A Novel Role for Aquaporin-5 in Enhancing Microtubule Organization and Stability

Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function

Probing native macromolecular complexes using single-molecule pull-down

Macromolecular complexes consisting of proteins, lipids, and/ or nucleic acids are ubiquitous in biological processes. Their composition, stoichiometry, order of assembly, and conformations can be heterogeneous or can change dynamically, making single-molecule studies best suited to measure these properties accurately. These interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. Recent single-molecule pull-down (SiMPull) has combined the principles of conventional co-immunoprecipitation assay with the quantitative power of single-molecule fluorescence microscopy to probe native macromolecular complexes. We have extended the SiMPull technique to investigate various macromolecular complexes and biological systems from different cellular contexts. Using lipid vesicles, instead of antibodies, we are able to capture lipid-binding proteins from whole cell extracts, thus, abrogating the step of protein purification and maintain the native physiological state of the proteins. Additionally, by optimizing the sample preparation, we could extend the application of SiMPull to the study of protein complexes directly from leaf tissues. We study the protein complex involved in organization of heterochromatin, and investigate the presence of heterogeneous population of multiprotein complexes. We also apply SiMPull to multiprotein complexes involved in the cytokinesis of Dictyostelium. Finally, we investigate the kinase activity and assembly mechanism of mechanistic target of rapamycin complexes (mTOR complexes)

Ligand modulated parallel mechanical unfolding pathways of Maltose binding proteins (MBPs)

Protein folding/unfolding are complex phenomena and it is accepted that multidomain proteins generally follow multiple pathways. Maltose binding protein is a large (a two-domain 370 amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of maltose binding protein (MBP) and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) while the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events and the remaining 38% follow path II. In the case of maltose bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) to that of maltose. The precursor protein preMBP also shows a similar behaviour upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively for preMBP

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space

The START domain potentiates HD-ZIPIII transcriptional activity

Abstract The CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) transcription factors (TFs) were repeatedly deployed over 725 million years of evolution to regulate central developmental innovations. The START domain of this pivotal class of developmental regulators was recognized over twenty years ago, but its putative ligands and functional contributions remain unknown. Here, we demonstrate that the START domain promotes HD-ZIPIII TF homodimerization and increases transcriptional potency. Effects on transcriptional output can be ported onto heterologous TFs, consistent with principles of evolution via domain capture. We also show the START domain binds several species of phospholipids, and that mutations in conserved residues perturbing ligand binding and/or its downstream conformational readout, abolish HD-ZIPIII DNA-binding competence. Our data present a model in which the START domain potentiates transcriptional activity and uses ligand-induced conformational change to render HD-ZIPIII dimers competent to bind DNA. These findings resolve a long-standing mystery in plant development and highlight the flexible and diverse regulatory potential coded within this widely distributed evolutionary module.</jats:p