Parth Mangrolia

B.S. Chemical Engineering - The University of Texas at Austin (2009 - 2013)

Ph.D. Chemical Engineering - The University of Wisconsin - Madison (2013 - present)

Aggregation of β-amyloid (Aβ), 4.3-kDa monomer proteolytic cleavage product of transmembrane APP, is widely believed to cause neuronal dysfunction in Alzheimer’s disease (AD). Transthyretin (TTR) binds to Aβ and inhibits its aggregation and neurotoxicity. TTR is a 55-kDa homotetrameric protein, with each monomer containing a short α-helix and two anti-parallel β-sheets. Dimers pack into tetramers to form a hydrophobic cavity. Our group has extensively investigated the interaction between Aβ and TTR. The binding domains on TTR for Aβ oligomers include the EF loop-helix region (L82) and G β-strand (L110). An engineered monomeric TTR variant, mTTR (F87M/L110M), inhibits Aβ aggregation substantially stronger than wild-type (WT) TTR. Increased binding of Aβ to TTR via mutagenesis, chemical modification, or TTR tetramer dissociation directly correlates to enhanced suppression of Aβ aggregation.

Initial work in the group during my time focused on enhancing TTR’s protective ability against Aβ while maintaining the quaternary structure via mutagenesis or chemical modification. My research led to the discovery of a TTR variant, N98A, that demonstrated superior effectiveness at inhibiting Aβ aggregation than WT TTR. The N98A mutation is located on a flexible loop distant from the putative Aβ-binding sites and does not alter secondary and tertiary structures nor prevent correct assembly into tetramers. Under non-physiological conditions, N98A tetramers were kinetically and thermodynamically less stable than WT, suggesting a difference in the tetramer folded structure. We attributed the enhanced activity of N98A to the slightly weakened tetramer core stability, since N98A and WT bound Aβ equally. Our data indicates that even a subtle weakening change in TTR tetramer structure measurably increases TTR’s ability to inhibit Aβ aggregation!

Transthyretin, present in both blood (3-7 mM) and CSF (0.1-0.4 uM), is the major transporter of retinol-binding protein (RBP; 21-kDa; 2-4 mM) in plasma and thyroxine (T4) in cerebrospinal fluid (CSF). The interaction and effect of both ligands on TTR have been established in literature. RBP coordinates to three subunits of the TTR tetramer including two EF-helix regions, and T4 binds within the hydrophobic channel of TTR. Both ligands reportedly stabilize the quaternary structure of TTR and do not compete for binding since the binding domains are orthogonally positioned. To date, however, research into the effect of Aβ on TTR-ligand interactions have not been explored. Approximately half of the TTR present in plasma is bound to holo-RBP (retinol bound RBP) and 80% is bound to T4 in CSF. The goal of my current work is to investigate potential ligand binding competition and ligand stability effects on TTR. Specifically, I want to examine whether either ligand (RBP or T4) interferes with the other role of TTR: protection against Aβ. This work will also provide insight to the implications of Aβ on retinol transport.

My research primarily centers on characterizing protein-protein interactions using a vast toolbox of techniques. Protein aggregation kinetics and behavior were observed via dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and thioflavin T (ThT) fluorescence. Protein-ligand binding was measured using enzyme-linked immunosorbent assays (ELISA), hetero- and homo-FRET, fluorescence anisotropy, and western blots. Protein kinetic and thermodynamic stability was monitored using intrinsic tryptophan fluorescence or kinetic trapping of quaternary structure loss under high temperature, low pH, chemical denaturants or physiological conditions. Protein structure determination was performed using circular dichroism (CD), intrinsic aromatic fluorescence, and SDS-PAGE. My experience extends into protein purification and separation as well using chitin affinity column chromatography, size-exclusion chromatography (SEC), and high-performance liquid chromatography (HPLC).