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Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses
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Published on: April 1, 2022

Specific lipids modulate the transporter associated with antigen processing (TAP).

Christian Schölz1, David Parcej, Christer S Ejsing

  • 1Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt am Main, Germany.

The Journal of Biological Chemistry
|March 2, 2011
PubMed
Summary
This summary is machine-generated.

The transporter associated with antigen processing (TAP) is crucial for adaptive immunity. Its activity is influenced by lipids, with phosphatidylinositol and phosphatidylethanolamine stimulating it, while cholesterol inhibits it.

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Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • The transporter associated with antigen processing (TAP) is essential for adaptive immunity.
  • TAP translocates proteasomal degradation products into the endoplasmic reticulum for MHC class I loading.
  • Understanding TAP function requires robust methods for its isolation and analysis.

Purpose of the Study:

  • To establish an overexpression system for functional and structural analysis of TAP.
  • To investigate the influence of the lipid environment on TAP activity.
  • To identify specific lipids affecting TAP function.

Main Methods:

  • Overexpression of TAP in Pichia pastoris.
  • Optimization of solubilization and purification protocols.
  • Functional assays in membrane, solubilized, purified, and reconstituted states.
  • Lipid profiling using liquid chromatography Fourier transform mass spectrometry.

Main Results:

  • Successfully overexpressed and purified heterodimeric TAP complex (30 mg/L).
  • Demonstrated that the native lipid environment significantly impacts TAP activity.
  • Identified phosphatidylinositol and phosphatidylethanolamine as stimulators of antigen translocation.
  • Showed that cholesterol negatively affects TAP activity.

Conclusions:

  • Pichia pastoris is a suitable host for TAP overexpression and purification.
  • Lipid composition is critical for regulating TAP-mediated antigen transport.
  • Specific phospholipids and cholesterol modulate TAP function, offering insights into immune regulation.