Austin Huang

The Effect of a ΔK280 Mutation on the Unfolded State of a Microtubule-Binding Repeat in Tau

Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimers disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW), samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2) in wild-type (WT) tau and a ΔK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and ΔK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of β-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates.

Residual structure within the disordered C‐terminal segment of p21Waf1/Cip1/Sdi1 and its implications for molecular recognition

Probably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein‐protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets. How does this small, unstructured protein recognize each of these targets with high affinity? Here, we characterize residual structural elements of the C‐terminal segment of p21 encompassing residues 145–164 using a combination of NMR measurements and molecular dynamics simulations. The N‐terminal half of the peptide has a significant helical propensity which is recognized by calmodulin while the C‐terminal half of the peptide prefers extended conformations that facilitate binding to the proliferating cell nuclear antigen (PCNA). Our results suggest that the final bound conformations of p21 (145–164) pre‐exist in the free peptide even without its binding partners. While the conformational flexibility of the p21 peptide is essential for adapting to diverse binding environments, the intrinsic structural preferences of the free peptide enable promiscuous yet high affinity binding to a diverse array of molecular targets.

Finding order within disorder: elucidating the structure of proteins associated with neurodegenerative disease

A number of neurodegenerative disorders, such as Alzheimers disease and Parkinsons disease, involve the formation of protein aggregates. The primary constituent of these aggregates belongs to a unique class of heteropolymers known as intrinsically disordered proteins (IDPs). While many proteins fold to a unique conformation that is determined by their amino acid sequence, IDPs do not adopt a single well-defined conformation in solution. Instead, they populate a heterogeneous set of conformers under physiological conditions. Despite this intrinsic propensity for disorder, a number of these proteins can form ordered aggregates both in vitro and in vivo. As the formation of these aggregates may play an important role in disease pathogenesis, a detailed structural characterization of these proteins and their mechanism of aggregation is of critical importance. However, new methods are needed to understand the diversity of structures that make up the unfolded ensemble of these systems. In this review, we discuss recent advances in the structural analysis and modeling of IDPs involved in neurodegenerative diseases. While there are challenges in both the experimental characterization and the modeling of such proteins, a comprehensive understanding of the structure of IDPs will likely facilitate the development of effective therapies for a number of neurodegenerative diseases.

An EGFR autocrine loop encodes a slow-reacting but dominant mode of mechanotransduction in a polarized epithelium

The mechanical landscape in biological systems can be complex and dynamic, with contrasting sustained and fluctuating loads regularly superposed within the same tissue. How resident cells discriminate between these scenarios to respond accordingly remains largely unknown. Here, we show that a step increase in compressive stress of physiological magnitude shrinks the lateral intercellular space between bronchial epithelial cells, but does so with strikingly slow exponential kinetics (time constant ~110 s). We confirm that epidermal growth factor (EGF)‐family ligands are constitutively shed into the intercellular space and demonstrate that a step increase in compressive stress enhances EGF receptor (EGFR) phosphorylation with magnitude and onset kinetics closely matching those predicted by constant‐rate ligand shedding in a slowly shrinking intercellular geometry. Despite the modest degree and slow nature of EGFR activation evoked by compressive stress, we find that the majority of transcriptomic responses to sustained mechanical loading require ongoing activity of this autocrine loop, indicating a dominant role for mechanotransduction through autocrine EGFR signaling in this context. A slow deformation response to a step increase in loading, accompanied by synchronous increases in ligand concentration and EGFR activation, provides one means for cells to mount a selective and context‐appropriate response to a sustained change in mechanical environment.—Kojic, N., Chung, E., Kho, A. T., Park, J.‐A., Huang, A., So, P. T. C., Tschumperlin, D. J. An EGFR autocrine loop encodes a slow‐reacting but dominant mode of mechano‐transduction in a polarized epithelium. FASEB J. 24, 1604‐1615 (2010). www.fasebj.org

A 3-D model of ligand transport in a deforming extracellular space

Cells communicate through shed or secreted ligands that traffic through the interstitium. Force-induced changes in interstitial geometry can initiate mechanotransduction responses through changes in local ligand concentrations. To gain insight into the temporal and spatial evolution of such mechanotransduction responses, we developed a 3-D computational model that couples geometric changes observed in the lateral intercellular space (LIS) of mechanically loaded airway epithelial cells to the diffusion-convection equations that govern ligand transport. By solving the 3-D fluid field under changing boundary geometries, and then coupling the fluid velocities to the ligand transport equations, we calculated the temporal changes in the 3-D ligand concentration field. Our results illustrate the steady-state heterogeneities in ligand distribution that arise from local variations in interstitial geometry, and demonstrate that highly localized changes in ligand concentration can be induced by mechanical loading, depending on both local deformations and ligand convection effects. The occurrence of inhomogeneities at steady state and in response to mechanical loading suggest that local variations in ligand concentration may have important effects on cell-to-cell variations in basal signaling state and localized mechanotransduction responses.

Quantification of three-dimensional dynamics of intercellular geometry under mechanical loading using a weighted directional adaptive-threshold method

Capturing and quantifying dynamic changes in three-dimensional cellular geometries on fast time scales is a challenge because of mechanical limitations of imaging systems as well as of the inherent tradeoffs between temporal resolution and image quality. We have combined a custom high-speed two-photon microscopy approach with a novel image segmentation method, the weighted directional adaptive-threshold (WDAT), to quantify the dimensions of intercellular spaces of cells under compressive stress on timescales previously inaccessible. The adaptation of a high-speed two-photon microscope addressed the need to capture events occurring on short timescales, while the WDAT method was developed to address artifacts of standard intensity-based analysis methods when applied to this system. Our novel approach is demonstrated by the enhanced temporal analysis of the three-dimensional cellular and extracellular deformations that accompany compressive loading of airway epithelial cells.