Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is characterized as a rare autosomal dominant disease that causes smooth muscle tumors and aggressive kidney cancer [1]. This condition could appear in up to 20% of families, but this could potentially be higher due to the variety of symptoms and difficulty in tracking with a pedigree [2]. HLRCC develops through a fumarate hydratase (FH) deficiency, which occurs when one copy of a chromosome is mutated FH gene [1, 3]. In HLRCC, the FH gene cannot encode fumarate hydratase, which lowers the energy needed to convert fumarate to malate in the Krebs cycle. This means the double bond in fumarate cannot break, which somehow affects cellular signaling pathways. When these signaling pathways are expressed in this way, cancer growth is promoted in the cell. In HLRCC, the role of the FH gene in mitochondrial function in the kidney is still unknown [1].
My primary goal is to learn more about fumarate hydratase's role in kidney (renal cells) and mitochondria signaling pathways. In this set of experiments, I will be using Danio rerio (zebrafish) as a model organism to look at renal cells that are affected by FH [4]. Zebrafish are transparent, so it is easy to see mutant phenotypes[5]. I hypothesize that FH mutants that have the most aggressive renal cell cancer will occur in response to imbalances of mitochondria homeostasis. My long-term goal is to better understand FH’s role in the development of renal cell cancer to find treatment options for HLRCC. Focusing on the fumarate hydratase enzyme, I will be pursuing the following three specific aims:
Aim 1: Identify the protein domain that contributes to mitochondria maintenance in the kidney.
Hypothesis: I expect mutations in the conserved domain, C terminal, of FH will cause a phenotype of cancerous cell growth in the kidneys in zebrafish.
Rationale: Not all FH mutations cause the development of renal cell cancer. Identifying regions of FH strongly correlated with kidney development allows for correlating genotypes to phenotypes. The C terminal is responsible for encoding the fumarate hydratase activity in the mitochondria [6]. Knockouts of the domain associated with the mutant FH should give a phenotype such as developmental effects in the kidneys and mitochondria.
Approach: First, I will use InterPro to find the location of any conserved domains that are part of the N and C terminals. Next, I will separate the zebrafish into three groups and use CRISPR to create knockouts of the N terminal in one group and the C terminal in another. The remaining group will be used as the control.
Aim 2: Identify the compound that rescues mitochondrial homeostasis in the kidney.
Hypothesis: I expect there is a compound associated with the C terminal that will rescue the mitochondrial homeostasis in the kidney.
Rationale: Since the C terminal is conserved and associated with mitochondrial maintenance, specific chemicals such as fumartate hydrataes-IN-1 and fumarate hydratase-IN-2 can inhibit C terminal expression. Modifying these chemicals can be used to rescue mutant FH effects, allowing the knockout to express the wild-type phenotype.
Approach: I will use the mutant knockout zebrafish and control created in Aim 1 for chemical screening. First, I used PubChem to find compounds Fumarate hydratase-IN-1 and Fumarate hydratase-IN2, that inhibit wild-type FH. I will modify these compounds with hydroxyl and acetyl groups to create a targeted protein assay. The chemical hits from this assay will be used to rescue the mutant knockout zebrafish.
Aim 3: Identify proteins that are a part of the signaling pathway concerning mitochondrial maintenance in the kidney.
Hypothesis: I expect the novel proteins identified to be associated with the N terminal to rescue the mitochondrial homeostasis in the kidney in the C terminal knockout zebrafish.
Rationale: Understanding and identifying the protein interactions will allow for a clear understanding of FH’s role in converting fumarate to malate. I will focus on the proteins expressed in the N terminal knockouts since this will show the proteins expressed only in the C terminal.
Approach: First, I will use the mutant knockout and control zebrafish created in Aim 1 to do TurboID tagging the proteins associated with each group (wildtypes and both knockout zebrafish). I will purify and introduce the proteins associated with the N terminal back into the knockout and control zebrafish. This should rescue the C terminal knockout zebrafish phenotype. Then, I will compare the rescue and control phenotypes.
