Current Search: Siddiqi, Shadab (x)
View All Items
Pages
- 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.
Show less - Date Issued
- 2015
- Identifier
- CFH0004736, ucf:45366
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004736
- 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.
Show less - Date Issued
- 2015
- Identifier
- CFH0004866, ucf:45491
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004866
- Title
- THE ROLE OF HSC-70 IN VERY LOW DENSITY LIPOPROTEIN TRANPORT VESICLE GOLGI FUSION COMPLEX FORMATION.
- Creator
-
Nafi-Valencia, Erika, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Excess production and secretion of very low-density lipoprotein (VLDL) by the liver into the circulatory system is directly related to atherosclerosis, a chronic cardiovascular disease that threatens the lives of many worldwide and continues to be a leading cause of death in the United States. The rate-limiting step in VLDL secretion is its transport from the site of biogenesis, the hepatic endoplasmic reticulum to the cis-Golgi. This step is mediated by a specialized ER- derived vesicle, the...
Show moreExcess production and secretion of very low-density lipoprotein (VLDL) by the liver into the circulatory system is directly related to atherosclerosis, a chronic cardiovascular disease that threatens the lives of many worldwide and continues to be a leading cause of death in the United States. The rate-limiting step in VLDL secretion is its transport from the site of biogenesis, the hepatic endoplasmic reticulum to the cis-Golgi. This step is mediated by a specialized ER- derived vesicle, the VLDL transport vesicle (VTV). Upon exit of the ER the VTV targets, fuses and delivers VLDL into the lumen of the Golgi. The targeting and fusion of the VTV with the Golgi is facilitated by specific set of soluable N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that form a SNARE complex, which is required for the VTV-Golgi fusion and thus delivery to the Golgi. Data from our laboratory indicates that the formation of the SNARE complex requires cytosolic factors. Through the purification of liver cytosol, chromatographic steps, detailed mass spectrometry, immunodepletion and western blotting data it was identified that the protein necessary for SNARE complex formation is Hsc-70. Although Hsc-70's identification is significant, the role it plays in SNARE complex formation for VTV -Golgi fusion is a predicament and yet to be unraveled. In this study we performed a series of co-immunoprecipitation reactions to identify its role in SNARE-complex assembly. Using western blot data we confirmed binding of Hsc-70 with Sec22b, the v-SNARE on the VTV. Moreover, we confirmed the interaction of Hsc-70 with t-SNAREs, (syn5, rBet1 and GOS28) on the Golgi membrane. Removal of Hsc-70 from the liver cytosol resulted in significant reduction of SNARE-complex formation. Ultimately, the identification proteins involved in the process of VLDL delivery to the Golgi would offer therapeutic targets to control VLDL secretion into the blood by the liver.
Show less - Date Issued
- 2012
- Identifier
- CFH0004322, ucf:45036
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004322
- Title
- Embryonic Stem Cell-Derived Exosomes Inhibit Doxorubicin-Induced Pyroptosis in Cell Culture Models.
- Creator
-
Tavakoli Dargani, Zahra, Singla, Dinender, Masternak, Michal, Siddiqi, Shadab, Steward, Robert, University of Central Florida
- Abstract / Description
-
Doxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture...
