Assembly of MHC class I molecules within the endoplasmic reticulum.
ABSTRACT MHC class I molecules bind cytosolically derived peptides within the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T cells. A major focus of our laboratory has been to understand the functions of the diverse proteins involved in the intracellular assembly of MHC class I molecules. These include the molecular chaperones calnexin and calreticulin, which enhance the proper folding and subunit assembly of class I molecules and also retain assembly intermediates within the ER; ERp57, a thiol oxidoreductase that promotes heavy chain disulfide formation and proper assembly of the peptide loading complex; tapasin, which recruits class I molecules to the TAP peptide transporter and enhances the loading of high affinity peptide ligands; and Bap31, which is involved in clustering assembled class I molecules at ER exit sites for export along the secretory pathway. This review describes our contributions to elucidating the functions of these proteins; the combined effort of many dedicated students and postdoctoral fellows.
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ABSTRACT: Hand, foot and mouth disease is mainly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16), but EV-A71 is also associated with severe neurological complications. Host factors may contribute to the different clinical outcomes of EV-A71 and CV-A16 infections. A neurovirulent EV-A71 strain (EV-A71/UH1) from a fatal case, a non-neurovirulent EV-A71 strain (EV-A71/Sha66) and a CV-A16 strain (CV-A16/22159) from cases of uncomplicated HFMD were used. Replication of the viruses in SK-N-MC (neuronal) and HT-29 (intestinal) cell lines correlated with the severity of clinical disease associated with each virus. EV-A71/UH1 showed the greatest replication in neuronal cells. In HT-29 cells, both EV-A71 strains replicated well, but CV-A16/22159 showed no effective replication. The proteomes of mock and infected SK-N-MC and HT-29 cell lines were compared by 2D-SDS-PAGE. The differentially expressed proteins were identified by MALDI-TOF/TOF analysis. There were 46 and 44 differentially expressed proteins identified from SK-N-MC and HT-29 cells, respectively, categorized under apoptosis, stress, cytoskeletal, energy metabolism proteins and others. Western blot validation showed that EV-A71/UH1 and CV-A16 also differentially induced proteins involved in viral RNA translation and host cell stress responses in neuronal and intestinal cell lines. This study compared the host cellular responses of both EV-A71 and CV-A16 in SK-N-MC neuronal and HT-29 intestinal cell lines. As the gene organization and replication cycle for EV-A71 and CV-A16 are very similar, we hypothesize that the different clinical manifestations resulting from these viruses may be due to differentially regulated proteins induced in a primary target host cell (gastrointestinal) and an end target host cell (neuronal). We demonstrated that the differential host responses of these enteroviruses may alter their pathogenesis and virulence. Copyright © 2015. Published by Elsevier B.V.Journal of proteomics 05/2015; 125. DOI:10.1016/j.jprot.2015.05.016 · 3.93 Impact Factor
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ABSTRACT: Calnexin (IP90/P88) is an integral membrane protein of the endoplasmic reticulum that binds newly synthesized N-linked glycoproteins during their folding in the ER including MHC class I molecule. This manuscript reports the identification of two unique cDNA clones of calnexin in rainbow trout. Both encode putative mature proteins of 579 and 592 aa respectively in addition to a 24 aa signal peptide. Sequence analysis revealed that only one of the two cDNA clones encodes a putative ER retention signal, K/QEDDL, followed by a serine phosphorylation site conserved with mammalian homologs. Amino acid sequence alignment illustrated conservation of the calnexin luminal domain, which consists of a globular and a P domain, in both copies. Southern blotting revealed that there are at least two copies of the calnexin gene in the trout genome and northern blotting showed a wide tissue distribution of an estimated 3 kbp calnexin transcript with an additional minor transcript of 2.3 kbp expressed only in head kidney, spleen PBLs and strongly in RTS11. Importantly, the smaller transcript was predominantly upregulated in RTS11 after a 24 h treatment with the calcium ionophore A23187. In western blots, calnexin was detected primarily as a 120 kDa protein and upon A23187 treatment; a 100 kDa band was most prominently expressed. These results suggest that in salmonids there are two differentiated versions of the calnexin gene which encode proteins that may have diverged to perform unique biological functions.Developmental and comparative immunology 09/2013; 42(2). DOI:10.1016/j.dci.2013.09.005 · 3.71 Impact Factor
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ABSTRACT: To understand the interaction of dendritic cells (DCs) with cancer cells, we investigated molecular changes in DCs following co-culture with cancer cells. DCs co-cultured with Jurkat cancer cells showed remarkable down-regulation of MHC class I molecules, while DCs co-cultured with MCF-7 cancer cells showed minimal changes. Interestingly, down-regulation of MHC class I on DCs was not observed upon treatment with Jurkat cell lysate or culture supernatant, suggesting the importance of direct cell-cell interactions. The expressions of CD40, CD80, CD83, MHC class II, and IL-12p40 on DCs co-cultured with Jurkat cells were only slightly affected. In contrast, DCs co-cultured with MCF-7 cells showed increased expressions of CD80, CD83, CD86, and IL-12p40. Furthermore, DCs co-cultured with Jurkat cells showed a down-regulation of low molecular weight polypeptides (LMP) 7, and of transporter associated with antigen processing (TAP) 1 and 2 at the mRNA expression level. LMP7, TAP2 and β2-microglobulin (β2M) were also down-regulated at the protein level. We further demonstrated how altered expression of MHC class I on DCs caused by co-culture with cancer cells affected autologous CD8(+) T cells, using the model MHC class I-presented HSV antigen. We found that DCs that had been HSV-treated and co-cultured with Jurkat cells showed a reduced potency to activate CD8(+) T cells. In contrast, HSV-treated DCs that had been co-cultured with MCF-7 cells induced activation of CD8(+) T cells, including high expression of CD25, CD69, granzyme B and cytokines, TNF-α and IFN-γ.Molecular Immunology 06/2011; 48(15-16):1744-52. DOI:10.1016/j.molimm.2011.04.018 · 3.00 Impact Factor