly. Conversely, incubation with MA increased the VTV budding, suggesting recruitment of SVIP on ER surface facilitates the VTV budding. We conclude that SVIP recruits Sar1 on ER membrane and makes an intricate COPII coat leading to the formation of a large vesicle, the VTV. Overall, the data presented in this thesis, determines the role of CideB and SVIP in regulating VLDL sorting and VTV biogenesis.
Show less - Date Issued
- 2013
- Identifier
- CFE0005270, ucf:50553
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005270
- Title
- Nano-pipette as nanoparticle analyzer and capillary gated ion transistor.
- Creator
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Rudzevich, Yauheni, Chow, Lee, Heinrich, Helge, Schulte, Alfons, Yuan, Jiann-Shiun, University of Central Florida
- Abstract / Description
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The ability to precisely count inorganic and organic nanoparticles and to measure their size distribution plays a major role in various applications such as drug delivery, nanoparticles counting, and many others. In this work I present a simple resistive pulse method that allows translocations, counting, and measuring the size and velocity distribution of silica nanoparticles and liposomes with diameters from 50 nm to 250 nm. This technique is based on the Coulter counter technique, but has...
Show moreThe ability to precisely count inorganic and organic nanoparticles and to measure their size distribution plays a major role in various applications such as drug delivery, nanoparticles counting, and many others. In this work I present a simple resistive pulse method that allows translocations, counting, and measuring the size and velocity distribution of silica nanoparticles and liposomes with diameters from 50 nm to 250 nm. This technique is based on the Coulter counter technique, but has nanometer size pores. It was found that ionic current drops when nanoparticles enter the nanopore of a pulled micropipette, producing a clear translocation signal. Pulled borosilicate micropipettes with opening 50 ~ 350 nm were used as the detecting instrument. This method provides a direct, fast and cost-effective way to characterize inorganic and organic nanoparticles in a solution. In this work I also introduce a newly developed Capillary Ionic Transistor (CIT). It is presented as a nanodevice which provides control of ionic transport through nanochannel by gate voltage. CIT is Ionic transistor, which employs pulled capillary as nanochannel with a tip diameter smaller than 100 mm. We observed that the gate voltage applied to gate electrode, deposited on the outer wall of a capillary, affect a conductance of nanochannel, due to change of surface charge at the solution/capillary interface. Negative gate voltage corresponds to lower conductivity and positive gate increases conductance of the channel. This effect strongly depends on the size of the channel. In general, at least one dimension of the channel has to be small enough for electrical double layer to overlap.
Show less - Date Issued
- 2014
- Identifier
- CFE0005880, ucf:50882
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005880
- Title
- Alpha-Tocopherol Reduces VLDL Secretion Through Modulation of the VLDL Transport Vesicle.
- Creator
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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.
Show less - Date Issued
- 2019
- Identifier
- CFE0007617, ucf:52538
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007617
- Title
- Molecular Regulators of Post-Golgi VLDL Transport Vesicle (PG-VTV) Biogenesis.
- Creator
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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.
Show less - Date Issued
- 2013
- Identifier
- CFE0005236, ucf:50602
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005236