Show moreDoxorubicin (Dox) is a potent chemotherapeutic drug used for the treatment of various cancers. Unfortunately, its use is limited as Dox induces adverse cardiotoxicity (DIC) and muscle toxicity (DIMT), which are mediated through oxidative stress, ER stress, and inflammation. However, it remains unknown whether Dox induces an inflammation mediated cell death, called (")pyroptosis("). The current study is designed to determine whether Dox induces pyroptosis in cardiac and muscle cell culture models. Moreover, the protective effects of embryonic stem cell-derived exosomes (ES-Exos) in inhibiting pyroptosis will also be determined. For this purpose, we designed two different cell culture models using H9c2 cadiomyoblasts and Sol 8 cells. For the DIC model, H9c2 were exposed to Dox to induce pyroptosis and then treated with exosomes. Cells were divided into 4 groups: Control, Dox, Dox+ES-Exos, and Dox+MEF-Exos (negative control). Furthermore, to generate the DIMT model, Sol 8 cells were incubated with Dox+THP-1 conditioned medium (TCM) to induce toxicity and inflammation, which was followed by exosomes treatment. We assigned cells into 5 groups: Control, Dox+TCM, Dox+TCM+ES-Exos, Dox+TCM+MEF-Exos (negative control), and Dox+TCM+ES-Exos+GW4869 compound (exosomes inhibitor, negative control). Our data shows that Dox treatment significantly increased pyroptotic marker expression including TLR-4, NLRP3, caspase-1, IL1-?, Caspase-11, and gasdermin-D as well as increased pro-inflammatory TNF-? and IL-6 expression in H9c2 cells. There was also a significant increase in caspase-1, IL1-?, and IL-18 expression in Dox+TCM treated Sol 8 cells. Conversely, increased pyroptosis and inflammation post-Dox treatment were inhibited by ES-Exos in both culture models. No significant changes observed upon MEF-Exos and GW4869 compound treatments. In conclusion, our data shows Dox induces pyroptosis and inflammation within cardiac and skeletal muscle cells, which can be inhibited following treatment with ES-exosomes. This is a novel study with new mechanistic observations on the pathophysiological role of pyroptosis in Dox-induced cardio and muscle toxicities.
Show less - Date Issued
- 2018
- Identifier
- CFE0007416, ucf:52700
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007416
- Title
- PG-VTV Biogenesis Requires ATP to Facilitate Phosphorylation of Syntaxin 17.
- Creator
-
Saxena, Anika, Siddiqi, Shadab, Jewett, Travis, Tigno-Aranjuez, Justine, University of Central Florida
- Abstract / Description
-
The uptake of cytotoxic free fatty acids (FFA) and their conversion to physiologically expedient triglycerides (TAG) which are later on assimilated to very low density lipoproteins (VLDL) is of utmost value among the multifarious tasks performed by the liver. Inflated concentration of VLDL in the blood stream directly correlates with the reinforcement of atherosclerosis. VLDL is synthesized in the hepatic endoplasmic reticulum (ER) and transported to the Golgi where it encounters several...
Show moreThe uptake of cytotoxic free fatty acids (FFA) and their conversion to physiologically expedient triglycerides (TAG) which are later on assimilated to very low density lipoproteins (VLDL) is of utmost value among the multifarious tasks performed by the liver. Inflated concentration of VLDL in the blood stream directly correlates with the reinforcement of atherosclerosis. VLDL is synthesized in the hepatic endoplasmic reticulum (ER) and transported to the Golgi where it encounters several alterations. It is then enclosed in distinct post-Golgi VLDL transport vesicles (PG-VTVs) and released into the blood. The data generated in our lab has proved the requirement of ATP for PG-VTV biogenesis however, ATP substitution with non-hydrolyzable ATP analogue (ATP?S) had no effect on this process. Therefore, the present study is based on the hypothesis that ATP mediated protein phosphorylation regulates PG-VTV biogenesis. First, hepatic subcellular organelles were isolated and their purity was determined by performing Western blot. A cell-free in vitro budding assay was performed in presence or absence of ATP, GTP and cytosol using 3[H]-TAG labelled hepatic Golgi to generate PG-VTVs. We performed Western blotting to confirm distinct protein phosphorylation at tyrosine residue during PG-VTV formation however, protein phosphorylation event did not occur when PG-VTV budding was blocked. Two-dimensional gel electrophoresis identified Syntaxin 17 (STX17) as the phosphorylated protein required for PG-VTV formation. ATP mediated phosphorylation of STX17 during biogenesis of PG-VTVs was confirmed by its presence on PG-VTVs. PG-VTV budding was found to be significantly reduced on performing budding assay using STX17 immunodepleted cytosol compared to positive control. RNAi mediated knockdown of STX17 in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3[H]-TAG liquid scintillation counter. Based on these results, it can be justified that STX17 plays a vital role in regulating PG-VTV budding and overall VLDL secretion from hepatocytes.
