Research ArticleCELL BIOLOGY

Oncoprotein SND1 hijacks nascent MHC-I heavy chain to ER-associated degradation, leading to impaired CD8+ T cell response in tumor

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Science Advances  29 May 2020:
Vol. 6, no. 22, eaba5412
DOI: 10.1126/sciadv.aba5412
  • Fig. 1 SND1 is physically associated and colocalized with MHC-I HC.

    (A) Immunopurification and mass spectrometry of SND1-containing protein complexes. Cellular extracts from HeLa cells stably expressing SND1-FLAG were immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The elutes were resolved on SDS-PAGE and silver-stained. The protein bands on the gel were recovered by trypsinization and analyzed by mass spectrometry. (B) Co-IP analysis of the association between SND1 and HLA-A. Whole-cell extracts from HeLa cells with SND1-FLAG expression were immunoprecipitated with anti-FLAG beads, followed by Western blot with antibodies against the HLA-A. (C) Co-IP analysis of the association between SND1 and HC10. Whole-cell extracts from HeLa cells were immunoprecipitated with anti-HC10, followed by immunoblot (IB) with antibodies against the SND1. (D) Cellular extracts from HeLa cells were immunoprecipitated with anti-SND1 antibody, followed by Western blot with antibodies against the indicated proteins. (E) Duolink in situ PLA was adopted for detecting the association between SND1 and HLA-A. Two PLA probes were designed to respectively recognize either mouse or rabbit antibody against SND1 or HLA-A. Immunoglobulin G (IgG) was used as staining control. Scale bar, 20 μm. (F) Immunostaining and confocal microscopic analysis of subcellular colocalization of SND1 and HLA-A–FLAG (C terminus) in HeLa cells. HeLa cells were fixed and immunostained with antibodies against the indicated proteins. Scale bar, 10 μm. (G) GST pull-down analysis of the bacterially produced GST-fusion protein containing full-length SND1 (GST-SND1), SN domain (GST-SN), and TSN domain (GST-TSN) involved in the interaction with in vitro–translated HLA-A from rabbit reticulocytes. Coomassie blue staining for GST-fusion proteins refers to fig. S1A. aa, amino acid. (H) GST pull-down analysis of the different domains of HLA-A involved in the interaction with SND1. The His-SND1 and sample of GST-tagged different domains of HLA-A were purified from E. coli bacteria cells. Coomassie blue staining for GST-fusion proteins refers to fig. S1B. (I) Immunoprecipitation analysis of the domains involved in the interaction between SND1 and HLA-A with FLAG-tagged deletion mutants of SND1 purified from HeLa SND1-KO cells. The immunoprecipitation of FLAG refers to fig. S1C. (J) The spatial conformation of SND1-HLA-A complex predicted by the database of ZDOCK (http://zdock.umassmed.edu/) was further analyzed using the Gromacs package. The structural stability and binding energy refer to fig. S1 (E and F).

  • Fig. 2 SND1 is a novel ER-associated protein interacting with SEC61A on ER membrane via N-terminal peptide.

    (A) Immunostaining for cellular colocalizations, followed by confocal microscopic analysis by using antibody against SND1, RRBP1, SEC61A, and HLA-A–FLAG. Scale bar, 10 μm. (B) HeLa cells were transfected with the ER reporter plasmids, GFG, HLA-SP-GFG, UGGT1-SP-GFG, GAPDH-NP-GFG, and SND1-NP-GFG, respectively. Western blot for molecular weight of these GFG-tagged fusion proteins expressed in HeLa cells. (C) Colocalizations of these GFG-tagged fusion proteins with SEC61A were detected by confocal microscopy. UGGT1 was used as a positive control for ER-associating protein, while GAPDH was used as a negative control. Scale bar, 20 μm. Fluorescence intensity profiles of regions indicated by short lines are shown in the bottom. (D) Co-IP by antibody against SEC61A for interaction with SND1-GFP or SND1-NP−/−-GFP in HeLa cells transfected with either SND1-GFP (lane 3) or SND1-NP−/−-GFP vector (lane 4). (E) Co-IP by antibody against FLAG for interaction with SND1-NP in HeLa cells transfected with either GFG (lane 3) or SND1-NP-GFG vector (lane 4). (F) Ectopically increased expression of either SND1-GFP or SND1-NP−/−-GFP in SND1-KO HeLa cells followed by Western blot for SND1 and HLA-A expression. WT, wild type.

