In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry.

Autor: Overall SA; Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland., Price LE; Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland., Albert BJ; Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA., Gao C; Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland., Alaniva N; Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland.; Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA., Judge PT; Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA.; Department of Biochemistry, Biophysics and Structural Biology, Washington University in St. Louis, St. Louis, MO 63110, USA., Sesti EL; Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA., Wender PA; Departments of Chemistry and Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA., Kyei GB; Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO 63110, USA.; Noguchi Memorial Institute for Medical Research, University of Ghana, Accra 00233, Ghana., Barnes AB; Laboratory of Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2020 Jun 30; Vol. 21 (13). Date of Electronic Publication: 2020 Jun 30.
DOI: 10.3390/ijms21134649
Abstrakt: We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15 N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15 N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP- cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR.
Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje