Volume 12, Issue 2 (March & April 2021)
BCN 2021, 12(2): 213-222 |
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1- Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.
2- Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.
3- Department of Pharmacology and Toxicology, Department of Neurology, Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, USA.
2- Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.
3- Department of Pharmacology and Toxicology, Department of Neurology, Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, USA.
Abstract:
Introduction: Profilin1 (PFN1) is a ubiquitously expressed protein known for its function as a regulator of actin polymerization and dynamics. A recent discovery linked mutant PFN1 to Amyotrophic Lateral Sclerosis (ALS), which is a fatal and progressive motor neuron disease. We have also demonstrated that Gly118Val mutation in PFN1 is a cause of ALS, and the formation of aggregates containing mutant PFN1 may be a mechanism for motor neuron death. Hence, we were interested in investigating the aggregation of PFN1 further and searching for co-aggregated proteins in our mouse model overexpressing mutant PFN1.
Methods: We investigated protein aggregation in several tissues of transgenic and no-transgenic mice using western blotting. To further understand the neurotoxicity of mutant PFN1, we conducted a pull-down assay using an insoluble fraction of spinal cord lysates from hPFN1G118V transgenic mice. For this assay, we expressed His6-tagged PFN1WT and PFN1G118V in E. coli and purified these proteins using the Ni-NTA column.
Results: In this study, we demonstrated that mutant PFN1 forms aggregate in the brain and spinal cord of hPFN1G118V mice, while WT-PFN1 remains soluble. Among these tissues, spinal cord lysates were found to have PFN1 bands at higher molecular weights recognized with anti-PFN1. Moreover, the pull-down assay using His6-PFN1G118V showed that Myelin Binding Protein (MBP) was present in the insoluble fraction.
Conclusion: Our analysis of PFN1 aggregation in vivo revealed further details of mutant PFN1 aggregation and its possible complex formation with other proteins, providing new insights into the ALS mechanism.
Methods: We investigated protein aggregation in several tissues of transgenic and no-transgenic mice using western blotting. To further understand the neurotoxicity of mutant PFN1, we conducted a pull-down assay using an insoluble fraction of spinal cord lysates from hPFN1G118V transgenic mice. For this assay, we expressed His6-tagged PFN1WT and PFN1G118V in E. coli and purified these proteins using the Ni-NTA column.
Results: In this study, we demonstrated that mutant PFN1 forms aggregate in the brain and spinal cord of hPFN1G118V mice, while WT-PFN1 remains soluble. Among these tissues, spinal cord lysates were found to have PFN1 bands at higher molecular weights recognized with anti-PFN1. Moreover, the pull-down assay using His6-PFN1G118V showed that Myelin Binding Protein (MBP) was present in the insoluble fraction.
Conclusion: Our analysis of PFN1 aggregation in vivo revealed further details of mutant PFN1 aggregation and its possible complex formation with other proteins, providing new insights into the ALS mechanism.
Type of Study: Original |
Subject:
Cellular and molecular Neuroscience
Received: 2019/12/15 | Accepted: 2020/01/23 | Published: 2021/03/1
Received: 2019/12/15 | Accepted: 2020/01/23 | Published: 2021/03/1
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