Antigenic Peptide Recognition on the Human ABC Transporter TAP Resolved by DNP-Enhanced Solid-State NMR Spectroscopy

The human transporter associated with antigen processing (TAP) is a 150 kDa heterodimeric ABC transport complex that selects peptides for export into the endoplasmic reticulum and subsequent loading onto major histocompatibility complex class I molecules to trigger adaptive immune responses against virally or malignantly transformed cells. To date, no atomic-resolution information on peptide–TAP interactions has been obtained, hampering a mechanistic understanding of the early steps of substrate translocation catalyzed by TAP. Here, we developed a mild method to concentrate an unstable membrane protein complex and combined this effort with dynamic nuclear polarization enhanced magic angle spinning solid-state NMR to study this challenging membrane protein–substrate complex. We were able to determine the atomic-resolution backbone conformation of an antigenic peptide bound to human TAP. Our NMR data also provide unparalleled insights into the nature of the interactions between the side chains of the antigen peptide and TAP. By combining NMR data and molecular modeling, the location of the peptide binding cavity has been identified, revealing a complex scenario of peptide–TAP recognition. Our findings reveal a structural and chemical basis of substrate selection rules, which define the crucial function of this ABC transporter in human immunity and health. This work is the first NMR study of a eukaryotic transporter protein and presents the power of solid-state NMR in this growing field

IFNα-activated signalling pathways in THP1 and A375 cells.

<p>THP1 (A) and A375 (B) cells were stimulated with the indicated concentrations of IFNα. As controls, THP-1 cells were additionally stimulated with IL-6/sIL-6R and LPS; A375 cells were stimulated with OSM or IL-1β. The phosphorylation levels of STAT1, STAT3, ERK1/2 and Akt were detected via Western blot analysis. Displayed are mean values of at least three independent experiments.</p

The inhibition of Janus kinases abrogates the IFNα-upregulated TAP1 expression in THP1 and A375 cells.

<p>(A) THP1 (left) or A375 (right) cells were pre-incubated with the indicated amounts of JAK inhibitor I (JI-1), followed by stimulation with 100 U/ml or 1000 U/ml IFNα, respectively. Western blot analysis was performed using a specific antibody against phosphorylated STAT1 (STAT1-pY701), a STAT1 antibody recognizing the protein irrespective of its phosphorylation status and with an α-tubulin antibody. (B) Phosphorylation intensities were quantified by chemiluminescence analysis and normalization to loading controls. Shown are the means (n = 3) with standard error of mean (SEM, two-tailed, paired Student’s t-test, * p<0.05, ** p<0.01, *** p<0.001). (C) THP1 (left) or A375 (right) cells were pretreated with JI-1, and subsequently exposed to 100 U/ml or 1000 U/ml IFNα. Relative expression levels of TAP1 were analyzed by qRT-PCR and normalized to 18SrRNA as internal reference. Shown are -fold changes relative to unstimulated control = SEM (n = 3). Statistical significance was evaluated by performing a two-tailed, paired Student’s t-test (** p<0.01, *** p<0.001).</p

IFNα stimulates TAP1 expression in mouse blood and tumor tissue and suppresses subcutaneous melanoma metastasis in a murine model for malignant melanoma.

<p>C57BL/6 mice were inoculated subcutaneously with B16F1 malignant melanoma cells (day 0). The mice received either no treatment (control group, n = 3) or recombinant murine IFNα (n = 3). IFNα treatment (10.000 IU) started on day +3 after application of B16F1 cells for 5 consecutive days. (A) Measurement of tumor growth in control mice and IFNα treated mice. (B) On day +7.5, three IFNα treated and three control mice were sacrificed and blood was collected for qRT-PCR analysis of TAP1 expression. (C) Three days after the last injection of IFNα (day +11), three mice of the IFNα treated and three mice of the control group were sacrificed and tumors were excised for qRT-PCR analysis of TAP1 expression. Statistical analysis was performed using unpaired Student’s t-test (* p<0.05; not significant (n.s.)).</p

IFNα-2b (IntronA) stimulates TAP1 expression in peripheral blood mononuclear cells (PBMC) of patients (n = 18) with malignant melanoma receiving adjuvant high-dose immunotherapy.

<p>(A) The actual administered dose of IFNα-2b was about 20 million IU/m² dependent on the clinically observed side effects. Only days of IFNα treatment are shown. (B) mRNA expression of TAP1 in PBMCs of patients treated with adjuvant high-dose IFNα-2b. Statistical analysis of TAP expression was performed using the <i>Statistical Analysis System</i> of the SAS Institute Inc. (Cary, NC, USA).</p

Stimulatory effects of IFNα on TAP expression, peptide binding and transport.

<p>THP1 cells and A375 cells were treated with the indicated concentrations of IFNα for 3 hours (A, B). After stimulation, TAP1 mRNA and 18SrRNA mRNA (as internal reference) were measured by qRT-PCR analysis. Depicted are -fold changes relative to untreated cells = standard error of mean (SEM) of three independent experiments. (C) TAP1 protein expression is induced by IFNα in THP1 cells measured by western blot analysis. (D) TAP dependent peptide-binding sites are increased significantly by IFNα in THP1 cells (p = 0.0072). (E) ATP-dependent peptide transport is stimulated significantly by IFNα in THP1 cells (p = 0.0006). Statistical analysis was performed using unpaired Student’s t-test (** p<0.01, *** p<0.001).</p