Biology subjects

Leach, B. I.

Publications and source records attributed to Leach, B. I..

3 recordsLinked to original sources

bioRxiv2024

Characterization of an amyloidogenic intermediate of transthyretin by NMR relaxation dispersion

The aggregation pathway of transthyretin (TTR) proceeds through rate-limiting dissociation of the tetramer and partial misfolding of the monomers, which assemble into amyloid structures through a downhill polymerization mechanism. The structural features of the aggregation-prone monomeric intermediate are poorly understood. Characterization of amyloidogenic intermediates is challenging due to their propensity to aggregate at concentrations necessary for structural studies. NMR relaxation dispersion offers a unique opportunity to characterize these intermediates when they exchange on favorable timescales with NMR-visible ground states. To characterize the structural transitions associated with tetramer dissociation, we have analyzed ground-state chemical shift differences between the native tetramer and an engineered monomer in which the critical F87 side chain is replaced by glutamate. The secondary structure and overall fold of the F87E monomer is similar to that of the tetramer except for {beta}-strand H. This strand populates two conformations, where it is either docked on the protein core or is displaced from the edge of the {beta}-sheet formed by {beta}-strands D, A, G, and H (DAGH {beta}-sheet) and is dynamically disordered. Chemical shift differences derived from analysis of 1H/15N single, double and zero quantum relaxation dispersion data provide insights into the structure of a low-lying excited state that exchanges with the ground state of the F87E monomer at a rate of 3800 s-1. Disruption of the subunit interfaces of the TTR tetramer leads to destabilization of edge strands in both {beta}-sheets of the F87E monomer. Conformational fluctuations are propagated through the entire hydrogen bonding network of the DAGH {beta}-sheet, from the inner {beta}-strand H, which forms the strong dimer interface in the TTR tetramer, to outer strand D which is unfolded in TTR fibrils. Fluctuations are also propagated from the AB loop in the weak dimer interface to the EF helix, which undergoes structural remodeling in fibrils. The conformational fluctuations in both regions are enhanced at acidic pH where amyloid formation is most favorable. The relaxation dispersion data provide insights into the conformational dynamics of the amyloidogenic state of monomeric TTR that predispose it for structural remodeling and progression to amyloid fibrils.

biophysics↗

bioRxiv2023

Probing the dissociation pathway of a kinetically labile transthyretin mutant

Aggregation of transthyretin (TTR) is associated with devastating TTR amyloid disease. Amyloidosis begins with dissociation of the native tetramer to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the dissociation and reassembly of TTR at neutral pH remains poorly understood, due to the low population of intermediates. We use NMR studies with a highly sensitive 19F probe that allows deconvolution of relative populations of a destabilized A25T mutant at concentrations as low as 2 {micro}M. The A25T mutation, located at the weak dimer interface, perturbs both the weak and strong dimer interfaces. A tetramer-dimer-monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. All thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by vant Hoff and 19F NMR line-shape analysis. The conversion from T to T*, the slowest first-order kinetic step, shows anti-Arrhenius behavior. The 19F and methyl chemical shifts of probes close to the strong dimer interface in the dimer and T* species are degenerate, implicating interfacial perturbation as a common structural feature of these intermediate species. Molecular dynamics (MD) simulations further suggest more frequent F87 ring flipping on the nanoscale timescale in the A25T dimer than in the tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

biophysics↗

bioRxiv2019

Tankyrase inhibition sensitizes cells to CDK4 blockade

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the {beta}-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.

cancer biology↗