Protein misfolding studies on TDP-43 protein involved in Amyotrophic Lateral Sclerosis (ALS) disease

Prasad, Archana and Patel, Basant Kumar (2017) Protein misfolding studies on TDP-43 protein involved in Amyotrophic Lateral Sclerosis (ALS) disease. PhD thesis, Indian institute of technology Hyderabad.

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Several devastating diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS) are associated with the conversion and deposition of native soluble protein into insoluble, highly ordered, fibrillar aggregates termed amyloid. Amyotrophic Lateral Sclerosis (ALS) is a fatal disease associated with motor neuron degeneration and thus far, there is no cure or effective treatment for ALS. Notably, presence of TDP-43 aggregates in ALS cases of diverse etiology suggests its importance in ALS pathology. TDP-43 is a 43 kDa, 414 amino-acid-long, versatile RNA/DNA-binding protein involved in cellular process such as RNAmetabolism, apoptosis, cell division, embryo development, and stress response, etc. Full-length TDP-43 has been shown to form structurally stable spherical oligomers with high neurotoxicity and reduced DNA binding capability. The aberrant cleavage of TDP-43 by caspases releases C-terminal fragments causing cellular toxicity and enhanced aggregation. The 25-35 kDa fragments are more prone to phosphorylation than full-length TDP-43, and form ubiquitinated, hyper-phosphorylated cytoplasmic inclusions in affected neuron cells. Several abnormal post-translational modifications such as acetylation and deamidation, etc. have been identified in ALS patients. Further, dimers of full-length TDP-43 and C-terminal fragments have also been identified in the ALS-affected human brains but their role in ALS pathogenesis is unknown. Here, we recombinantly expressed and purified human TDP-432C protein (23 kDa, aa 193-414) and achieved aggregation into amyloid using a modified method. New information about TDP-432C amyloid aggregation was obtained by investigating the effect of kosmotropic and chaotropic salts on amyloid aggregation rate. TDP-43 was chemically acetylated and its in vitro aggregation was then examined. Furthermore, dimeric TDP-432C was purified by size-exclusion chromatography, and its aggregation behaviour was also observed for amyloid formation. Small acridine-based compounds were used to evaluate inhibitory potential in vitro on TDP-432C aggregation. After identification of a promising candidate AIM4, further in vivo aggregation inhibition studies were carried out in an erg6 deletion mutant of S. cerevisiae yeast, using full-length TDP-43-YFP protein. We observed that TDP-432C aggregates showed amyloid-like binding with Thioflavin-T and Congo red. Far-UV CD spectra showed negative ellipticity at 218 nm further indicating that TDP-432C aggregates were β-sheet rich, as seen in amyloids. AFM images showed fibrillar aggregates several hundred nanometers long with heights of about 3-4 nm. Kosmotropic anions increased TDP-432C aggregation rate and decreased the lag period, with increasing salt concentration, while the opposite trend was observed with chaotropic anions. Novel lysine acetylation sites on TDP-43 were identified by mass spectrometry. Acetylation abolished TDP-43 amyloid aggregation indicating the importance of lysine residues in the aggregation process. TDP-432C dimers were isolated and were found to be disulfide-linked. Amyloid-like aggregation from TDP-432C dimer was also achieved, providing new insight into the TDP-432C aggregation process. Among, the different compounds investigated for inhibitory potential against in vitro TDP-432C aggregation, AIM4 displayed the highest inhibition arresting the aggregation of TDP-432C into small, non-amyloidogenic, oligomers. Additionally, in S. cerevisiae erg6 deletion strain, AIM4 treated yeast cells after induction with galactose displayed diffused fluorescence and fewer TDP-43-YFP dots per cell compared to untreated cells. AIM4 treated cells also displayed relatively fewer number of TDP-43-YFP punctate foci per cell compared to untreated control, suggesting dissolution of the TDP-43-YFP aggregates. These observations support the ability of AIM4 to influence the aggregation of TDP-43-YFP in yeast cells.

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IITH Creators:
IITH CreatorsORCiD
Patel, Basant Kumar
Item Type: Thesis (PhD)
Subjects: Others > Biotechnology
Divisions: Department of Biotechnology
Depositing User: Team Library
Date Deposited: 20 May 2019 03:48
Last Modified: 21 Sep 2019 08:02
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