Walker A lysine mutations of TAP1 and TAP2 interfere with peptide translocation but not peptide binding

We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1.TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M).TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.     

Physical and functional interactions of the cytomegalovirus US6 glycoprotein with the transporter associated with antigen processing

The endoplasmic reticulum-resident human cytomegalovirus glycoprotein US6 (gpUS6) inhibits peptide translocation by the transporter associated with antigen processing (TAP) to prevent loading of major histocompatibility complex class I molecules and antigen presentation to CD8+ T cells. TAP is formed by two subunits, TAP1 and TAP2, each containing one multispanning transmembrane domain (TMD) and a cytosolic nucleotide binding domain. Here we reported that the blockade of TAP by gpUS6 is species-restricted, i.e. gpUS6 inhibits human TAP but not rat TAP. Co-expression of human and rat subunits of TAP demonstrates independent binding of gpUS6 to human TAP1 and TAP2, whereas gpUS6 does not bind to rat TAP subunits. gpUS6 associates with preformed TAP1/2 heterodimers but not with unassembled TAP subunits. To locate domains of TAP required for gpUS6 binding and function, we took advantage of reciprocal human/rat intrachain TAP chimeras. Each TAP subunit forms two contact sites within its TMD interacting with gpUS6. The dominant gpUS6-binding site on TAP2 maps to an N-terminal loop, whereas inhibition of peptide transport is mediated by a C-terminal loop of the TMD. For TAP1, two gpUS6 binding domains are formed by loops of the C-terminal TMD. The domain required for TAP inactivation is built by a distal loop of the C-terminal TMD, indicating a topology of TAP1 comprising 10 endoplasmic reticulum transmembrane segments. By forming multimeric complexes, gpUS6 reaches the distant target domains to arrest peptide transport. The data revealed a nonanalogous multipolar bridging of the TAP TMDs by gpUS6.     

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37 degrees C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.     

Tapasin interacts with the membrane-spanning domains of both TAP subunits and enhances the structural stability of TAP1 x TAP2 Complexes

The transporter associated with antigen processing (TAP) proteins are involved in transport of peptides from the cytosol into the endoplasmic reticulum. Two subunits, TAP1 and TAP2, are necessary and sufficient for peptide binding and peptide translocation across the endoplasmic reticulum membrane. TAP1 and TAP2 contain an N-terminal hydrophobic membrane-spanning region and a C-terminal nucleotide binding domain. Tapasin is an endoplasmic reticulum resident protein that has been found associated with the TAP subunits and shown to increase expression levels of TAP. Here we investigated TAP-tapasin interactions and their effects on TAP function in insect cells. We show tapasin binding to both TAP1 and TAP2 and to the corresponding nucleotide binding domain-exchanged chimeras as well as to a truncated TAP1.TAP2 complex containing just the membrane-spanning regions of TAP1 and TAP2. However, tapasin interactions with either the truncated TAP construct containing just the nucleotide binding domain are not observed. Tapasin is not required for high affinity peptide binding to TAP1.TAP2 complexes, and in fact, the presence of tapasin slightly reduces the affinity of TAP complexes for peptides. However, at near physiological temperatures, both tapasin and nucleotides stabilize the peptide binding site of TAP1.TAP2 complexes against inactivation, and enhanced thermostability of both TAP subunits is observed in the presence of tapasin. The enhanced structural stability of TAP1.TAP2 complexes in the presence of tapasin might explain the observations that tapasin increases TAP protein expression levels in mammalian cells.     

Cadmium induces hypertrophy accompanied by increased myc mRNA accumulation in NRK-49F cells