  • Fig. 3 SND1 promotes the degradation of MHC-I HC.

    (A) Two clones of HeLa cells with stable depletion of SND1 by CRISPR-Cas9 system were analyzed by flow cytometry for human MHC-I using antibody simultaneously against HLA-A/B/C. (B) Two clones of HeLa cells with stable depletion of SND1 by CRISPR-Cas9 system and HeLa cells stably expressing SND1-FLAG were collected, followed by Western blot using antibodies against HLA-A. (C) The effect of KO SND1 on the half-life of HLA-A was evaluated in HeLa cells treated with CHX (50 μg/ml) and harvested at the indicated time point, followed by Western blot. The protein half-life curves were obtained by quantifying relative intensities. (D) HeLa cells with ectopic HLA-A expression were pretreated with proteasome inhibitor MG132 (10 mM) or lysosomal inhibitor chloroquine (100 mM) for 8 hours and subjected to Western blot with anti–HLA-A antibody. DMSO, dimethyl sulfoxide. (E) WT and SND1-KO HeLa cells were transfected with HLA-A–FLAG and treated with MG132 (10 mM) for 8 hours. Cellular extracts were immunoprecipitated with anti-FLAG, followed by Western blot with anti-ubiquitin (Ub) antibody. (F) HeLa cells were cotransfected with control vector or SND1-HA and HLA-A–FLAG or with control small interfering RNA (siRNA) or SND1 siRNA and HLA-A–FLAG, and whole-cell lysates were collected and immunoprecipitated with anti-FLAG, followed by Western blot with indicating antibodies. (G) The Duolink in situ PLA was adopted for detecting the direct association between HLA-A and calnexin or β2-microglobulin (β2m) in the presence of SND1-HA or in the absence of SND1. Scale bar, 20 μm. The signal dots were calculated and plotted. *P < 0.05 and ****P < 0.0001, by unpaired t test.

  • Fig. 4 SND1 hijacks the nascent MHC-I HC to ERAD process in tumor cells.

    (A) Immunopurification and mass spectrometry of HLA-A–containing protein complexes. Cellular extracts from HeLa cells stably expressing HLA-A–FLAG were immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The elutes were resolved on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and silver-stained. The protein bands on the gel were recovered by trypsinization and analyzed by mass spectrometry. HLA-A–interacted proteins were highlighted. (B) HeLa cells were coimmunoprecipitated by HLA-A antibody and subjected to Western blot by antibody against VCP. (C) HeLa cells were coimmunoprecipitated by SND1 antibody and subjected to Western blot by antibody against VCP. (D) Duolink assay followed by confocal microscopic analysis for direct molecular interactions among SND1, VCP, and HLA-A. IgG was used as a negative control. Scale bar, 20 μm. (E) HeLa cells were cotransfected with control vector or SND1-HA and HLA-A–FLAG or cotransfected with control siRNA or SND1 siRNA and HLA-A–FLAG, and whole-cell lysates were collected and immunoprecipitated with anti-FLAG, followed by Western blot with anti-SND1, anti-VCP, anti-VIMP, and anti-HRD1 antibodies. Results of input were shown in fig. S4D. (F) HeLa cells were cotransfected with vector or HRD1-HA and HLA-A–FLAG with the treatment of MG132. Cellular extracts were immunoprecipitated with anti-FLAG, followed by Western blot with anti-ubiquitin antibody.

  • Fig. 5 Loss of SND1 in murine tumors limits tumor size with more CD8+ T cell infiltration in vivo.

