Final antigenic Melan-A peptides produced directly by the proteasomes are preferentially selected for presentation by HLA-A*0201 in melanoma cells

The melanoma-associated protein Melan-A contains the immunodominant CTL epitope Melan-A(26/27-35)/HLA-A*0201 against which a high frequency of T lymphocytes has been detected in many melanoma patients. In this study we show that the in vitro degradation of a polypeptide encompassing Melan-A(26/27-35) by proteasomes produces both the final antigenic peptide and N-terminally extended intermediates. When human melanoma cells expressing the corresponding fragments were exposed to specific CTL, those expressing the minimal antigenic sequence were recognized more efficiently than those expressing the N-terminally extended intermediates. Using a tumor-reactive CTL clone, we confirmed that the recognition of melanoma cells expressing an N-terminally extended intermediate of Melan-A is inefficient. We demonstrated that the inefficient cytosolic trimming of N-terminally extended intermediates could offer a selective advantage for the preferred presentation of Melan-A peptides directly produced by the proteasomes. These results imply that both the proteasomes and postproteasomal peptidases limit the availability of antigenic peptides and that the efficiency of presentation may be affected by conditions that alter the ratio between fully and partially processed proteasomal products.     

Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.     

A HLA-Cw*0701 restricted Melan-A/MART1 epitope presented by melanoma tumor cells to CD8+ tumor infiltrating lymphocytes

Melan-A/MART1 is a melanocytic differentiation antigen recognized on melanoma tumor cells by CD8+ and CD4+ T cells. In this study, we describe a new epitope of this protein recognized in the context of HLA-Cw*0701 molecules by a CD8+ tumor infiltrating lymphocyte (TIL) clone. This CD8+ TIL clone specifically recognized and killed a fraction of melanoma cells lines expressing Melan-A/MART1 and HLA-Cw*0701. We further show that the Melan-A/MART1_51–61 peptide is the optimal peptide recognized by this clone. Together, these data significantly enlarge the fraction of melanoma patients susceptible to benefit from a Melan-A/MART1 vaccine approach.     

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.     

Lentiviral vector expression of tumour antigens in dendritic cells as an immunotherapeutic strategy

Therapeutic cancer vaccines need to stimulate a refractory immune system to make an effective anti-tumour response. We have explored the use of lentiviral vectors to deliver tumour antigen genes to dendritic cells (DC) as a possible mechanism of immune stimulation. Direct injection of a lentiviral vector encoding the melanoma antigen NY-ESO-1 in HLA-A2 transgenic mice primed NY-ESO-1-specific CD8+ cells that could be expanded by boosting with an NY-ESO-1 vaccinia virus. The expanded cells could kill NY-ESO-1_157–165 peptide-pulsed targets in vivo. In order to examine the priming step directly, we constructed another lentiviral vector expressing the melanoma antigen Melan-A (MART-1). Here we show that Melan-A protein is also efficiently expressed after transduction of human DC cultured from peripheral blood mononuclear cells. When these transduced DC are co-cultured with autologous naïve T cells, they cause the expansion of cells that recognise the HLA-A2 restricted Melan-A_27–35 epitope. The expanded cells are functional in that they release IFN-γ upon antigen stimulation. Melan-A lentiviral vector transduced DC caused a similar level of naïve T-cell expansion to Melan-A_27–35 peptide-pulsed DC in four experiments using different HLA-A2 positive donors. These data suggest that a vaccine based either on DC transduced with a lentiviral vector ex vivo, or on direct lentiviral vector injection, should be assessed in a phase I clinical trial.This article is a symposium paper from the “Robert Baldwin Symposium: 50 years of Cancer Immunotherapy”, held in Nottingham, Great Britain, on 30th June 2005.     

The activatory receptor 2B4 is expressed in vivo by human CD8+ effector alpha beta T cells.

The membrane receptor 2B4 is a CD2 family member that is involved in lymphocyte activation. A fraction of human CD8+ alphabeta T cells up-regulate 2B4 in vivo, and here we demonstrate that this correlates with the acquisition of effector cell properties such as granzyme B and perforin expression, rapid IFN-gamma production, and down-regulation of the lymph node homing chemokine receptor CCR7. In PBLs from healthy donors, cytomegalovirus-specific effector T cells were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag recognized by T cells-1)-specific T cells were 2B4 negative. In melanoma patients, Melan-A-specific T cells up-regulated 2B4 in parallel with in vivo differentiation. This occurred in PBLs after vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes, likely through disease-associated activation of Melan-A-specific T cells. Thus, 2B4 expression correlates with CD8+ T cell differentiation in vivo.     

Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8(+) T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8(+) T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-gamma and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A(27-35) in three of five HLA-A*0201 melanoma patients, and of CEA(571-579) and EpCAM(263-271) in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8(+) T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.     

