This web page was produced as an assignment for Genetics 564, an undergraduate capstone course at UW-Madison.
Hepatic Lipase Deficiency (HLD) is an autosomal recessive disorder that is characterized by elevated triglyceride and cholesterol levels in the blood of affected patients [1, 2]. HLD is caused by mutations in Lipase C gene (LIPC) which encodes the enzyme hepatic lipase (HL). Hepatic lipase is predominantly involved in the conversion of intermediate-density lipoproteins into low-density lipoproteins and triglyceride-rich high-density lipoproteins (HDL) found during the well-fed state into triglyceride-poor HDL found in the fasting state [2]. There are multiple isoforms of LIPC. The full-length isoform is expressed and secreted by the liver, where it carries out the functions mentioned above. The truncated isoform of LIPC is expressed in steroidogenic tissues where it acts intracellularly and may play a role in reproductive processes. Hepatic lipase deficiency has been associated with reduced reproductive potential in mice, though the exact mechanism for this problem has not been investigated [3]. Determining how reduced LIPC activity contributes to lowered reproductive potential will uncover potential treatments for patients with hepatic lipase deficiency.
The long-term goal of this project is to identify how LIPC is involved in reproduction to improve infertility treatments for patients with hepatic lipase deficiency. The goal of this specific project is to identify how LIPC isoforms interact to promote cholesterol substrate uptake. My hypothesis is that the extracellular form of LIPC promotes remodeling of intermediate-density lipoproteins into low-density lipoproteins that are taken up by the granulosa cells while the intracellular truncated isoform of LIPC mobilizes cholesteryl esters from lipid droplets to free the cholesterol for the synthesis of progesterone and other reproductive hormones. Rattus norvegicus will be used as a model organism due to its similar progesterone synthesis mechanism to humans and the presence of a close ortholog to human LIPC [4, 5].
AIM 1: Investigate the effects of 3 glycosylated residues in exons one and two of full-length hepatic lipase.
Approach: Domain analysis has revealed the existence of three conserved glycosylated residues in exons one and two of LIPC. To investigate the purpose of these residues, I will mutagenize them using CRISPR-Cas9 to prevent their glycosylation. The effects of these changes on LIPC activity and localization can then be measured through triglyceride lipase activity assays and fluorescence microscopy.
Rationale: Glycosylation is important for the function of secreted proteins. The presence of these conserved residues in only the full-length LIPC isoform suggest that they are important for extracellular LIPC function.
Hypothesis: Mutagenesis of these conserved glycosylation residues will reduce LIPC function and potentially inhibit secretion. If these sites are the drivers for full-length LIPC function, I may see a complete switch to the truncated LIPC isoform activity.
Approach: Domain analysis has revealed the existence of three conserved glycosylated residues in exons one and two of LIPC. To investigate the purpose of these residues, I will mutagenize them using CRISPR-Cas9 to prevent their glycosylation. The effects of these changes on LIPC activity and localization can then be measured through triglyceride lipase activity assays and fluorescence microscopy.
Rationale: Glycosylation is important for the function of secreted proteins. The presence of these conserved residues in only the full-length LIPC isoform suggest that they are important for extracellular LIPC function.
Hypothesis: Mutagenesis of these conserved glycosylation residues will reduce LIPC function and potentially inhibit secretion. If these sites are the drivers for full-length LIPC function, I may see a complete switch to the truncated LIPC isoform activity.
AIM 2: Characterize differentially expressed genes in rats that only express truncated LIPC or full-length LIPC.
Approach: I will create two mutant rat lines. One will have a mutation in the exon two splice site that prevents the truncated version of LIPC from being created. In other words, this line will only express full-length LIPC. The other line will have a deletion of exons one and two, meaning all the LIPC it expresses will be in the truncated isoform. Once these lines are created, I will extract the livers and ovaries from each line for RNA-sequencing. I will then compare the results to those of a wildtype mouse line to detect changes in gene expression between wildtype mice and the mutants. Genes with differential expression will be subject to gene ontology analysis to determine pathways that are affected by the presence of only full-length LIPC or only truncated LIPC.
Rationale: RNA sequencing will determine differential gene expression of genes that are affected by the pathways involving both full-length and truncated LIPC. Gene ontology analysis can further pin down pathways involved in LIPC function.