Show less - Date Issued
- 2018
- Identifier
- CFE0007094, ucf:51931
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007094
- Title
- Role of Kruppel-like Factor 8 (KLF8) in Cancer and Cardiomyopathy.
- Creator
-
Lahiri, Satadru, Zhao, Jihe, Parthasarathy, Sampath, Masternak, Michal, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Cancer and cardiovascular diseases are two most fatal diseases causing innumerable death each year. Understanding the mechanisms underlying these diseases is critical for developing proper therapeutic approach. Kr(&)#252;ppel-like factor 8 (KLF8) is a member of Kr(&)#252;ppel-like family transcription factors that is overexpressed in many types of cancers. There is no report on role of KLF8 in cardiovascular diseases to date. KLF8 transcriptionally activates or represses a host of target...
Show moreCancer and cardiovascular diseases are two most fatal diseases causing innumerable death each year. Understanding the mechanisms underlying these diseases is critical for developing proper therapeutic approach. Kr(&)#252;ppel-like factor 8 (KLF8) is a member of Kr(&)#252;ppel-like family transcription factors that is overexpressed in many types of cancers. There is no report on role of KLF8 in cardiovascular diseases to date. KLF8 transcriptionally activates or represses a host of target genes to promote cancer cell proliferation, migration, invasion and epithelial to mesenchymal transition during tumor progression. Studies proposed in this thesis identified a novel posttranslational modification of KLF8 essential for its role in promoting cancer cell migration and discovered a novel function of KLF8 in cardiomyopathy. In our first study, we identified serine 48 (S48) as a novel phosphorylation site on KLF8. Pharmacological and genetic manipulations of various potential kinases further revealed ERK2 as the kinase responsible for this novel phosphorylation. Functional studies indicated that this phosphorylation is crucial for protecting KLF8 protein from degradation in the nucleus and promoting cancer cell migration. Preclinical xenograft models have indicated an important role of KLF8 for tumor progression. To investigate role of KLF8 in spontaneous tumorigenesis better recapitulating pathology in patients, we established the first Cre-regulated conditional KLF8 transgenic mouse model. Upon induction of global expression of the KLF8 transgene, spontaneous mammary and testicular tumors were formed in a small population of the mice by their mid-age, as expected considering the long latency required for tumor progression. Surprisingly, however, nearly 100% of KLF8 the mice died with a significantly enlarged heart, which did not occur to any littermate control mouse. Further characterization of the mice revealed that the global expression of the transgene caused striking systolic dysfunction leading to fatal dilated cardiomyopathy. Importantly, these similar phenotypes were reproduced in heart-specific KLF8 transgenic mice. Cardiovascular disease PCR array identified a number of genes potentially mediating KLF8-induced cardiac pathology. These results identified a previously unimagined function of KLF8 in the heart, shed new light on the mechanisms of cardiac diseases and provide novel preclinical mouse models for future translational research.
Show less - Date Issued
- 2016
- Identifier
- CFE0006692, ucf:51914
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006692
- Title
- Apolipoprotein-AI Regulates Hepatic VLDL Secretion by Controlling Intracellular VLDL-Trafficking.
- Creator
-
Gurwani, Bhavesh, Siddiqi, Shadab, Masternak, Michal, Naser, Saleh, University of Central Florida
- Abstract / Description
-
Cardiovascular diseases cause 17 million deaths annually, which is estimated to increase to 23 million deaths by the year 2030. One of the major risk factors for the pathogenesis of cardiovascular diseases is increased secretion of very-low density lipoproteins (VLDL) by the liver; however, reduced VLDL-secretion causes fatty liver disease. Synthesis and secretion of VLDL by the liver plays an important role in maintaining overall lipoprotein homeostasis. Assembly of VLDL occurs along with...
