DOI: 10.5061/dryad.w9ghx3g31
title: T cell cholesterol transport links intestinal immune responses to dietary lipid absorption

description: The intrinsic pathways that control membrane organization in immune cells
 and the impact of such pathways on cellular functions are not well
 defined. Here we show that the nonvesicular cholesterol transporter
 Aster-A links plasma membrane (PM) cholesterol availability in T cells to
 immune signaling and systemic metabolism. Aster-A is recruited to the PM
 during T-cell receptor (TCR) activation, where it facilitates the removal
 of “accessible” cholesterol. Loss of Aster-A leads to PM cholesterol
 accumulation, resulting in enhanced TCR nano-clustering and signaling, and
 Th17 cytokine production. Furthermore, Aster-A associates with STIM1 and
 negatively regulates STIM1-dependent Ca2+ flux during activation of mouse
 and human T cells. Finally, mucosal Th17 response towards commensals is
 restrained by PM cholesterol remodeling. Ablation of Aster-A in T cells
 stimulates IL-22 production, which reduces intestinal fatty acid
 absorption and confers resistance to diet-induced obesity. These findings
 delineate a multi-tiered regulatory scheme linking immune cell lipid flux
 to nutrient absorption and systemic physiology.
description: <em>Biotin affinity purification and sample preparation for
 proximity labeling</em> EL-4 cell lines stably
 transduced with pRetroX-3xHA-TurboID-Aster-A or pRetroX-3HA-TurboID (empty
 vector) were constructed as described above. Tight TRE promoter has a
 leakage expression in EL-4 cell line, which resulted in moderate
 expression of 3x-HA-TurboID-Aster-A and stable cell lines were therefore
 used without doxycycline induction. TurboID or
 TurboID-Aster-A expressing EL-4 cells were cultured in 10% FCS complete
 culture media or starved with a cholesterol-depletion media containing 1%
 Lipoprotein-deficient serum, 5 μM Simvastatin, 50 µM Mevalonate for 18-24
 h. Depleted cells were either loaded with 100 μΜ MβCD-Cholesterol or
 maintained in fresh cholesterol-depletion media for 1 h and 15 minutes.
 Cholesterol-rich samples are maintained in 10% FCS complete media for the
 same time. 50 μM Biotin was added to the culture media for the last 30
 minutes to facilitate labeling. Isolation of biotinylated proteins were
 performed based on a modified protocol (<em>41</em>). Cells
 were collected and washed extensively with cold PBS and incubated with
 lysis buffer (RIPA buffer containing 1X Halt protease and phosphatase
 inhibitor and 1 mM PMSF) with gentle agitation for 30 minutes on ice.
 Lysate were cleared by centrifugation at 12,000 xg for 15 minutes and
 protein concentration was determined by BCA assay. Equal quantities
 (900 μg) of cleared lysates in lysis buffer were incubated with 75 μl
 Streptavidin magnetic beads (Thermo Fisher Pierce) overnight at 4 °C with
 gentle rotation. The beads were then washed at room temperature twice in
 lysis buffer for 5 minutes each, once with 1 mL 1 M KCl for 2 minutes,
 once with 1 mL 0.1 M Na2CO3 for ~10 seconds, once with 1 mL 2 M urea in 10
 mM Tris-HCl (pH 8.0) for ~10 seconds, and again twice with 1 mL lysis
 buffer for 2 minutes.  <em>In solution tryptic
 digestion for mass spectrometry</em> The samples
 were heated at 95 °C in elution buffer (12 mM sodium lauroyl sarcosine,
 0.5% sodium deoxycholate, 50 mM triethylammonium bicarbonate (TEAB)
 containing tris(2-carboxyethyl)phosphine (10 mM) and chloroacetamide (40
 mM) for 20 minutes. The samples were then diluted 5-fold with 50 mM TEAB
 and digested with trypsin (1 μg) for 16 h at 37 °C. A 1:1 (volume/volume)
 ratio of ethyl acetate plus 1% trifluoroacetic acid (TFA) was added to the
 samples and samples were vortexed for 5 minutes. Samples were centrifuged
 at 16000 xg for 5 minutes at room temperature and the supernatant was
 discarded. The samples were desalted using a modified version of
 Rappsilber's protocol (<em>91</em>) in which the dried
 samples were reconstituted in acetonitrile/water/TFA (solvent A, 100 μL,
 2/98/0.1, v/v/v) and then loaded onto a small portion of a C18-silica disk
 (3M, Maplewood, MN) placed in a 200 μL pipette tip. Prior to sample
 loading the C18 disk was prepared by sequential treatment with methanol
 (20 μL), acetonitrile/water/TFA (solvent B, 20 μL, 80/20/0.1, v/v/v) and
 finally with solvent A (20 μL). After loading the sample, the disc was
 washed with solvent A (20 μL, eluent discarded) and eluted with solvent B
 (40 μL). The collected eluent was dried in a centrifugal vacuum
