Tapasin assembly surveillance by the RNF185/Membralin ubiquitin ligase complex regulates MHC-I surface expression

Abstract

Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance.

Fig. 1. RNF185/MBRL-deficient cells display elevated levels of the MHC-I chaperone TPSN.

a Schematic overview of the workflow for substrate identification in MBRL-deficient mouse astrocytes. Primary cortical astrocytes from 5 parental and 5 MBRL KO P1–P3 mouse pups were cultured ex vivo for 21 days. Astrocytes were harvested, lysed and protein extracts digested. The resulting peptide samples were labelled with TMT-10-plex reagent and analysed by LC-MS/MS. Moderated t-tests, with patient accounted for in the linear model, were performed using Limma, where proteins with p-value < 0.05 were considered as statistically significant. b Identification of proteins increased and decreased in MBRL KO mouse astrocytes compared to wildtype astrocytes. Volcano plot showing the relation of log2 fold-change and -log10 p-value of protein levels in MBRL KO vs. wildtype mouse astrocytes. Proteins that are significantly decreased in MBRL KO astrocytes are on the left and labelled in Orange. Proteins that are significantly increased in MBRL KO astrocytes are on the right and labelled in Blue. Astrocytes from 5 animals were analysed for each condition. c Validation of the proteins identified in b. Protein extracts from wildtype and MBRL KO mouse astrocytes were analysed by SDS–PAGE and immunoblotting with the indicated antibodies. Of note, the anti-RNF185 antibody cross-reacts with RNF5, as indicated. The asterisk (*) indicates a non-specific background band. d, e TPSN protein levels are increased in RNF185 and MBRL KO human iPSCs and HEK293 cells. Extracts from parental, RNF185 and MBRL KO iPSCs (d) and HEK293 Flp-In T-Rex cells (e) were analysed by SDS–PAGE and immunoblotting with the indicated antibodies. The asterisk (*) indicates a non-specific background band.

Fig. 2. TPSN-sfGFP-3xHA is functional and associates both with PLC and RNF185/MBRL ERAD complex.

a TPSN-sfGFP-3xHA is functional. Cell surface expression of MHC-I was analysed with the conformation-sensitive W6/32 antibody in Parental, TPSN KO, and TPSN KO HEK cells stably expressing TPSN-sfGFP-3xHA using flow cytometry. b Proteins co-precipitating with TPSN-sfGFP-3xHA were analysed by mass spectrometry. The x-axis shows enrichment of proteins associated with TPSN-sfGFP-3xHA over an untagged control. The −log₁₀ of the p-value is shown on the y-axis. p-values were calculated in Perseus using a two-sample t-test for significance. Proteins significantly enriched (FDR < 0.05) are displayed in black, orange (PLC components) or green (quality control factors). Three independent replicates were analysed. c Validation of the proteins co-precipitating with TPSN-sfGFP-3xHA from (b) as analysed by immunoblotting. Cells were lysed in buffer containing 1% DMNG and TPSN-sfGFP-3xHA was immunoprecipitated. Selected proteins were analysed by immunoblotting with the antibodies indicated.

Fig. 3. TPSN is a substrate of the RNF185/MBRL complex.

a TPSN levels are increased in RNF185 and MBRL knockout cells. Clonal RNF185, MBRL, RNF5, and HRD1 knockout lines were established from Flp-In T-Rex HEK293 cells expressing TPSN-sfGFP-3xHA. TPSN-sfGFP-3xHA levels were assessed by flow cytometry (based on GFP fluorescence). b RNF185/MBRL complex is essential for TPSN turnover. Degradation of TPSN was analysed in parental, RNF5, RNF185, and MBRL KO Flp-In T-Rex HEK293 cells expressing TPSN-sfGFP-3xHA after inhibition of protein synthesis by cycloheximide (CHX). Cell extracts were analysed by SDS–PAGE and immunoblotting. TPSN was detected with an anti-HA antibody. GAPDH was used as a loading control and detected with an anti-GAPDH antibody. Quantifications of 3 independent experiments are shown in the graph below as mean; error bars represent the standard deviation. c RNF185/MBRL complex promotes TPSN ubiquitination. TPSN-sfGFP-3xHA was immunoprecipitated from parental cells or cells lacking the indicated proteins and analysed by SDS–PAGE followed by immunoblotting with anti-HA and anti-ubiquitin antibodies. Parental cells were used as a negative control. d The steady-state levels of TPSN 4K-to-A tail mutant are high and largely unresponsive to ERAD inhibition. Flp-In T-Rex HEK293 cells expressing GFP-HA-tagged WT or 4K-to-A tail mutant TPSN were analysed by flow cytometry (based on GFP fluorescence) in the absence or presence of the p97 inhibitor CB-5083 (2.5 µM). e RNF185/MBRL complex promotes the ubiquitination of lysines in the cytosolic tail of TPSN. WT or 4K-to-A tail mutant TPSN expressed as GFP-HA fusion proteins were immunoprecipitated from the indicated cell lines and analysed by SDS–PAGE followed by immunoblotting with anti-HA and anti-ubiquitin antibodies. Parental cells were used as a negative control. f MBRL binds TPSN independently of its partners RNF185, TMUB1/2. Endogenous MBRL or HRD1 were immunoprecipitated from the indicated cells and precipitated proteins were analysed by immunoblotting with anti-HA (for TPSN-sfGFP-3xHA), anti-HRD1 and anti-SEL1L antibodies.