Show moreCardiovascular diseases cause 17 million deaths annually, which is estimated to increase to 23 million deaths by the year 2030. One of the major risk factors for the pathogenesis of cardiovascular diseases is increased secretion of very-low density lipoproteins (VLDL) by the liver; however, reduced VLDL-secretion causes fatty liver disease. Synthesis and secretion of VLDL by the liver plays an important role in maintaining overall lipoprotein homeostasis. Assembly of VLDL occurs along with the expression of apolipoproteinB-100 (apoB100) and its lipidation at the endoplasmic reticulum (ER) level. Once formed in the ER lumen, the nascent VLDL is transported to the Golgi for its maturation. In the Golgi compartment, the nascent VLDL acquires apolipoproteinAI (apoAI), more triglycerides, and its apoB100 undergoes phosphorylation and glycosylation. These modifications are necessary for VLDL-exit from the trans-Golgi network (TGN) and this step is mediated by post-Golgi VLDL transport vesicle (PG-VTV). The transport of mature VLDL from the TGN to the plasma membrane (PM) is required for its secretion by the liver but remains to be studied. Our group has shown that the nascent VLDL particles do not contain apoAI, however, VLDL acquires apoAI in the cis-Golgi compartment. Interestingly, apoAI comes off the VLDL as soon as VLDL is secreted into the blood. We hypothesised that apoAI plays an important role in post-TGN VLDL trafficking and thus controls VLDL secretion by the liver. To determine the role of apoAI in the formation of PG-VTV and VLDL secretion, we knocked down apoAI in the hepatocytes using apoAI specific siRNA. The deficiency of apoAI did not have any effect on the expression of apoB100 and other apolipoprotein synthesis that are involved in VLDL synthesis; however, VLDL secretion was significantly reduced. Next, we overexpressed apoAI using plasmid with apoAI gene sequence and checked for the effects in VLDL secretion from the hepatocytes. We observed a significant increase in VLDL secretion from apoAI-overexpressing hepatocytes which is consistent with knockdown results. To determine the role of apoAI in post-TGN trafficking of the mature VLDLs, we isolated sub-cellular organelles from apoAI knockout (apoAI KO) and control mice. Subsequently, we performed in vitro PG-VTV budding assays to assess the effect of apoAI silencing on PG-VTV formation from the TGN. Our results strongly suggest that the deficiency of apoAI increases PG-VTV formation (i.e. TGN-exit of mature VLDL) but significantly reduces VLDL-triglyceride secretion from the hepatocytes. We conclude that apoAI controls VLDL secretion by the liver by regulating post-TGN trafficking of mature VLDL.
Show less - Date Issued
- 2016
- Identifier
- CFE0006685, ucf:51908
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006685
- Title
- Metabolic Effects of 17a-Estradiol are Growth Hormone Independent and Sex Specific.
- Creator
-
Sidhom, Silvana, Masternak, Michal, Altomare, Deborah, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Aging is a major risk factor for metabolic syndromes and type two diabetes. With growing elderly populations worldwide and increasing incidence of age-related diseases there is a great need to develop pharmacological interventions that would delay aging and protect from age-related diseases. 17-alpha estradiol (17?-E2) is an epimer of the primary female sex hormone estradiol and has been shown to extend lifespan and downregulate markers of age-related metabolic dysfunction in male mice....
Show moreAging is a major risk factor for metabolic syndromes and type two diabetes. With growing elderly populations worldwide and increasing incidence of age-related diseases there is a great need to develop pharmacological interventions that would delay aging and protect from age-related diseases. 17-alpha estradiol (17?-E2) is an epimer of the primary female sex hormone estradiol and has been shown to extend lifespan and downregulate markers of age-related metabolic dysfunction in male mice. Because 17?-E2 does not induce feminization in males it holds potential as a novel therapeutic in humans for age-related metabolic dysfunction. Importantly, we have previously shown that 17?-E2 causes an increase of circulating and hepatic IGF-1 in aged mice, without any changes in GH release in treated animals. Based on this we propose a new hypothesis that 17?-E2 acts through a novel, GH-independent pathway stimulating production of IGF-1 and positively modulating metabolic function in a sex-specific manner. Here we studied 17?-E2 treated long-lived growth hormone receptor knockout (GHRKO) mice, characterized by severely reduced circulating and hepatic IGF-1 due to GH-resistance. We found increases in circulating IGF-1 after treatment in normal and GHRKO male mice, with no effect in female mice, which supports our hypothesis that 17?-E2 induces GH independent IGF-1 production. To determine novel genetic pathways activated by 17?-E2 we performed sequencing of hepatic RNA. Our analysis indicated differential regulation of steroid biosynthesis and insulin signaling pathways. The validation of our sequencing data using qPCR showed significant upregulation of genes involved in insulin action. Importantly, differential regulation of these pathways was present in normal male mice, with no changes in normal females or either male or female GHRKO animals. In summary, this new data supports our hypothesis of a sex-specific effect of 17?-E2 treatment and differing mechanisms of action by which 17?-E2 upregulates IGF-1 independently of GH action.