 concentrator and reconstituted in 0.1% Formic Acid (10 μL) for LC-MS/MS
 analysis. <em>LC-MS/MS
 analysis</em> Aliquots of each sample (5 μL) were
 injected onto a reverse phase nanobore HPLC column (AcuTech Scientific,
 C18, 1.8μm particle size, 360 μm x 20 cm, 150 μm ID), equilibrated in
 solvent A (water/acetonitrile/FA, 98/2/0.1, v/v/v) and eluted (300
 nL/minute) with an increasing concentration of solvent B
 (acetonitrile/water/FA, 98/2/0.1, v/v/v: min/% F; 0/0, 5/3, 18/7, 74/12,
 144/24, 153/27, 162/40, 164/80, 174/80, 176/0, 180/0) using an EASY-nLC II
 (Thermo Fisher Scientific). The effluent from the column was directed to a
 nanospray ionization source connected to a hybrid quadrupole-Orbitrap mass
 spectrometer (Q Exactive Plus, Thermo Fisher Scientific) acquiring mass
 spectra in a data-dependent mode alternating between a full scan (m/z
 350-1700, automated gain control (AGC) target 3 × 106, 50 ms maximum
 injection time, FWHM resolution 70,000 at m/z 200) and up to 15 MS/MS
 scans (quadrupole isolation of charge states 2-7, isolation window 0.7
 m/z) with previously optimized fragmentation conditions (normalized
 collision energy of 32, dynamic exclusion of 30 s, AGC target 1 × 105, 100
 ms maximum injection time, FWHM resolution 35,000 at m/z 200).
description: # T cell cholesterol transport links intestinal immune responses to
 dietary lipid absorption Dataset DOI:
 [10.5061/dryad.w9ghx3g31](10.5061/dryad.w9ghx3g31) ## Description of the
 data and file structure ### Files and variables #### File:
 wc_QE_080622_Tontonoz_Yajing_5_A-LPDS.raw **Description:** Raw mass
 spectrometry peptide identification data for pull-down of biotinylated
 proteins from EL-4 cells overexpressing TurboID-Aster-A, starved overnight
 in media containing lipoprotein deficient serum (LPDS), simvastatin and
 mevalonate.  #### File: wc_QE_080622_Tontonoz_Yajing_3_Turbo-Chol.raw
 **Description:** Raw mass spectrometry peptide identification data for
 pull-down of biotinylated proteins from EL-4 cells overexpressing only
 TurboID, starved overnight in media containing lipoprotein deficient serum
 (LPDS), simvastatin and mevalonate, and reloaded with MbCD-Cholesterol. 
 #### File: wc_QE_080622_Tontonoz_Yajing_2_Turbo-LPDS.raw
 **Description:** Raw mass spectrometry peptide identification data for
 pull-down of biotinylated proteins from EL-4 cells overexpressing only
 TurboID, starved overnight in media containing lipoprotein deficient serum
 (LPDS), simvastatin and mevalonate.  #### File:
 wc_QE_080622_Tontonoz_Yajing_4_A-FCS.raw **Description:** Raw mass
 spectrometry peptide identification data for pull-down of biotinylated
 proteins from EL-4 cells overexpressing TurboID-Aster-A, cultured in media
 with 10% lipoprotein-sufficient fetal bovine serum. #### File:
 wc_QE_080622_Tontonoz_Yajing_6_A-Chol.raw **Description:** Raw mass
 spectrometry peptide identification data for pull-down of biotinylated
 proteins from EL-4 cells overexpressing TurboID-Aster-A, starved overnight
 in media containing lipoprotein deficient serum (LPDS), simvastatin and
 mevalonate, and reloaded with MbCD-Cholesterol. #### File:
 wc_QE_080622_Tontonoz_Yajing_1_Turbo-FCS.raw **Description:** Raw mass
 spectrometry peptide identification data for pull-down of biotinylated
 proteins from EL-4 cells overexpressing only TurboID, cultured in media
 with 10% lipoprotein-sufficient fetal bovine serum. ## Code/software For
 experimental mass spectrometry data acquisition and analysis workflow, the
 raw proteomic data were searched against a Uniprot database containing the
 complete mouse proteome using SEQUEST-HT (Version 2.4, Thermo Scientific),
 which provided measurements of relative abundance of the identified
 peptides. Decoy database searching was used to generate high-confidence
 tryptic peptides (FDR &lt; 1%). Tryptic peptides containing amino acid
 sequences unique to individual proteins were used to identify and provide
 relative quantification between different proteins in each sample. We
 performed protein identification of all treatment conditions in both empty
 vector (TurboID only) and TurboID-Aster-A expressing cells.  The raw data
 can also be analyzed using open-source proteomics analysis softwares such
 as MaxQuant ([https://www.maxquant.org/](https://www.maxquant.org/)) or
 ProteoWizard
 ([https://proteowizard.sourceforge.io/](https://proteowizard.sourceforge.io/)). ## Access information Other publicly accessible locations of the data: * NONE Data was derived from the following sources: * NONE