Through these aims, I expect to better understand how the mutation in FH causes tumor proliferation in kidneys due to mitochondrial maintenance. With the results of my aims, I could find compounds that can be used as a treatment for HLRCC and have new data discussing how loss of mitochondrial homeostasis affects kidney tissue. In the future, more genomic research, such as single-cell transcriptomics on wild-type and knockout zebrafish, can be performed to further understand this complex relationship.
My primary goal is to learn more about fumarate hydratase's role in kidney (renal cells) and mitochondria signaling pathways. In this set of experiments, I will be using Danio rerio (zebrafish) as a model organism to look at renal cells that are affected by FH [4]. Zebrafish are transparent, so it is easy to see mutant phenotypes[5]. I hypothesize that FH mutants that have the most aggressive renal cell cancer will occur in response to imbalances of mitochondria homeostasis. My long-term goal is to better understand FH’s role in the development of renal cell cancer to find treatment options for HLRCC. Focusing on the fumarate hydratase enzyme, I will be pursuing the following three specific aims:
Aim 1: Identify the protein domain that contributes to mitochondria maintenance in the kidney.
Hypothesis: I expect mutations in the conserved domain, C terminal, of FH will cause a phenotype of cancerous cell growth in the kidneys in zebrafish.
Rationale: Not all FH mutations cause the development of renal cell cancer. Identifying regions of FH strongly correlated with kidney development allows for correlating genotypes to phenotypes. The C terminal is responsible for encoding the fumarate hydratase activity in the mitochondria [6]. Knockouts of the domain associated with the mutant FH should give a phenotype such as developmental effects in the kidneys and mitochondria.
Approach: First, I will use InterPro to find the location of any conserved domains that are part of the N and C terminals. Next, I will separate the zebrafish into three groups and use CRISPR to create knockouts of the N terminal in one group and the C terminal in another. The remaining group will be used as the control.
Aim 2: Identify the compound that rescues mitochondrial homeostasis in the kidney.
Hypothesis: I expect there is a compound associated with the C terminal that will rescue the mitochondrial homeostasis in the kidney.
Rationale: Since the C terminal is conserved and associated with mitochondrial maintenance, specific chemicals such as fumartate hydrataes-IN-1 and fumarate hydratase-IN-2 can inhibit C terminal expression. Modifying these chemicals can be used to rescue mutant FH effects, allowing the knockout to express the wild-type phenotype.
Approach: I will use the mutant knockout zebrafish and control created in Aim 1 for chemical screening. First, I used PubChem to find compounds Fumarate hydratase-IN-1 and Fumarate hydratase-IN2, that inhibit wild-type FH. I will modify these compounds with hydroxyl and acetyl groups to create a targeted protein assay. The chemical hits from this assay will be used to rescue the mutant knockout zebrafish.
Aim 3: Identify proteins that are a part of the signaling pathway concerning mitochondrial maintenance in the kidney.
Hypothesis: I expect the novel proteins identified to be associated with the N terminal to rescue the mitochondrial homeostasis in the kidney in the C terminal knockout zebrafish.
Rationale: Understanding and identifying the protein interactions will allow for a clear understanding of FH’s role in converting fumarate to malate. I will focus on the proteins expressed in the N terminal knockouts since this will show the proteins expressed only in the C terminal.
Approach: First, I will use the mutant knockout and control zebrafish created in Aim 1 to do TurboID tagging the proteins associated with each group (wildtypes and both knockout zebrafish). I will purify and introduce the proteins associated with the N terminal back into the knockout and control zebrafish. This should rescue the C terminal knockout zebrafish phenotype. Then, I will compare the rescue and control phenotypes.
Through these aims, I expect to better understand how the mutation in FH causes tumor proliferation in kidneys due to mitochondrial maintenance. With the results of my aims, I could find compounds that can be used as a treatment for HLRCC and have new data discussing how loss of mitochondrial homeostasis affects kidney tissue. In the future, more genomic research, such as single-cell transcriptomics on wild-type and knockout zebrafish, can be performed to further understand this complex relationship.