Show less - Date Issued
- 2019
- Identifier
- CFE0007726, ucf:52424
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007726
- 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.
Show less - Date Issued
- 2019
- Identifier
- CFE0007617, ucf:52538
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007617
- Title
- Expression and functional evaluation of exendin 4 fused to cholera toxin B subunit in tobacco chloroplasts to treat type 2 diabetes.
- Creator
-
Nityanandam, Ramya, Daniell, Henry, Naser, Saleh, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4)...
Show moreThe prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the ability of CTB pentamer to bind the GM1 receptors on the intestinal epithelium and to bioencapsulate EX4 within plant cells to confer protection in the digestive system. The LAMD tobacco leaves were bombarded with chloroplast vectors expressing modified EX4. The transgene integration was confirmed by PCR analysis and Southern blot analysis. Densitometric analysis revealed expression level of the protein varied from 9-13% of the total leaf protein depending on the developmental stage and time of harvest. The pentameric structure and functionality of CTB-EX4 fusion protein was confirmed by CTB-GM1 binding assay. The effect of transplastomic protein on insulin secretion was tested in ?-TC6, a mouse pancreatic cell line. The plant derived CTB-EX4, partially purified with anti-CTB antibody conjugated protein A beads, showed the increase of insulin ~ 2.5 fold increase when compared to untreated cells. The transplastomic protein showed a linear increase in insulin secretion comparable to the commercially available EX4. The current cost of treatment with EX4 varies between $1800-$2200, annually. Production of functional EX4 in plants should facilitate low cost orally deliverable form of this drug for treatment of type 2 diabetes.
Show less - Date Issued
- 2011
- Identifier
- CFE0004485, ucf:49306
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004485
- Title
- Bone Morphogenetic Protein-7 (BMP-7) Polarizes Monocytes into M2 Macrophages.
- Creator
-
Rocher, Crystal, Singla, Dinender, Siddiqi, Shadab, Sugaya, Kiminobu, University of Central Florida
- Abstract / Description
-
Atherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however,...
Show moreAtherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however, its role in monocyte polarization is yet to be revealed. In the current study, we hypothesize that monocyte treatment with BMP-7 will significantly result in increased polarization of monocytes into M2 macrophages and increased expression of anti-inflammatory cytokines. To that effect, we have established a stress induced cell culture system with monocytes (THP-1 cells) and apoptotic conditioned medium (ACM), simulating injury, to understand the effects of BMP-7 on M2 macrophage polarization from monocytes. Our data demonstrates that the BMP type 2 receptor (BMPR2) is found on monocytes and its activation is significantly (p(<)0.05) increased in both monocytes and M2 macrophages following treatment with BMP-7. Furthermore, a significant (p(<)0.05) increase of M2 macrophages in the BMP-7 treated group was shown following immunostaining with CD206 and arginase-1, two M2 macrophage markers, whereas a significant (p(<)0.05) decrease of iNOS expression, an M1 macrophage marker, was shown. Moreover, treatment with BMP-7 resulted in significantly (p(<)0.05) increased expression of IL-10 and IL-1ra, two anti-inflammatory cytokines, but significantly (p(<)0.05) decreased levels of the pro-inflammatory cytokines, MCP-1, IL-6 and TNF-?. We also hypothesize that polarization of monocytes to M2 macrophages occurs through activation of SMAD1/5/8 and PI3K-Akt-mTOR pathways. Upon BMP-7 binding to its receptor, BMPR2, activation of SMAD1/5/8 occurs which then activates the p85 subunit of PI3K resulting in downstream activation of Akt and mTOR. Our data shows that following treatment with BMP-7, expression of p-SMAD1/5/8, p-PI3K, p-Akt and p-mTOR is significantly (p(<)0.05) increased compared to controls whereas p-PTEN, an inhibitor of the PI3K pathway, is significantly (p(<)0.05) decreased in the BMP-7 treated group compared to controls. In conclusion, our data reveals that BMP-7 polarizes monocytes into M2 macrophages and it achieves this through activation of the PI3K-Akt-mTOR pathway, which will have significant applications for atherosclerosis treatment.
