Investigation of the role of prolyl isomerase A in protein misfolding associated with neurodegenerative diseases
by Maria Babu
Date of Examination:2023-01-20
Date of issue:2023-06-26
Advisor:Prof. Dr. Markus Zweckstetter
Referee:Prof. Dr. Markus Zweckstetter
Referee:Prof. Dr. Tiago Fleming Outeiro
Persistent Address:
http://dx.doi.org/10.53846/goediss-9950
Files in this item
Name:maria_babu_thesis.pdf
Size:5.28Mb
Format:PDF
Description:Ph.D. Thesis
The following license files are associated with this item:
Abstract
English
Misfolded proteins and disrupted homeostasis are key characteristics of neurodegenerative diseases. There is a strong association between intrinsically disordered proteins and pathogenic misfolding in neurodegenerative diseases. As a result of their exposed amino acid chain, intrinsically disordered proteins are highly vulnerable to aberrant interactions, which are frequently triggered by mutations or any cellular perturbations that modulate their functional conformation. Although abnormal interactions and misfolding start with a few intrinsically disordered proteins in most diseases, many interactor proteins essential for proper cellular function eventually co-aggregate with the misfolding proteins. Loss of crucial proteins into aggregates leads to disrupted homeostasis and neuronal death. Prolyl isomerases are a class of chaperones that bind to proline residues and enhance their cis/trans isomerization rate. Through their unique action on proline residues, prolyl isomerases play an essential role in maintaining protein homeostasis: they assist in the de novo protein folding of globular proteins and regulate the misfolding of intrinsically disordered proteins. In this work, we investigated the involvement of prolyl isomerase A (PPIA)-the most abundant kind of prolyl isomerase- in two different cases of pathogenic protein misfolding: a) protein misfolding in C9orf72-Amyotrophic Lateral Sclerosis (ALS)/Frontotemporal Dementia (FTD) and b) protein misfolding from biomolecular condensates. In the first part of the thesis, we uncovered a novel neurotoxic pathway that results from the inhibition of PPIA in the neurodegenerative diseases-ALS and FTD. Hexanucleotide repeat expansion mutation in the C9orf72 gene is the most common cause of familial ALS and FTD. The toxicity of this mutation is attributed to the aberrant interactions of the disordered dipeptide repeat polymers that are translated from the repeat expanded region. Here, we showed that proline/arginine repeat polymers bind to the catalytic activity site of PPIA. As a result, these repeats inhibited PPIA’s de novo protein folding activity, which is essential in maintaining cellular homeostasis. NMR spectroscopy and X-Ray crystallography studies revealed the structural basis of proline/arginine repeat polymer binding to PPIA. It provided insights into the importance of the specific combination of proline and arginine residues in the inhibition of PPIA activity. In summary, our data revealed a pathway that can lead to disrupted homeostasis in C9orf72-ALS/FTD due to the loss of function of PPIA. In the second part, we investigated the regulatory role of prolyl isomerase on biomolecular condensation. Biomolecular condensates are enriched in proline-rich intrinsically disordered proteins, many of which are involved in neurodegeneration associated misfolding. The high concentration of proteins inside the condensate puts them at increased risk of undergoing pathogenic interactions, emphasizing the need for tight regulation. Despite the abundance of proline residues in intrinsically disordered sequences, the regulatory role of prolyl isomerases on the liquid-liquid phase separation of intrinsically disordered proteins remains unknown. Our study shows that PPIA can migrate into self-assembled liquid-like droplets of Alzheimer’s disease protein Tau and complex coacervates of a proline/arginine repeat polymer with RNA. At increased concentrations of PPIA, the two proline-rich condensates dissolved back into the mixed state. Binding and isomerization studies of PPIA with the proline/arginine repeat polymer and Tau protein provided information into the mechanism of chaperoning of liquid-liquid phase separation by PPIA. Our studies established a regulatory function of PPIA on the liquid-liquid phase separation of proline-rich intrinsically disordered proteins.
Keywords: PPIA, dipeptide repeats, Tau, intrinsically disordered proteins, proline isomerization, liquid-liquid phase separation, neurodegenerative diseases