Previous studies showed that Cd⁺⁺ inhibits EGF-induced DNA synthesis that not EGF-induced myc mRNA accumulation or amino acid incorporation into protein in serum-starved NRK-49F cells. In this study, flow cytometry was used to analyze the DNA and protein content of individual cells stimulated with Cd⁺⁺ and/or epidermal growth factor (EGF). myc oncogene expression in these cells was also measured. It was found that, in both parental NRK-49F cells and in a clonal subpopulation, N1, Cd⁺⁺ induces an hypertrophic response. In parental NRK-49F cells, however, lower doses of Cd⁺⁺ (0.5 M) induced more pronounced hypertrophic responses than did higher doses (4 M); whereas in N1 cells, the Cd⁺⁺-induced hypertrophic response shows a pattern of increasing response with doses of Cd⁺⁺ from 0.5 to 4 M. myc mRNA accumulation measured 2 hours after stimulation correlated with the hypertrophic responses in both NRK-49F cells and in N1 cells. The results show that Cd⁺⁺-induced hypertrophy in NRK-49F cells is associated with increased myc oncogene mRNA accumulation, indicating that cell proliferation and cell hypertrophy may in part share common activation pathways.Abbreviations EGF Epidermal growth factor - FCM Flow cytometric analysis - FITC fluorescein isothiocyanate - PI propidium iodide     

Distinct functions of the ATP binding cassettes of transporters associated with antigen processing: a mutational analysis of Walker A and B sequences

The transporters associated with antigen processing (TAP1/TAP2) provide peptides to MHC class I molecules in the endoplasmic reticulum. Like other ATP-binding cassette proteins, TAP uses ATP hydrolysis to power transport. We have studied peptide binding to as well as translocation by TAP proteins with mutations in the Walker A and B sequences that are known to mediate ATP binding and hydrolysis. We show that a mutation in the TAP1 Walker B sequence reported to abrogate class I expression by a lung tumor does not affect ATP binding affinity, suggesting a defect restricted to ATP hydrolysis. This mutation reduces peptide transport by only 50%, suggesting that TAP function can be highly limiting for antigen presentation in non-lymphoid cells. Single substitutions in Walker A sequences (TAP1K544A, TAP2K509A), or their complete replacements, abrogate nucleotide binding to each subunit. Although all of these mutations abrogate peptide transport, they reveal distinct roles for nucleotide binding to the two transporter subunits in TAP folding and in regulation of peptide substrate affinity, respectively. Alteration of the TAP1 Walker A motif can have strong effects on TAP1 and thereby TAP complex folding. However, TAP1 Walker A mutations compatible with correct folding do not affect peptide binding. In contrast, abrogation of the TAP2 nucleotide binding capacity has little or no effect on TAP folding but eliminates peptide binding to TAP at 37 degrees C in the presence of nucleotides. Thus, nucleotide binding to TAP2 but not to TAP1 is a prerequisite for peptide binding to TAP. Based on these results, we propose a model in which nucleotide and peptide release from TAP are coupled and followed by ATP binding to TAP2, which induces high peptide affinity and initiates the transport cycle.     

Inhibition of 4E-BP1 phosphorylation promotes tubular cell escaping from G2/M arrest and ameliorates kidney fibrosis

Upon occurrence of kidney injury, tubular cells arrested in G2/M stage may promote interstitial fibroblast activation and kidney fibrosis through producing large amounts of pro-fibrotic cytokines. MTORC1 signaling is essential for controlling cell growth, however, the role and mechanisms for mTORC1 in regulating tubular cell cycle progression during kidney fibrosis are not clear. Here we reported that p-S6 abundance was increased at 15 min, reached peak at 1 h and declined from 3 h to 24 h, while the abundance of p-4E-BP1 and p-Histone H3 was increased from 15 min to 24 h in tubular epithelial cells at the similar pattern after serum stimulation. The phosphorylation of 4E-BP1 was prohibited in NRK-52E cells by the transfection of 4E-BP1 plasmid with four phospho-sites mutation (4E-BP1A4). 4E-BP1A4 transfection led to less G2/M cell arrest as well as the production of pro-fibrotic cytokine and extracellular matrix in NRK-52E cells. In addition, aristolochic acid (AA)-induced tubular cell G2/M arrest induced by treatment was also largely attenuated in NRK-52E cells transfected with 4E-BP1A4. In mouse kidneys with UUO nephropathy, p-4E-BP1 abundance was markedly elevated in the mitotic tubular cells. Therefore, these data indicates that suppressing 4E-BP1 phosphorylation may inhibit tubular cell G2/M-arrest and kidney fibrosis.     

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.     

Stable binding of the herpes simplex virus ICP47 protein to the peptide binding site of TAP.