Hypothesis: I expect to see differential expression of genes involved in lipoprotein remodeling in the mutant line that only expresses truncated LIPC. Likewise, for the line that only expresses full-length LIPC I would expect to see differential expression of genes involved in lipid droplet remodeling
Approach: I will create two mutant rat lines. One will have a mutation in the exon two splice site that prevents the truncated version of LIPC from being created. In other words, this line will only express full-length LIPC. The other line will have a deletion of exons one and two, meaning all the LIPC it expresses will be in the truncated isoform. Once these lines are created, I will extract the livers and ovaries from each line for RNA-sequencing. I will then compare the results to those of a wildtype mouse line to detect changes in gene expression between wildtype mice and the mutants. Genes with differential expression will be subject to gene ontology analysis to determine pathways that are affected by the presence of only full-length LIPC or only truncated LIPC.
Rationale: RNA sequencing will determine differential gene expression of genes that are affected by the pathways involving both full-length and truncated LIPC. Gene ontology analysis can further pin down pathways involved in LIPC function.
Hypothesis: I expect to see differential expression of genes involved in lipoprotein remodeling in the mutant line that only expresses truncated LIPC. Likewise, for the line that only expresses full-length LIPC I would expect to see differential expression of genes involved in lipid droplet remodeling
AIM 3: Identifying protein interactions of both truncated and full-length LIPC isoforms
Approach: The truncated-only and the full-length-only LIPC mutant rat lines will be used to study the protein interactions of each isoform. I will make lysates from the liver and ovaries of rats from each mutant line and perform a tandem-affinity purification (TAP) against LIPC. The resulting samples will be run through mass spectrometry analysis to identify proteins interacting with truncated or full-length LIPC.
Rationale: Performing TAP and mass spectrometry will provide direct evidence for the proteins that interact with the full-length and truncated isoforms of LIPC.
Hypothesis: I expect that proteins involved with lipoprotein and chylomicron remodeling interact with the full-length LIPC isoform while proteins involved in lipid droplet remodeling interact with the truncated isoform of LIPC.
Approach: The truncated-only and the full-length-only LIPC mutant rat lines will be used to study the protein interactions of each isoform. I will make lysates from the liver and ovaries of rats from each mutant line and perform a tandem-affinity purification (TAP) against LIPC. The resulting samples will be run through mass spectrometry analysis to identify proteins interacting with truncated or full-length LIPC.
Rationale: Performing TAP and mass spectrometry will provide direct evidence for the proteins that interact with the full-length and truncated isoforms of LIPC.
Hypothesis: I expect that proteins involved with lipoprotein and chylomicron remodeling interact with the full-length LIPC isoform while proteins involved in lipid droplet remodeling interact with the truncated isoform of LIPC.
References
[1] Ng, D.M., et al., Update on the diagnosis, treatment and management of rare genetic lipid disorders. Pathology, 2019. 51(2): p. 193-201.
[2] Kobayashi, J., et al., Hepatic Lipase: a Comprehensive View of its Role on Plasma Lipid and Lipoprotein Metabolism. J Atheroscler Thromb, 2015. 22(10): p. 1001-11.
[3] Wade, R.L., et al., Hepatic lipase deficiency attenuates mouse ovarian progesterone production leading to decreased ovulation and reduced litter size. Biol Reprod, 2002. 66(4): p. 1076-82.
[4] Verhoeven, A.J., D. Carling, and H. Jansen, Hepatic lipase gene is transcribed in rat adrenals into a truncated mRNA. J Lipid Res, 1994. 35(6): p. 966-75.
[5] Verhoeven, A.J. and H. Jansen, Hepatic lipase mRNA is expressed in rat and human steroidogenic organs. Biochim Biophys Acta, 1994. 1211(1): p. 121-4.
[1] Ng, D.M., et al., Update on the diagnosis, treatment and management of rare genetic lipid disorders. Pathology, 2019. 51(2): p. 193-201.
[2] Kobayashi, J., et al., Hepatic Lipase: a Comprehensive View of its Role on Plasma Lipid and Lipoprotein Metabolism. J Atheroscler Thromb, 2015. 22(10): p. 1001-11.
[3] Wade, R.L., et al., Hepatic lipase deficiency attenuates mouse ovarian progesterone production leading to decreased ovulation and reduced litter size. Biol Reprod, 2002. 66(4): p. 1076-82.
[4] Verhoeven, A.J., D. Carling, and H. Jansen, Hepatic lipase gene is transcribed in rat adrenals into a truncated mRNA. J Lipid Res, 1994. 35(6): p. 966-75.
[5] Verhoeven, A.J. and H. Jansen, Hepatic lipase mRNA is expressed in rat and human steroidogenic organs. Biochim Biophys Acta, 1994. 1211(1): p. 121-4.
Drafts
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