    (A) Three clones of B16F10 cells with stable depletion of SND1 by CRISPR-Cas9 system were collected, followed by IB for murine MHC-I (H2Kb). (B) Two clones of B16F10 cells with stable depletion of SND1 by CRISPR-Cas9 system were analyzed by flow cytometry for murine MHC-I (H2Kb/H2Db) using antibody simultaneously against H2Kb/H2Db. (C to E) 5 × 105 of either WT or SND1-KO B16F10 cells were subcutaneously transplanted into C57BL/6 mice. The tumor growth was monitored at the indicated times. C57BL/6 mice were sacrificed at day 11. Tumors were removed and photographed. The tumor tissues were weighed and plotted. Data are presented as means ± SD; n = 5 tumors for each group. *P < 0.05, two-tailed t test. (F) Immunofluorescence images of CD4+ T and CD8+ T cells in B16F10 tumor sections (scale bar, 20 μm). (G) C57BL/6 mice injected with equal numbers of WT or SND1-KO B16F10 cells were sacrificed at day 11. The digested tumor suspensions stained with antibodies against CD8 and CD45.2 (pan-leukocyte marker) were subjected to flow cytometry. (H to J) Percentages of infiltrating CD45.2+ cells and CD8+ T cells among total tumor tissue–derived cells and the percentage of infiltrating CD8+ T cells among total CD45+ leucocytes. n = 5 tumors for each group. *P < 0.05 and **P < 0.01, by unpaired t test. The experiments were performed and repeated at least three times, independently. (K) The percentage of infiltrating PD-1+ CD8+ T cells among total CD8+ T cells. n = 5 tumors for each group. n.s., not significant.

  • Fig. 6 SND1 deletion promotes specific antigen presentation and enhances antitumor immunity.

    (A) B16F10 cells with stable depletion of SND1 by CRISPR-Cas9 system were stably transfected with OVA vector, followed by IB. (B) B16F10-OVA with SND1 deficiency was analyzed by flow cytometry for murine MHC-I (H2Kb/H2Db). (C to E) OT-I mice were injected with equal numbers of WT or SND1-KO B16F10-OVA cells, and tumor growth was observed over time. Then tumors were removed, photographed, and weighted. *P < 0.05 and **P < 0.01. (F) Flow cytometry was used for the analysis of CD45.2+ leucocyte and CD8+ T cell infiltration in tumor tissues. (G to I) Percentages of infiltrating CD45.2+ leucocytes and CD8+ T cells among total tumor tissue–derived cells and the percentage of infiltrating CD8+ T cells among total CD45.2+ leucocytes. n = 5 tumors for each group. **P < 0.01 and ***P < 0.001, by unpaired t test. (J) CD8+ T cells were purified from spleens of tumor-bearing OT-I mice and stimulated with 257 to 264 (SIINFEKL) peptide of OVA for 24 hours. Percentages of IFNγ+CD8+ T cells among total CD8+ T cells in the culture system were measured by flow cytometry. (n = 5, **P < 0.01). The experiments were repeated two times independently. (K) CD8+ T cells recognizing specific peptide of OVA (SIINFEKL) were purified from spleens of OT-I and then cocultured with WT or SND1-KO B16F10 cells stably expressing OVA (CD8+ T:B16F10-OVA, 10:1). Representative images were taken under a bright field at different time points. Scale bar, 20 μm. (L) In vitro comparison of cytolysis rates against CD8+ T cells purified from spleens of OT-I mice between WT and SND1-KO B16F10-OVA cells at different cell rates of CD8+ T (effector cells) to B16F10 (target cells) with/without anti–MHC class I antibodies (Ab) (E:T, 5:1, 10:1, 15:1, or 20:1). A lactate dehydrogenase–releasing cytotoxicity assay was performed to measure the cytolysis efficiency of CD8+ T cells on tumor cells. Each bar represents mean ± SD for biological triplicate experiments. ****P < 0.0001, two-way analysis of variance (ANOVA).

Supplementary Materials

  • Supplementary Materials

    Oncoprotein SND1 hijacks nascent MHC-I heavy chain to ER-associated degradation, leading to impaired CD8+ T cell response in tumor

    Yuan Wang, Xinting Wang, Xiaoteng Cui, Yue Zhuo, Hongshuai Li, Chuanbo Ha, Lingbiao Xin, Yuanyuan Ren, Wei Zhang, Xiaoming Sun, Lin Ge, Xin Liu, Jinyan He, Tao Zhang, Kai Zhang, Zhi Yao, Xi Yang, Jie Yang

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