Show less - Date Issued
- 2013
- Identifier
- CFE0004922, ucf:49617
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004922
- Title
- Cathepsin B Regulates VLDL Secretion Through LFABP Cleavage.
- Creator
-
Thibeaux, Simeon, Siddiqi, Shadab, Kim, Yoon-Seong, Teter, Kenneth, University of Central Florida
- Abstract / Description
-
The liver is tasked with managing the concentration of various metabolites in the blood, and of particular importance is the uptake of free fatty-acid (FFA), as elevated concentrations of FFA are toxic to cells. FFAs are transported across the cell membrane by CD36 and distributed by LFABP to the endoplasmic reticulum (ER), where they are esterified to glycerol, yielding more chemically inert triglyceride (TAG), which is essential to the process of VLDL assembly. VLDL secretion distributes...
Show moreThe liver is tasked with managing the concentration of various metabolites in the blood, and of particular importance is the uptake of free fatty-acid (FFA), as elevated concentrations of FFA are toxic to cells. FFAs are transported across the cell membrane by CD36 and distributed by LFABP to the endoplasmic reticulum (ER), where they are esterified to glycerol, yielding more chemically inert triglyceride (TAG), which is essential to the process of VLDL assembly. VLDL secretion distributes energy rich TAG to peripheral tissues, and its dysfunction leads to hepatic steatosis, which may progress into hepatocellular carcinoma. The present study examined the role of cathepsin B (CatB) in regulating very-low density lipoprotein (VLDL) secretion through liver fatty-acid binding protein (LFABP) cleavage as well as CD36 expression in response to 0.5 mM oleic acid:BSA treatment, which has been reported to redistribute CatB from the lysosome to the cytosol, where the majority of cellular LFABP is localized. Genetic knock-down of CatB in McA-RH7777 cells resulted in increased VLDL secretion as measured by 3H TAG DPM counting and immunoblot for ApoB in cell culture media, due to increased expression of LFABP and CD36 and increased FFA uptake. Knock-down of CatB also resulted in decreased cellular TAG as measured by 3H DPM counting due to increased VLDL secretion. CatB over-expression in McA-RH7777 cells resulted in decreased FFA uptake leading to decreased VLDL secretion, which was due to increased cleavage of LFABP. Co-localization of LFABP and CatB was observed exclusively under conditions of 0.5 mM oleic acid:BSA treatment. Based on these results, we can conclude that CatB plays a distinct physiological role in the turnover of LFABP and CD36 protein, which leads to suppressed uptake of FFA, and thus, reduced TAG synthesis and VLDL secretion.
Show less - Date Issued
- 2017
- Identifier
- CFE0006669, ucf:51236
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006669
- Title
- Evaluation of Intestinal Microbial Diversity and a New Antibiotic Regimen in Crohn's Disease Patients.
- Creator
-
Alcedo, Karel, Naser, Saleh, Cheng, Zixi, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three...
Show moreCrohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs (-) Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.
Show less - Date Issued
- 2015
- Identifier
- CFE0005917, ucf:50831
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005917
- 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.
Show less - Date Issued
- 2015
- Identifier
- CFE0005916, ucf:50829
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005916
- Title
- Role of KLF8-CXCR4 signaling in Breast Cancer Metastasis.
- Creator
-
Mukherjee, Debarati, Zhao, Jihe, Khaled, Annette, Altomare, Deborah, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
Kr(&)#252;ppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. In this study we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of...
