76 research outputs found
Thermophoretic melting curves quantify the conformation and stability of RNA and DNA
Measuring parameters such as stability and conformation of biomolecules, especially of nucleic acids, is important in the field of biology, medical diagnostics and biotechnology. We present a thermophoretic method to analyse the conformation and thermal stability of nucleic acids. It relies on the directed movement of molecules in a temperature gradient that depends on surface characteristics of the molecule, such as size, charge and hydrophobicity. By measuring thermophoresis of nucleic acids over temperature, we find clear melting transitions and resolve intermediate conformational states. These intermediate states are indicated by an additional peak in the thermophoretic signal preceding most melting transitions. We analysed single nucleotide polymorphisms, DNA modifications, conformational states of DNA hairpins and microRNA duplexes. The method is validated successfully against calculated melting temperatures and UV absorbance measurements. Interestingly, the methylation of DNA is detected by the thermophoretic amplitude even if it does not affect the melting temperature. In the described setup, thermophoresis is measured all-optical in a simple setup using a reproducible capillary format with only 250 nl probe consumption. The thermophoretic analysis of nucleic acids shows the techniqueâs versatility for the investigation of nucleic acids relevant in cellular processes like RNA interference or gene silencing
TAp73 is a central transcriptional regulator of airway multiciliogenesis.
Motile multiciliated cells (MCCs) have critical roles in respiratory health and disease and are essential for cleaning inhaled pollutants and pathogens from airways. Despite their significance for human disease, the transcriptional control that governs multiciliogenesis remains poorly understood. Here we identify TP73, a p53 homolog, as governing the program for airway multiciliogenesis. Mice with TP73 deficiency suffer from chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance. Organotypic airway cultures pinpoint TAp73 as necessary and sufficient for basal body docking, axonemal extension, and motility during the differentiation of MCC progenitors. Mechanistically, cross-species genomic analyses and complete ciliary rescue of knockout MCCs identify TAp73 as the conserved central transcriptional integrator of multiciliogenesis. TAp73 directly activates the key regulators FoxJ1, Rfx2, Rfx3, and miR34bc plus nearly 50 structural and functional ciliary genes, some of which are associated with human ciliopathies. Our results position TAp73 as a novel central regulator of MCC differentiation
Designer Lipid-Like Peptides: A Class of Detergents for Studying Functional Olfactory Receptors Using Commercial Cell-Free Systems
A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012)Massachusetts Institute of Technology. Undergraduate Research Opportunities Progra
Rational identification of a Cdc42 inhibitor presents a new regimen for long- term hematopoietic stem cell mobilization
Mobilization of hematopoietic stem cells (HSCs) from bone marrow (BM) to peripheral blood (PB) by cytokine granulocyte colony-stimulating factor (G-CSF) or the chemical antagonist of CXCR4, AMD3100, is important in the treatment of blood diseases. Due to clinical conditions of each application, there is a need for continued improvement of HSC mobilization regimens. Previous studies have shown that genetic ablation of the Rho GTPase Cdc42 in HSCs results in their mobilization without affecting survival. Here we rationally identified a Cdc42 activity-specific inhibitor (CASIN) that can bind to Cdc42 with submicromolar affinity and competitively interfere with guanine nucleotide exchange activity. CASIN inhibits intracellular Cdc42 activity specifically and transiently to induce murine hematopoietic stem/progenitor cell egress from the BM by suppressing actin polymerization, adhesion, and directional migration of stem/progenitor cells, conferring Cdc42 knockout phenotypes. We further show that, although, CASIN administration to mice mobilizes similar number of phenotypic HSCs as AMD3100, it produces HSCs with better long-term reconstitution potential than that by AMD3100. Our work validates a specific small molecule inhibitor for Cdc42, and demonstrates that signaling molecules downstream of cytokines and chemokines, such as Cdc42, constitute a useful target for long-term stem cell mobilization
Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes
Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interac- tions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin g1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs
Molecular Interaction Studies Using Microscale Thermophoresis
The use of infrared laser sources for creation of localized temperature fields has opened new possibilities for basic research and drug discovery. A recently developed technology, Microscale Thermophoresis (MST), uses this temperature field to perform biomolecular interaction studies. Thermophoresis, the motion of molecules in temperature fields, is very sensitive to changes in size, charge, and solvation shell of a molecule and thus suited for bioanalytics. This review focuses on the theoretical background of MST and gives a detailed overview on various applications to demonstrate the broad applicability. Experiments range from the quantification of the affinity of low-molecular-weight binders using fluorescently labeled proteins, to interactions between macromolecules and multi-component complexes like receptor containing liposomes. Information regarding experiment and experimental setup is based on the Monolith NT.115 instrument (NanoTemper Technologies GmbH)
Peptide surfactants for cell-free production of functional G proteincoupled receptors
Two major bottlenecks in elucidating the structure and function of membrane proteins are the difficulty of producing large quantities of functional receptors, and stabilizing them for a sufficient period of time. Selecting the right surfactant is thus crucial. Here we report using peptide surfactants in commercial Escherichia coli cell-free systems to rapidly produce milligram quantities of soluble G protein-coupled receptors (GPCRs). These include the human formyl peptide receptor, human trace amine-associated receptor, and two olfactory receptors. The GPCRs expressed in the presence of the peptide surfactants were soluble and had α-helical secondary structures, suggesting that they were properly folded. Microscale thermophoresis measurements showed that one olfactory receptor expressed using peptide surfactants bound its known ligand heptanal (molecular weight 114.18). These short and simple peptide surfactants may be able to facilitate the rapid production of GPCRs, or even other membrane proteins, for structure and function studies.United States. Defense Advanced Research Projects Agency (HR0011-09-C-0012)China University of PetroleumNanoSystems Initiative MunichUniversitaÌt MuÌnchen (Center for NanoScience
Transcription factor TAp73 and microRNA-449 complement each other to support multiciliogenesis.
Motile cilia serve vital functions in development, homeostasis, and regeneration. We recently demonstrated that TAp73 is an essential transcriptional regulator of respiratory multiciliogenesis. Here, we show that TAp73 is expressed in multiciliated cells (MCCs) of diverse tissues. Analysis of TAp73 mutant animals revealed that TAp73 regulates Foxj1, Rfx2, Rfx3, axonemal dyneins Dnali1 and Dnai1, plays a pivotal role in the generation of MCCs in male and female reproductive ducts, and contributes to fertility. However, the function of MCCs in the brain appears to be preserved despite the loss of TAp73, and robust activity of cilia-related networks is maintained in the absence of TAp73. Notably, TAp73 loss leads to distinct changes in ciliogenic microRNAs: miR34bc expression is reduced, whereas the miR449 cluster is induced in diverse multiciliated epithelia. Among different MCCs, choroid plexus (CP) epithelial cells in the brain display prominent miR449 expression, whereas brain ventricles exhibit significant increase in miR449 levels along with an increase in the activity of ciliogenic E2F4/MCIDAS circuit in TAp73 mutant animals. Conversely, E2F4 induces robust transcriptional response from miR449 genomic regions. To address whether increased miR449 levels in the brain maintain the multiciliogenesis program in the absence of TAp73, we deleted both TAp73 and miR449 in mice. Although loss of miR449 alone led to a mild ciliary defect in the CP, more pronounced ciliary defects and hydrocephalus were observed in the brain lacking both TAp73 and miR449. In contrast, miR449 loss in other MCCs failed to enhance ciliary defects associated with TAp73 loss. Together, our study shows that, in addition to the airways, TAp73 is essential for generation of MCCs in male and female reproductive ducts, whereas miR449 and TAp73 complement each other to support multiciliogenesis and CP development in the brain
Detection of Thermoresponsive Polymer Phase Transition in Dilute Low-Volume Format by Microscale Thermophoretic Depletion
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