This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Discussion
The results from my bioinformatic analyses show that mutations in PARK2 can have deleterious effects on parkin’s function as a ubiquitin ligase. The more critical point mutations that impair the protein are those that arise in either of the protein’s RING finger domains and the arginine 42-proline mutation in the UBQ domain. Recent studies suggest that parkin relies upon the PINK1 protein for localizing to damaged mitochondria, to where parkin assists in the degradation of these damaged organelles (1). Further, the Chung et al paper (see Drug Interactions page) showed that parkin’s ability to attenuate cell death was inhibited by free radical stressors on it.
While a link has been shown between parkin mutations and autosomal recessive juvenile Parkinson’s disease, there has yet to be a connection between parkin defects and late onset PD. Late onset PD is the predominant form of the disease and generally occurs in people aged 50 years and older. It is my hypothesis that the aging process may have an effect on parkin activity in neuron cells. This effect would decrease parkin activity, which would lead to higher rates of cell death. The expression profile that I analyzed supports the correlation between age and parkin expression (see Microarray Expression).
A family of proteins known as SIRTUINS has been implicated in the cellular aging process (2). One member of this family, SIRT3, is a soluble protein that localizes to the mitochondrial matrix. The protein is thought to mediate the levels of reactive oxygen species in the mitochondria, thereby protecting the organelle and cell from damage. When looking at the SIRT3 protein interaction network (Figure1), one sees its association with FOXO3A, a transcriptional factor of autophagy in mitochondria. Indeed, a study showed that over expression of SIRT3 increased FOXO3A DNA binding activity (2). I am interested in seeing whether FOXO3A is a transcriptional activator of parkin. Because parkin is involved in autophagy of mitochondria, its effects could somehow be mitigated by FOXO3A, either directly through PARK2 DNA binding, or indirectly by altering the expression of PINK1, for example.
While a link has been shown between parkin mutations and autosomal recessive juvenile Parkinson’s disease, there has yet to be a connection between parkin defects and late onset PD. Late onset PD is the predominant form of the disease and generally occurs in people aged 50 years and older. It is my hypothesis that the aging process may have an effect on parkin activity in neuron cells. This effect would decrease parkin activity, which would lead to higher rates of cell death. The expression profile that I analyzed supports the correlation between age and parkin expression (see Microarray Expression).
A family of proteins known as SIRTUINS has been implicated in the cellular aging process (2). One member of this family, SIRT3, is a soluble protein that localizes to the mitochondrial matrix. The protein is thought to mediate the levels of reactive oxygen species in the mitochondria, thereby protecting the organelle and cell from damage. When looking at the SIRT3 protein interaction network (Figure1), one sees its association with FOXO3A, a transcriptional factor of autophagy in mitochondria. Indeed, a study showed that over expression of SIRT3 increased FOXO3A DNA binding activity (2). I am interested in seeing whether FOXO3A is a transcriptional activator of parkin. Because parkin is involved in autophagy of mitochondria, its effects could somehow be mitigated by FOXO3A, either directly through PARK2 DNA binding, or indirectly by altering the expression of PINK1, for example.
Figure 1: Protein network for SIRT3 (3).
My hypothesis proposes that the following signaling cascade will occur in a cell when SIRT3 levels decrease with age:
↓SIRT3 → ↓FOXO3A → ↓PARK2
To test my hypothesis, my experimental setup involves an in vitro assay using Cos-7 cells that contain wild type-FOXO3A expression vectors. Further, siRNA targeting FOXO3A will be transfected into the cells. As a control, I will use cells that have not been transfected with any siRNA. Next, the mRNA levels of parkin from the transfected cells will be quantified using RT-PCR and measured against the parkin mRNA levels of the control group. Expected results for this experiment would be that the parkin mRNA levels in the transfected cells are significantly lower than the parkin mRNA in the control group. By showing that parkin decreases with FOXO3A reductions, my experiment could shed light on the molecular basis for late onset PD, with parkin playing a critical role in the process.
↓SIRT3 → ↓FOXO3A → ↓PARK2
To test my hypothesis, my experimental setup involves an in vitro assay using Cos-7 cells that contain wild type-FOXO3A expression vectors. Further, siRNA targeting FOXO3A will be transfected into the cells. As a control, I will use cells that have not been transfected with any siRNA. Next, the mRNA levels of parkin from the transfected cells will be quantified using RT-PCR and measured against the parkin mRNA levels of the control group. Expected results for this experiment would be that the parkin mRNA levels in the transfected cells are significantly lower than the parkin mRNA in the control group. By showing that parkin decreases with FOXO3A reductions, my experiment could shed light on the molecular basis for late onset PD, with parkin playing a critical role in the process.
References
1. Matsuda, N., Sato, S., Shiba, K., Okatsu, K., Saisho, K., Gautier, C. A., Sou, Y. S., Saiki, S., Kawajiri, S., Sato, F., Kimura, M., Komatsu, M., Hattori, N., Tanaka, K. (2010). PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J Cell Biol, 189(2), 211-21. Doi: 10.1083/jcb.200910140
2. Jacobs, K. M., Pennington, J. D., Bisht, K. S., Aykin-Burns, N., Kim, H., Mishra, M., Sun, L., Nguyen, P., Ahn, B., Leclerc, J., Deng, C., Spitz, D. R., Gius D. (2008). SIRT3 interacts with the daf-16 homolog FOXO3a in the Mitochondria, as well as increases FOXO3a Dependent Gene expression. Int J Biol Sci, 4(5), 291–299. Retreived from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2532794/?tool=pubmed
3. STRING. http://string.embl.de/
2. Jacobs, K. M., Pennington, J. D., Bisht, K. S., Aykin-Burns, N., Kim, H., Mishra, M., Sun, L., Nguyen, P., Ahn, B., Leclerc, J., Deng, C., Spitz, D. R., Gius D. (2008). SIRT3 interacts with the daf-16 homolog FOXO3a in the Mitochondria, as well as increases FOXO3a Dependent Gene expression. Int J Biol Sci, 4(5), 291–299. Retreived from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2532794/?tool=pubmed
3. STRING. http://string.embl.de/