Show moreKr(&)#252;ppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. In this study we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Furthermore, CXCR4-CXCL12 engagement downstream of KLF8 leads to the feed-forward activation of FAK. Interestingly, KLF8 expression, through CXCR4 engagement, triggered the formation of filopodium-like protrusions (FLP) and thereby enhanced the proliferation rate of breast cancer cells in 3D Matrigel-on-Top culture, under prolonged treatment with CXCL12. This indicates that KLF8 plays a major role in promoting aggressive colonization of tumor cells in a CXCL12-enriched foreign tissue microenvironment, thereby aiding in secondary macrometastasis formation. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. Apart from lung, KLF8 overexpression also induced spontaneous secondary metastasis to other CXCL12-rich organs through CXCR4 signaling. These results collectively suggest a critical role for KLF8 and the CXCR4-CXCL12 pathway in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies.
Show less - Date Issued
- 2016
- Identifier
- CFE0006149, ucf:51127
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006149
- Title
- Is breakdown of fatty acid peroxides involved in the induction of apolipoprotein A1?.
- Creator
-
Gupta, Rajat, Parthasarathy, Sampath, Siddiqi, Shadab, Jewett, Mollie, University of Central Florida
- Abstract / Description
-
Over the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the...
Show moreOver the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the susceptibility of serum lipoproteins to undergo oxidation, thereby contributing to the progression of atherosclerosis. The intestinal cells are responsible for the absorption of dietary fatty acid peroxides (FAOOH) which has been reported to enhance anti-atherosclerotic effects by inducing apolipoprotein A1 (apoA1) gene and protein levels. Therefore, there is a void in the knowledge of when to expect (")harmful(") or (")beneficial(") effects of dietary lipid peroxides. The formation of toxic products like aldehydes from the decomposition of FAOOH is well documented. On the other hand, carboxylic acids particularly azelaic acid, formed as an end product of FAOOH decomposition has been reported to have anti-atherosclerotic effects. Hence, we hypothesize that intestinal cells may decompose FAOOH to aldehydes, which might get converted to carboxylic acids that can be transported across the intestine. Linoleic acid is the most abundant polyunsaturated fatty acid (PUFA) present in the diet. So, we will use peroxidized linoleic acid (13-HPODE) and incubate with intestine derived cells or Caco -2 cells as an in-vitro model for determining its decomposition to aldehydes and carboxylic acids. We propose that the decomposition products of FAOOH in the presence of intestinal cells might be responsible for causing an increase in apoA1 levels, which might suggest that lipid peroxidation derived products might actually be beneficial for reducing the progression of atherosclerosis as compared to the absorption of intact FAOOH.
Show less - Date Issued
- 2013
- Identifier
- CFE0004856, ucf:49700
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004856
- 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.
Show less - Date Issued
- 2013
- Identifier
- CFE0005236, ucf:50602
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005236
- Title
- Identification of proteins regulating VLDL sorting into the VLDL Transport Vesicle (VTV) and involved in the biogenesis of the VTV.
- Creator
-
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
- A major double strand repair pathway and cancer-associated circulating proteins are effecters of epigenetic revision.
- Creator
-
Allen, Brittany, Masternak, Michal, Khaled, Annette, Zhao, Jihe, Muller, Mark, Siddiqi, Shadab, University of Central Florida
- Abstract / Description
-
DNA methylation is a vital epigenetic process that acts as a major control mechanism for gene expression. In addition to its essential role in many normal cellular processes, it is also implicated in a wide variety of disease states and processes including cancer. Along with genetic mutations, aberrant DNA methylation patterns, specifically the inappropriate DNA methylation or demethylation of CpG residues, may activate oncogenes or suppress tumor suppressor genes, respectively. These changes...