Fig. 4. TPSN assembly and degradation depends on evolutionary conserved charge in its transmembrane domain.

a Reduced TPSN steady-state levels in TAP1 depleted HEK293 cells depend on the RNF185/MBRL complex. RNF5, RNF185 and MBRL were deleted in TAP1 knockout cells. Cells were lysed, and protein extracts were subjected to SDS–PAGE followed by immunoblotting with the indicated antibodies. Parental cells were used as control. b TPSN steady-state levels are increased by overexpression of its binding partner TAP1. Parental and MBRL KO cells expressing TPSN-sfGFP-3xHA were transduced with a TAP1 overexpressing vector or an empty vector. TPSN-sfGFP-3xHA levels were assessed by flow cytometry (based on GFP fluorescence). c TPSN stability is increased by overexpression of its binding partner TAP1. Degradation of TPSN was analysed after inhibition of protein synthesis by cycloheximide (CHX) in Parental and MBRL KO cells expressing TPSN-sfGFP-3xHA in the presence and absence of a TAP1 overexpressing vector. Cell extracts were resolved by SDS–PAGE and subjected to immunoblotting for the proteins indicated. Quantifications for TPSN-sfGFP-3xHA and overexpressed TAP1 protein levels of 3 independent experiments are shown in the graph below as mean; error bars represent the standard deviation. d Schematic representation of the interaction between TAP1/2 and TPSN mediated through an intramembrane salt bridge as described by Blees et al. (Reference #16). The lysine at position 428 of TPSN forms a salt bridge with an aspartic acid of TAP1 and TAP2, at position 32 and 16, respectively. e Degradation of TPSN and TPSN K428A was examined after inhibition of protein synthesis by cycloheximide (CHX). Cell extracts were analysed by SDS–PAGE and immunoblotting. TPSN and TPSN K428A were detected with anti-HA antibodies. GAPDH was used as a loading control and detected with an anti-GAPDH antibody. The graph shows the average of 3 experiments as the mean; error bars represent the standard deviation. f Immunoprecipitation of TPSN-sfGFP-3xHA and the indicated mutants. Cells expressing the indicated HA-tagged constructs were lysed in a buffer containing 1% DMNG, subjected to immunoprecipitation using anti-HA beads. Proteins were eluted and subjected to SDS–PAGE, followed by immunoblotting with the indicated antibodies.

Fig. 5. Increased MHC-I surface expression in professional APCs upon loss of RNF185/MBRL ERAD complex.

a Deletion of RNF185 and MBRL in human iPSC-derived macrophages results in elevated TPSN levels. Parental, RNF185, and MBRL KO iPSCs from Fig. 1c were differentiated into macrophages. Macrophages were either left untreated or were treated for 16 h with IFN gamma (100 ng/mL). Protein extracts were analysed by SDS–PAGE and immunoblotting with the indicated antibodies. The asterisk (*) indicates a non-specific background band. b RNF185 and MBRL knockout iPSC-derived macrophages display elevated cell surface MHC-I levels as detected by flow cytometry with the conformation-sensitive W6/32 antibody. Macrophages were either left untreated or were treated for 16 h with IFN gamma (100 ng/mL). The identity of cells was confirmed with the macrophage markers CD14 and CD45.

Fig. 6. Model for TPSN regulation by the RNF185/MBRL ERAD complex.

The RNF185/MBRL complex degrades a pool of unassembled TPSN, thereby limiting the amount available for assembly onto the PLC. RNF185/MBRL-dependent degradation involves the recognition of evolutionary conserved lysine residue in the TPSN transmembrane segment that is also essential for its assembly with TAP1 and TAP2. The absence of RNF185/MBRL results in elevated TPSN levels. These stabilize its partners TAP1 and TAP and ultimately result in increased levels of MHC-I. See text for details.