Specific Aims
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References:
[1] Ooi, A. (2020, April). Advances in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) research. Seminars in cancer biology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078051/
[2] Menko, F. H., Maher, E. R., Schmidt, L. S., Middelton, L. A., Aittomäki, K., Tomlinson, I., Richard, S., & Linehan, W. M. (2014, December). Hereditary leiomyomatosis and renal cell cancer (HLRCC): Renal cancer risk, surveillance and treatment. Familial cancer. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574691/
[3] HLRCC Foundation. (2023, February 19). Hereditary leiomyomatosis and renal cell cancer (HLRCC) - overview of genetic kidney cancer syndrome. YouTube. https://www.youtube.com/watch?v=rX1BWQvjqN4
[4] Bellomo, R., Bingham, C., Bisgrove, B. W., Coffinier, C., Coxam, B., Drummond, I., Drummond, I. A., Duenas-Gonzalez, A., Hegde, R., Hiesberger, T., Huang, J., Hwang, D. Y., Lameire, N., Malicki, J., McCampbell, K. K., Nonaka, S., Outeda, P., Paavola, J., Pennekamp, P., … Gerlach, G. F. (2017, January 4). Using zebrafish to study kidney development and disease. Current Topics in Developmental Biology. https://www.sciencedirect.com/science/article/pii/S0070215316301946#:~:text=Zebrafish%20is%20an%20excellent%20system,kidney%20mutants%20have%20been%20identified.
[5] Teame, T., Zhang, Z., Ran, C., Zhang, H., Yang, Y., Ding, Q., Xie, M., Gao, C., Ye, Y., Duan, M., & Zhou, Z. (2019, June 25). The use of zebrafish (danio rerio) as biomedical models. Animal frontiers : the review magazine of animal agriculture. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6951987/#:~:text=Currently%2C%20zebrafish%20are%20considered%20as,variety%20of%20foods%20(euryphagous).
[6] William, S. (n.d.). N & C Terminal Sequencing: Amino Acid Sequence Analysis. BioPharmaSpec. https://biopharmaspec.com/protein-characterization-services/terminal-amino-acid-sequence/#:~:text=The%20free%20amine%20end%20of,naturally%20have%20different%20chemical%20properties.
[2] Menko, F. H., Maher, E. R., Schmidt, L. S., Middelton, L. A., Aittomäki, K., Tomlinson, I., Richard, S., & Linehan, W. M. (2014, December). Hereditary leiomyomatosis and renal cell cancer (HLRCC): Renal cancer risk, surveillance and treatment. Familial cancer. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574691/
[3] HLRCC Foundation. (2023, February 19). Hereditary leiomyomatosis and renal cell cancer (HLRCC) - overview of genetic kidney cancer syndrome. YouTube. https://www.youtube.com/watch?v=rX1BWQvjqN4
[4] Bellomo, R., Bingham, C., Bisgrove, B. W., Coffinier, C., Coxam, B., Drummond, I., Drummond, I. A., Duenas-Gonzalez, A., Hegde, R., Hiesberger, T., Huang, J., Hwang, D. Y., Lameire, N., Malicki, J., McCampbell, K. K., Nonaka, S., Outeda, P., Paavola, J., Pennekamp, P., … Gerlach, G. F. (2017, January 4). Using zebrafish to study kidney development and disease. Current Topics in Developmental Biology. https://www.sciencedirect.com/science/article/pii/S0070215316301946#:~:text=Zebrafish%20is%20an%20excellent%20system,kidney%20mutants%20have%20been%20identified.
[5] Teame, T., Zhang, Z., Ran, C., Zhang, H., Yang, Y., Ding, Q., Xie, M., Gao, C., Ye, Y., Duan, M., & Zhou, Z. (2019, June 25). The use of zebrafish (danio rerio) as biomedical models. Animal frontiers : the review magazine of animal agriculture. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6951987/#:~:text=Currently%2C%20zebrafish%20are%20considered%20as,variety%20of%20foods%20(euryphagous).
[6] William, S. (n.d.). N & C Terminal Sequencing: Amino Acid Sequence Analysis. BioPharmaSpec. https://biopharmaspec.com/protein-characterization-services/terminal-amino-acid-sequence/#:~:text=The%20free%20amine%20end%20of,naturally%20have%20different%20chemical%20properties.