PBMC are as good a source of tumor-reactive T lymphocytes as TIL after selection by Melan-A/A2 multimer immunomagnetic sorting

Choosing a reliable source of tumor-specific T lymphocytes and an efficient method to isolate these cells still remains a critical issue in adoptive cellular therapy (ACT). In this study, we assessed the capacity of MHC/peptide based immunomagnetic sorting followed by polyclonal T cell expansion to derive pure polyclonal and tumor-reactive Melan-A specific T cell populations from melanoma patient’s PBMC and TIL. We first demonstrated that this approach was extremely efficient and reproducible. We then used this procedure to compare PBMC and TIL-derived cells from three melanoma patients in terms of avidity for Melan-A A27L analog, Melan-A_26–35 and Melan-A_27–35, tumor reactivity (lysis and cytokine production) and repertoire. Regardless of their origin, i.e., fresh PBMC, peptide stimulated PBMC or TIL, all sorted populations (from the three patients) were cytotoxic against HLA-A2+ melanoma cell lines expressing Melan-A. Although some variability in peptide avidity, lytic activity and cytokine production was observed between populations of different origins in a given patient, it differed from one patient to another and thus no correlation could be drawn between T cell source and reactivity. Analysis of Vβ usage within the sorted populations showed the recurrence of Vβ3 and Vβ14 subfamilies in the three patients but differences in the rest of the Melan-A repertoire. In addition, in two patients, we observed major repertoire differences between populations sorted from the three sources. We especially documented that in vitro peptide stimulation of PBMC, used to facilitate the sort by enriching in specific T lymphocytes, could significantly alter their repertoire and reactivity towards tumor cells. We conclude that PBMC which are easily obtained from all melanoma patients, can be as good a source as TIL to derive high amounts of tumor-reactive Melan-A specific T cells, with this selection/amplification procedure. However, the conditions of peptide stimulation should be improved to prevent a possible loss of reactive clonotypes. Nathalie Labarrière and Nadine Gervois have equally contributed to this work.     

A HLA-DQ5 restricted Melan-A/MART-1 epitope presented by melanoma tumor cells to CD4+ T lymphocytes

Melan-A/MART1 is a melanocytic differentiation antigen expressed by tumor cells of the majority of melanoma patients and, as such, is considered as a good target for melanoma immunotherapy. Nonetheless, the number of class I and II restricted Melan-A epitopes identified so far remains limited. Here we describe a new Melan-A/MART-1 epitope recognized in the context of HLA-DQa1*0101 and HLA-DQb1*0501, -DQb1*0502 or -DQb1*0504 molecules by a CD4+ T cell clone. This clone was obtained by in vitro stimulation of PBMC from a healthy donor by the Melan-A51-73 peptide previously reported to contain a HLA-DR4 epitope. The Melan-A51-73 peptide, therefore contains both HLA-DR4 and HLA-DQ5 restricted epitope. We further show that Melan-A51-63 is the minimal peptide optimally recognized by the HLA-DQ5 restricted CD4+ clone. Importantly, this clone specifically recognizes and kills tumor cell lines expressing Melan-A and either HLA-DQb1*0501, -DQb1*0504 or -DQb1*0502 molecules. Moreover, we could detect CD4+ T cells secreting IFN-gamma in response to Melan-A51-63 and Melan-A51-73 peptides among tumor infiltrating and blood lymphocytes from HLA-DQ5+ patients. This suggests that spontaneous CD4+ T cell responses against this HLA-DQ5 epitope occur in vivo. Together these data significantly increase the fraction of melanoma patients susceptible to benefit from a Melan-A class II restricted vaccine approach.     

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.     

Spontaneous T cell responses to melanoma differentiation antigens from melanoma patients and healthy subjects

The spontaneous cytotoxic T cell responses to melanoma differentiation antigens and influenza matrix peptide were compared in 20 HLA-A2⁺ melanoma patients and 17 healthy A2⁺ individuals. Cytotoxic T lymphocyte (CTL) responses were determined by mixed lymphocyte peptide culture (MLPC) involving two stimulations of unfractionated peripheral blood lymphocytes (PBLs) with peptide in vitro. CTL responses to Melan-A 9-mer (amino acids 27–35, AAGIGILTV) peptide were detected in 4 out of 16 normal individuals, but in none of the melanoma patients. CTL specific for influenza matrix peptide were frequently found in both normal individuals and melanoma patients, suggesting that generalized immuno-suppression was not responsible for this difference. No significant responses were observed in either normal individuals or melanoma patients to Melan-A 10-mer (26–35, EAAGIGILTV), two gp100 epitopes (280–288, YLEPGPVTA; 457–466, LLDGTATLRL) and two tyrosinase epitopes (1–9, MLLAVLYCL; 368–376, YMDGMSQV). Melan-A (27–35)-specific CTL cells generated by normal individuals and melanoma patients recognized both synthetic peptide-pulsed T2 cells and two HLA-A2⁺, Melan-A⁺ melanoma cell lines (ME272, LAR1) in an antigen-specific, MHC class I restricted manner. T cells generated against Melan-A 9-mer were also able to recognize Melan-A 10-mer-pulsed target cells. Spontaneous CTL responses to Melan-A 9-mer from three known responder normal individuals were further evaluated over a prolonged time course (6–11 months). All 3 subjects demonstrated specific Melan-A 9-mer responses throughout the study period, although lytic activity fluctuated over time for a given individual. We found the MLPC assay to be reliable and easy to perform for monitoring T cell responses, although it may still not be sufficiently sensitive to detect low numbers of precursor T cells. Received: 21 May 1998 / Accepted: 23 July 1998     

Ubiquitylation of a melanosomal protein by HECT-E3 ligases serves as sorting signal for lysosomal degradation

The production of pigment by melanocytic cells of the skin involves a series of enzymatic reactions that take place in specialized organelles called melanosomes. Melan-A/MART-1 is a melanocytic transmembrane protein with no enzymatic activity that accumulates in vesicles at the trans side of the Golgi and in melanosomes. We show here that, in melanoma cells, Melan-A associates with two homologous to E6-AP C-terminus (HECT)-E3 ubiquitin ligases, NEDD4 and Itch, and is ubiquitylated. Both NEDD4 and Itch participate in the degradation of Melan-A. A mutant Melan-A lacking ubiquitin-acceptor residues displays increased half-life and, in pigmented cells, accumulates in melanosomes. These results suggest that ubiquitylation regulates the lysosomal sorting and degradation of Melan-A/MART-1 from melanosomes in melanocytic cells.     

Direct Formation of Vaccinia Virus Membranes from the Endoplasmic Reticulum in the Absence of the Newly Characterized L2-Interacting Protein A30.5

Crescents consisting of a single lipoprotein membrane with an external protein scaffold comprise the initial structural elements of poxvirus morphogenesis. Crescents enlarge to form spherical immature virions, which enclose viroplasm consisting of proteins destined to form the cores of mature virions. Previous studies suggest that the L2 protein participates in the recruitment of endoplasmic reticulum (ER)-derived membranes to form immature virions within assembly sites of cytoplasmic factories. Here we show that L2 interacts with the previously uncharacterized 42-amino-acid A30.5 protein. An open reading frame similar in size to the one encoding A30.5 is at the same genome location in representatives of all chordopoxvirus genera. A30.5 has a putative transmembrane domain and colocalized with markers of the endoplasmic reticulum and with L2. By constructing a complementing cell line expressing A30.5, we isolated a deletion mutant virus that exhibits a defect in morphogenesis in normal cells. Large electron-dense cytoplasmic inclusions and clusters of scaffold protein-coated membranes that resemble crescents and immature virions devoid of viroplasm were seen in place of normal structures. Crescent-shaped membranes were continuous with the endoplasmic reticulum membrane and oriented with the convex scaffold protein-coated side facing the lumen, while clusters of completed spherical immature-virion-like forms were trapped within the expanded lumen. Immature-virion-like structures were more abundant in infected RK-13 cells than in BS-C-1 or HeLa cells, in which cytoplasmic inclusions were decorated with scaffold protein-coated membrane arcs. We suggest that the outer surface of the poxvirus virion is derived from the luminal side of the ER membrane.     

The melanocytic protein Melan-A/MART-1 has a subcellular localization distinct from typical melanosomal proteins

To delineate the role of the melanocyte lineage-specific protein Melan-A/MART-1 in melanogenic functions, a set of biochemical and microscopical studies was performed. Biochemical analysis revealed that Melan-A/MART-1 is post-translationally acylated and undergoes a rapid turnover in a pigmented melanoma cell line. Immunofluorescence and immunoelectron microscopy analyses indicated that Melan-A/MART-1 is mainly located in the Golgi area and only partially colocalizes with melanosomal proteins. Quantitative immunoelectron microscopy showed that the highest proportion of the cellular content of Melan-A/MART-1 was found in small vesicles and tubules throughout the cell, whereas the concentration was maximal in the Golgi region, particularly the trans-Golgi network. Substantial labeling was also present on melanosomes, endosomes, ER, nuclear envelope, and plasma membrane. In early endosomes, Melan-A was enriched in areas of the limiting membrane covered by a bi-layered coat, a structural characteristic of melanosomal precursor compartments. Upon melanosome maturation, Melan-A concentration decreased and its predominant localization shifted from the limiting membrane to internal vesicle membranes. In conjunction with its acylation, the high expression levels of Melan-A in the trans-Golgi network, in dispersed vesicles, and on the limiting membrane of premelanosomes indicate that the protein may play a role during the early stage of melanosome biogenesis.     

