Thermophoresis for characterizing biomolecular interaction

Asmari, Mufarreh; Ratih, Ratih; Alhazmi, Hassan A.; Deeb, Sami El

doi:10.1016/j.ymeth.2018.02.003

Cited by 101 publications

(73 citation statements)

References 83 publications

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“…This method has been developed on the basis of the Soret effect (or thermophoresis), which describes the directed movement of particles in response to a temperature gradient in a fluid [17,18]. Particles with different sizes, structures or charges may exhibit different thermophoretic behaviors [19]. In this context we examined the thermophoresis of unbound and bound molecules through titrating D3 to fluorescein isothiocyanate (FITC)-Aβ42, or vice versa.…”

Section: Resultsmentioning

confidence: 99%

Interference with Amyloid-β Nucleation by Transient Ligand Interaction

et al. 2019

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Amyloid-β peptide (Aβ) is an intrinsically disordered protein (IDP) associated with Alzheimer’s disease. The structural flexibility and aggregation propensity of Aβ pose major challenges for elucidating the interaction between Aβ monomers and ligands. All-D-peptides consisting solely of D-enantiomeric amino acid residues are interesting drug candidates that combine high binding specificity with high metabolic stability. Here we characterized the interaction between the 12-residue all-D-peptide D3 and Aβ42 monomers, and how the interaction influences Aβ42 aggregation. We demonstrate for the first time that D3 binds to Aβ42 monomers with submicromolar affinities. These two highly unstructured molecules are able to form complexes with 1:1 and other stoichiometries. Further, D3 at substoichiometric concentrations effectively slows down the β-sheet formation and Aβ42 fibrillation by modulating the nucleation process. The study provides new insights into the molecular mechanism of how D3 affects Aβ assemblies and contributes to our knowledge on the interaction between two IDPs.

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Section: Resultsmentioning

confidence: 99%

Interference with Amyloid-β Nucleation by Transient Ligand Interaction

et al. 2019

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“…MST was used to characterize protein interactions as described previously ( Seidel et al, 2013 ; Asmari et al, 2018 ). Briefly, HEK293T cells were cultured at 37°C with 5% CO 2 in DMEM supplemented with 10% FBS (HyClone), 100 U/ml penicillin, and 100 mg/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

The amino acid transporter SLC-36.1 cooperates with PtdIns3P 5-kinase to control phagocytic lysosome reformation

Gan

Wang

Zhang

et al. 2019

Journal of Cell Biology

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Phagocytic removal of apoptotic cells involves formation, maturation, and digestion of cell corpse–containing phagosomes. The retrieval of lysosomal components following phagolysosomal digestion of cell corpses remains poorly understood. Here we reveal that the amino acid transporter SLC-36.1 is essential for lysosome reformation during cell corpse clearance in Caenorhabditis elegans embryos. Loss of slc-36.1 leads to formation of phagolysosomal vacuoles arising from cell corpse–containing phagosomes. In the absence of slc-36.1, phagosome maturation is not affected, but the retrieval of lysosomal components is inhibited. Moreover, loss of PPK-3, the C. elegans homologue of the PtdIns3P 5-kinase PIKfyve, similarly causes accumulation of phagolysosomal vacuoles that are defective in phagocytic lysosome reformation. SLC-36.1 and PPK-3 function in the same genetic pathway, and they directly interact with one another. In addition, loss of slc-36.1 and ppk-3 causes strong defects in autophagic lysosome reformation in adult animals. Our findings thus suggest that the PPK-3–SLC-36.1 axis plays a central role in both phagocytic and autophagic lysosome formation.

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“…MST is a technology used to determine binding affinities between macromolecules based on their directed movement along temperature gradients in solution (supplemental Figure 5A-C). 46 Other techniques that are often used to study molecular interactions, such as surface plasmon resonance and isothermal titration calorimetry, were impractical because of the elevated nonspecific surface binding of TPO, a highly hydrophobic protein.…”

Section: Ifn-g Disrupts the Low-affinity Binding Interaction Between mentioning

confidence: 99%

Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ

Alvarado

Huntsman

Cheng

et al. 2019

Blood

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The proinflammatory cytokine interferon-γ (IFN-γ) has been implicated in human hematopoietic stem and progenitor cell (HSPC) depletion in immune-mediated bone marrow failure syndromes. We show that IFN-γ specifically prevents full engagement of thrombopoietin (TPO), a primary positive regulator of HSPC survival, to its receptor (c-MPL) via steric occlusion of the low-affinity binding site, contributing to perturbation of TPO-induced signaling pathways and decreased survival of human HSPCs. Eltrombopag, a synthetic small molecule mimetic of TPO that interacts with c-MPL at a position distinct from the extracellular binding site of TPO, bypasses this inhibition, providing an explanation for its clinical activity in bone marrow failure, despite already elevated endogenous TPO levels. Thus, IFN-γ–mediated perturbation of TPO:c-MPL complex formation and the resulting inhibition of a critical pathway of growth factor cell signaling may represent a general mechanism by which IFN-γ impairs the function of human HSPCs. This understanding could have broad therapeutic implications for various disorders of chronic inflammation.

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Thermophoresis for characterizing biomolecular interaction

Cited by 101 publications

References 83 publications

Interference with Amyloid-β Nucleation by Transient Ligand Interaction

Interference with Amyloid-β Nucleation by Transient Ligand Interaction

The amino acid transporter SLC-36.1 cooperates with PtdIns3P 5-kinase to control phagocytic lysosome reformation

Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ

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