ASU Scholarship Showcase

Sliding clamps are ring-shaped oligomeric proteins that are essential for processive deoxyribonucleic acid replication. Although crystallographic structures of several clamps have been determined, much less is known about clamp structure and dynamics in solution. Here, we characterized the intrinsic solution stability and oligomerization dynamics of the homodimeric Escherichia coli β and the homotrimeric Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) clamps using single-molecule approaches. We show that E. coli β is stable in solution as a closed ring at concentrations three orders of magnitude lower than PCNA. The trimeric structure of PCNA results in slow subunit association rates and is largely responsible for the lower solution stability. Despite this large difference, the intrinsic lifetimes of the rings differ by only one order of magnitude. Our results show that the longer lifetime of the E. coli β dimer is due to more prominent electrostatic interactions that stabilize the subunit interfaces.

Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules. Herein, we demonstrate that AMPylation of Rho GTPases by VopS is a multifaceted virulence mechanism that counters several host immunity strategies. Activation of NFκB, Erk, and JNK kinase signaling pathways were inhibited in a VopS-dependent manner during infection with Vibrio parahaemolyticus. Phosphorylation and degradation of IKBα were inhibited in the presence of VopS as was nuclear translocation of the NFκB subunit p65. AMPylation also prevented the generation of superoxide by the phagocytic NADPH oxidase complex, potentially by inhibiting the interaction of Rac and p67. Furthermore, the interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a nucleic acid programmable protein array and discovered that Rac1 binds to the protein C1QA, a protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted by AMPylation. We conclude that AMPylation of Rho Family GTPases by VopS results in diverse inhibitory consequences during infection beyond the most obvious phenotype, the collapse of the actin cytoskeleton.

A new class of highly active solid base catalysts for biodiesel production was developed by creating hierarchically porous aluminosilicate geopolymer with affordable precursors and modifying the material with varying amounts of calcium. For the catalysts containing ≥8 wt% Ca, almost 100% conversion has been achieved in one hour under refluxing conditions with methanol solvent, and the high catalytic activity was preserved for multiple regeneration cycles. Temperature-programed desorption studies of CO2 indicate that the new base catalyst has three different types of base sites on its surface whose strengths are intermediate between MgO and CaO. The detailed powder X-ray diffraction (PXRD) and X-ray photoelectron spectroscopic (XPS) studies show that the calcium ions were incorporated into the aluminosilicate network of the geopolymer structure, resulting in a very strong ionicity of the calcium and thus the strong basicity of the catalysts. Little presence of CaCO3 in the catalysts was indicated from the thermogravimetric analysis (TGA), XPS and Fourier transform infrared spectroscopy (FT-IR) studies, which may contribute to the observed high catalytic activity and regenerability. The results indicate that new geopolymer-based catalysts can be developed for cost-effective biodiesel production.

Most current approaches for quantification of RNA species in their natural spatial contexts in single cells are limited by a small number of parallel analyses. Here we report a strategy to dramatically increase the multiplexing capacity for RNA analysis in single cells in situ. In this method, transcripts are detected by fluorescence in situ hybridization (FISH). After imaging and data storage, the fluorescence signal is efficiently removed by photobleaching. This enables the reinitiation of FISH to detect other RNA species in the same cell. Through reiterative cycles of hybridization, imaging and photobleaching, the identities, positions and copy numbers of a large number of varied RNA species can be quantified in individual cells in situ. Using this approach, we analyzed seven different transcripts in single HeLa cells with five reiterative RNA FISH cycles. This approach has the potential to detect over 100 varied RNA species in single cells in situ, which will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies.

Multilayer structures of TiO₂/Ag/TiO₂ have been deposited onto flexible substrates by room temperature sputtering to develop indium-free transparent composite electrodes. The effect of Ag thicknesses on optical and electrical properties and the mechanism of conduction have been discussed. The critical thickness (t_c) of Ag mid-layer to form a continuous conducting layer is 9.5 nm and the multilayer has been optimized to obtain a sheet resistance of 5.7 Ω/sq and an average optical transmittance of 90% at 590 nm. The Haacke figure of merit (FOM) for t_c has one of the highest FOMs with 61.4 × 10^-3 Ω^-1/sq.

Lineage-committed cells of many tissues exhibit substantial plasticity in contexts such as wound healing and tumorigenesis, but the regulation of this process is not well understood. We identified the Hippo transducer WWTR1/TAZ in a screen of transcription factors that are able to prompt lineage switching of mammary epithelial cells. Forced expression of TAZ in luminal cells induces them to adopt basal characteristics, and depletion of TAZ in basal and/or myoepithelial cells leads to luminal differentiation. In human and mouse tissues, TAZ is active only in basal cells and is critical for basal cell maintenance during homeostasis. Accordingly, loss of TAZ affects mammary gland development, leading to an imbalance of luminal and basal populations as well as branching defects. Mechanistically, TAZ interacts with components of the SWI/SNF complex to modulate lineage-specific gene expression. Collectively, these findings uncover a new role for Hippo signaling in the determination of lineage identity through recruitment of chromatin-remodeling complexes.

Many drugs are effective in the early stage of treatment, but patients develop drug resistance after a certain period of treatment, causing failure of the therapy. An important example is Herceptin, a popular monoclonal antibody drug for breast cancer by specifically targeting human epidermal growth factor receptor 2 (Her2). Here we demonstrate a quantitative binding kinetics analysis of drug-target interactions to investigate the molecular scale origin of drug resistance. Using a surface plasmon resonance imaging, we measured the in situ Herceptin-Her2 binding kinetics in single intact cancer cells for the first time, and observed significantly weakened Herceptin-Her2 interactions in Herceptin-resistant cells, compared to those in Herceptin-sensitive cells. We further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding. This effect of a third molecule on drug-receptor interactions cannot be studied using traditional purified protein methods, demonstrating the importance of the present intact cell-based binding kinetics analysis.

Nucleic Acid Programmable Protein Arrays (NAPPA) have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years.

Lithium-beryllium metal hydrides, which are structurally related to their parent compound, BeH₂, offer the highest hydrogen storage capacity by weight among the metal hydrides (15.93 wt. % of hydrogen for LiBeH₃). Challenging synthesis protocols have precluded conclusive determination of their crystallographic structure to date, but here we analyze directly the hydrogen hopping mechanisms in BeH₂ and LiBeH₃ using quasielastic neutron scattering, which is especially sensitive to single-particle dynamics of hydrogen. We find that, unlike its parent compound BeH₂, lithium-beryllium hydride LiBeH₃ exhibits a sharp increase in hydrogen mobility above 265 K, so dramatic that it can be viewed as melting of hydrogen sublattice. We perform comparative analysis of hydrogen jump mechanisms observed in BeH₂ and LiBeH₃ over a broad temperature range. As microscopic diffusivity of hydrogen is directly related to its macroscopic kinetics, a transition in LiBeH₃ so close to ambient temperature may offer a straightforward and effective mechanism to influence hydrogen uptake and release in this very lightweight hydrogen storage compound.

Rationale: Cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. However, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays.

Methods: In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot.

Results: Even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. Protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by M-NAPPA and non-multiplexed NAPPA arrays. An ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining M-NAPPA with a photolithography-based silicon nano-well platform. Finally, four new tuberculosis-related antigens in guinea pigs vaccinated with Bacillus Calmette-Guerin (BCG) were identified with M-NAPPA and validated with ELISA.

Conclusion: All data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research.