Show moreDNA methylation is a vital epigenetic process that acts as a major control mechanism for gene expression. In addition to its essential role in many normal cellular processes, it is also implicated in a wide variety of disease states and processes including cancer. Along with genetic mutations, aberrant DNA methylation patterns, specifically the inappropriate DNA methylation or demethylation of CpG residues, may activate oncogenes or suppress tumor suppressor genes, respectively. These changes can generate or facilitate the progression of tumorigenesis and tend to accumulate throughout the development of cancer. Although they play such a major role in cancer and in other diseases, it remains unclear what causes these epigenetic revisions to occur. This dissertation will focus on uncovering mechanisms that are sources of epigenetic revision, specifically as they relate to cancer. Due to rapid cell division and increased DNA damage, cells are increasingly dependent on DNA repair as they continue on a path of tumorigenic progression. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in inappropriate epigenetic revision. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we were able to demonstrate that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating one potential source for the altered methylation patterns seen in cancer cells.The Dim cells generated during this study are known to have a hypermethylated GFP gene that is correlated with reduced expression, allowing it to be used as a screening tool for hypomethylating agents. We used this tool to screen the blood serum of patients with head and neck squamous cell carcinoma (HNSCC). We found that the serum from HNSCC patients, but not from healthy individuals, contains some factor that causes hypomethylation in exposed cells. Further, we were able to identify this factor as a protein capable of effecting changes in DNA methylation, gene expression, and miRNA levels in the treated Dim cells. The novel concept presented in this study has immense implications on the study of cancer progression as it evidences circulating proteins, presumably released by cancer cells, which are able to effect gene expression in cells that are distal to the location of the cancer. Further, the fact that these proteins are in circulation makes them a potential target for use in diagnostics. Changes in DNA methylation play a major role in the development of cancer and understanding the mechanisms by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis. This dissertation presents potential sources of epigenetic revision in cancer and thus provides answers to a major question that has yet to be answered in the area of cancer research.
Show less - Date Issued
- 2017
- Identifier
- CFE0006555, ucf:51333
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006555
- Title
- The Role of Mitochondrial Omi/HtrA2 Protease in Protein Quality Control and Mitophagy.
- Creator
-
Ambivero, Camilla, Zervos, Antonis, Teter, Kenneth, Siddiqi, Shadab, Self, William, University of Central Florida
- Abstract / Description
-
Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors...
Show moreOmi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a Lys-63 deubiquitinating (DUB) complex. In addition, we found the mechanism by which Omi/HtrA2 protease participates in mitophagy by directly regulating the protein level of Mulan E3 ubiquitin ligase, especially during mitochondrial stress.Abro1 is a scaffold protein of the DUB complex known as BRISC (BRCC36 isopeptidase complex). In addition, Abro1 is involved in a cytoprotective pathway and is regulated by Omi/HtrA2. Three specific interactors of Abro1 protein were identified, ATF4, ATF5 and JunD, all members of the activating protein 1 (AP-1) family. We focused our studies on ATF4 since, like Abro1, it is ubiquitously expressed and is important in cell cycle regulation and survival. Abro1's interaction with ATF4 was specific and occurred only when cells were stressed. The significance of this interaction was the translocation of Abro1 from the cytoplasm to the cell nucleus. These results establish a new cytoprotective function of cytoplasmic Omi/HtrA2 as a regulator of the BRISC DUB complex.Furthermore, we have recently identified the mitochondrial Mulan E3 ubiquitin ligase as a substrate of Omi/HtrA2 protease. Mulan, along with MARCH5/MITOL and RNF185, are the only three mitochondrial E3 ubiquitin ligases identified thus far. The function of Mulan has been linked to cell growth, cell death, and autophagy/mitophagy. To investigate Mulan's function and its control by Omi/HtrA2, E2 conjugating enzymes that form a complex with Mulan E3 ligase were identified. Four specific interacting E2s were isolated, namely Ube2E2, Ube2E3, Ube2G2, and Ube2L3. To identify substrates for each unique Mulan-E2 complex, fusion baits were used in a modified yeast two-hybrid screen. Our results suggest that Mulan participates in various pathways, depending on the nature of its E2 conjugating enzyme partner. One of the interactors isolated against the Mulan-Ube2E3 bait was the GABARAP (GABAA receptor-associated protein), a member of the Atg8 family. We characterized this interaction both in vitro and in vivo and its potential role in mitophagy. Our studies defined a new pathway by which Mulan participates in mitophagy by recruiting GABARAP to the mitochondria.
Show less - Date Issued
- 2013
- Identifier
- CFE0004805, ucf:49752
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004805