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Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes

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Abstract

The phosphatidylinositol (PtdIns) family of lipids plays important roles in cell differentiation, proliferation, and migration. Abnormal expression, mutation, or regulation of their metabolic enzymes has been associated with various human diseases such as cancer, diabetes, and bipolar disorder. Recently, fluorescent derivatives have increasingly been used as chemical probes to monitor either lipid localization or enzymatic activity. However, the requirements of a good probe have not been well defined, particularly modifications on the diacylglycerol side chain partly due to challenges in generating PtdIns lipids. We have synthesized a series of fluorescent PtdIns(4,5)P2 (PIP2) and PtdIns (PI) derivatives with various lengths of side chains and tested their capacity as substrates for PI3KIα and PI4KIIα, respectively. Both capillary electrophoresis and thin-layer chromatography were used to analyze enzymatic reactions. For both enzymes, the fluorescent probe with a longer side chain functions as a better substrate than that with a shorter chain and works well in the presence of the endogenous lipid, highlighting the importance of hydrophobicity of side chains in fluorescent phosphoinositide reporters. This comparison is consistent with their interactions with lipid vesicles, suggesting that the binding of a fluorescent lipid with liposome serves as a standard for assessing its utility as a chemical probe for the corresponding endogenous lipid. These findings are likely applicable to other lipid enzymes where the catalysis takes place at the lipid-water interface.

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Acknowledgements

We thank the National Institutes of Health (GM086558 to Q.Z., CA177993 to N.A.), the American Foundation for Pharmaceutical Education (fellowship to J.W.), and the Eshelman Institute for Innovation (award to W.H.) for funding this work.

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Authors and Affiliations

  1. Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, 301 Pharmacy Lane, Chapel Hill, NC, 27599, USA

    Jarod Waybright, Weigang Huang, Xiaoyang Wang & Qisheng Zhang

  2. Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    Angela Proctor & Nancy L. Allbritton

  3. Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Campus Box 3290, Chapel Hill, NC, 27599, USA

    Nancy L. Allbritton

  4. North Carolina State University, Raleigh, NC, 27695, USA

    Nancy L. Allbritton

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  1. Jarod Waybright
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  2. Weigang Huang
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  3. Angela Proctor
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  4. Xiaoyang Wang
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  5. Nancy L. Allbritton
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Correspondence to Qisheng Zhang.

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Waybright, J., Huang, W., Proctor, A. et al. Required hydrophobicity of fluorescent reporters for phosphatidylinositol family of lipid enzymes. Anal Bioanal Chem 409, 6781–6789 (2017). https://doi.org/10.1007/s00216-017-0633-y

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  • DOI: https://doi.org/10.1007/s00216-017-0633-y

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