DSCR2, a Down syndrome critical region protein, is localized to the endoplasmic reticulum of mammalian cells

We used immunocytochemical and fluorescence assays to investigate the subcellular location of the protein encoded by Down syndrome critical region gene 2 (DSCR2) in transfected cells. It was previously suggested that DSCR2 is located in the plasma membrane as an integral membrane protein. Interestingly, we observed this protein in the endoplasmic reticulum (ER) of cells. We also studied whether the truncated forms of DSCR2 showed different subcellular distributions. Our observations indicate that DSCR2 probably is not inserted into the membrane of the endoplasmic reticulum since the fragments lacking the predicted transmembrane (TM) helices remained associated with the ER. Our analyses suggest that, although DSCR2 is associated with the endoplasmic reticulum, it is not an integral membrane protein and it is maintained on the cytoplasmic side of the ER by indirect interaction with the ER membrane or with another protein.     

Expression of the sarco/endoplasmic Ca(2+)-ATPase, SERCA1a, in fibroblasts induces the formation of organelle membrane arrays

Members of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) family are transmembrane proteins that are essential for the function of intracellular Ca(2+) storage organelles. We found that overexpression of avian muscle SERCA1a in transfected mouse fibroblasts led to the appearance of tubular membrane bundles that we termed plaques. These structures were generated in transfected cells when SERCA1a protein expression approached the endogenous level measured in chicken skeletal muscle. Plaque membranes had associated ribosomes and contained endoplasmic reticulum (ER) proteins. Endogenous ER protein levels were not elevated in SERCA1a-expressing cells, indicating that plaques were not generalized proliferations of ER but rather a reorganization of existing organelle membrane. Plaque formation also was observed in cells expressing a green fluorescent protein-SERCA1a fusion protein (GFP-SERCA1a). GFP-SERCA1a molecules displayed extensive lateral mobility between plaques, suggesting the presence of membrane continuities between these structures. Plaques were induced in cells expressing cDNA encoding a catalytically silent SERCA1a mutant indicating that ER redistribution was driven by a structural feature of the enzyme. SERCA1a-induced plaque formation shares some characteristics of sarcoplasmic reticulum (SR) biogenesis during muscle differentiation, and high-level SERCA1a expression in vivo may contribute to the formation of SR from ER during embryonic myogenesis.     

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).     

Cell surface expression and bile acid transport function of one topological form of m-epoxide hydrolase

The bifunctional hepatic protein, microsomal epoxide hydrolase (mEH), plays a central role in the metabolism of many xenobiotics as well as mediating the Na(+)-dependent uptake of bile acids in parallel with the Na(+)-taurocholate co-transporting protein (ntcp). Previous studies have established that mEH is expressed in the endoplasmic reticulum with two topological orientations, where the type II form is targeted to the plasma membrane. In this report the topology and transport properties of mEH as a function of plasma membrane expression in cultured hepatocytes, transfected Madin-Darby canine kidney cells expressing mEH (MDCK[mEH]), and the human hepatoma cell line, HepG2, were studied using confocal fluorescence microscopy and substrate uptake measurements. Analysis of mEH localization with an anti-mEH monoclonal antibody demonstrated the expression of one topological form on the plasma membrane of hepatocytes and MDCK[mEH] cells where both systems exhibited Na(+)-dependent bile acid uptake. In contrast, Na(+)-dependent bile acid transport in HepG2 cells and hepatocytes in culture (72 h) was substantially reduced as was the expression of ntcp. Although the total mEH level was undiminished, the decrease of bile acid transport was associated with the loss of mEH surface expression possibly resulting from an alteration in mEH endoplasmic reticulum topology and/or the plasma membrane protein targeting system in these de-differentiated cells.     

Membrane topology of the hepatitis C virus NS2 protein

The hepatitis C virus (HCV) NS2 protein is a hydrophobic protein. Previous studies indicate that this protein is an integral membrane protein, which is targeted to the membrane of the endoplasmic reticulum (ER) by the signal sequence located in its preceding p7 protein. In this report, we demonstrate that the membrane association of NS2 is p7-independent and occurs co-translationally. Further deletion-mapping studies suggest the presence of two internal signal sequences in NS2. These two internal signal sequences, which are located within amino acids 839-883 and amino acids 928-960, could target the alpha-globin reporter, a cytosolic protein, to the membrane compartments in HuH7 hepatoma cells. The presence of multiple signal sequences for its membrane association suggests that NS2 has multiple transmembrane domains. The glycosylation studies indicate that both amino and carboxyl termini of NS2 are located in the endoplasmic reticulum lumen. Based on these results, a model for the NS2 membrane topology is presented.