The herpes simplex virus (HSV) ICP47 protein inhibits the MHC class I antigen presentation pathway by inhibiting the transporter associated with antigen presentation (TAP) which translocates peptides across the endoplasmic reticulum membrane. At present, ICP47 is the only inhibitor of TAP. Here, we show that ICP47 produced in bacteria can block human, but not mouse, TAP, and that heat denaturation of ICP47 has no effect on its ability to block TAP. ICP47 inhibited peptide binding to TAP without affecting ATP binding, consistent with previous observations that the peptide binding and ATP binding sites of TAP are distinct. ICP47 bound to TAP with a higher affinity (KD approximately 5 x 10(-8) M) than did peptides, and ICP47 did not dissociate from TAP. ICP47 was not transported by TAP and remained sensitive to proteases added from the cytosolic surface of the membrane. Peptides acted as competitive inhibitors of ICP47 binding to TAP, and this inhibition required a 100- to 1000-fold molar excess of peptide. These results demonstrate that ICP47 binds to a site which includes the peptide binding domain of TAP and remains bound to this site in a stable fashion.     

Exploring the minimal functional unit of the transporter associated with antigen processing

TAP, an ABC transporter in the ER membrane, provides antigenic peptides derived from proteasomal degradation to MHC class I molecules for inspection by cytotoxic T lymphocytes at the cell surface so as to trace malignant or infected cells. To investigate the minimal number of transmembrane segments (TMs) required for assembly of the TAP complex based on hydrophobicity algorithms and alignments with other ABC transporters we generated N-terminal truncation variants of human TAP1 and TAP2. As a result, a 6+6 TM core-TAP complex represents the minimal functional unit of the transporter, which is essential and sufficient for heterodimer assembly, peptide binding, and peptide translocation into the ER. The TM1 of both, core-TAP1 and core-TAP2 are critical for heterodimerization of the complex.     

Functional roles of TAP and tapasin in the assembly of M3-N-formylated peptide complexes

H2-M3 is a MHC class Ib molecule with a high propensity to bind N-formylated peptides. Due to the paucity of endogenous Ag, the majority of M3 is retained in the endoplasmic reticulum (ER). Upon addition of exogenous N-formylated peptides, M3 trafficks rapidly to the cell surface. To understand the mechanism underlying Ag presentation by M3, we examined the role of molecular chaperones in M3 assembly, particularly TAP and tapasin. M3-specific CTLs fail to recognize cells isolated from both TAP-deficient (TAP(o)) and tapasin-deficient mice, suggesting that TAP and tapasin are required for M3-restricted Ag presentation. Impaired M3 expression in TAP(o) mice is due to instability of the intracellular pool of M3. Addition of N-formylated peptides to TAP(o) cells stabilizes M3 in the ER and partially restores surface expression. Surprisingly, significant amounts of M3 are retained in the ER in tapasin-deficient mice, even in the presence of N-formylated peptides. Our results define the role of TAP and tapasin in the assembly of M3-peptide complexes. TAP is essential for stabilization of M3 in the ER, whereas tapasin is critical for loading of N-formylated peptides onto the intracellular pool of M3. However, neither TAP nor tapasin is required for ER retention of empty M3.     

Herpes Simplex Virus Type 2 ICP47 Inhibits Human TAP but Not Mouse TAP

Herpes simplex virus serotype 1 (HSV-1) expresses an immediate-early protein, ICP47, that effectively blocks the major histocompatibility complex class I antigen presentation pathway. HSV-1 ICP47 (ICP47-1) binds with high affinity to the human transporter associated with antigen presentation (TAP) and blocks the binding of antigenic peptides. HSV type 2 (HSV-2) ICP47 (ICP47-2) has only 42% amino acid sequence identity with ICP47-1. Here, we compared the levels of inhibition of human and murine TAP, expressed in insect cell microsomes, by ICP47-1 and ICP47-2. Both proteins inhibited human TAP at similar concentrations, and the K_D for ICP47-2 binding to human TAP was 4.8 × 10⁻⁸ M, virtually identical to that measured for ICP47-1 (5.2 × 10⁻⁸ M). There was some inhibition of murine TAP by both ICP47-2 and ICP47-1, but this inhibition was incomplete and only at ICP47 concentrations 50 to 100 times that required to inhibit human TAP. Lack of inhibition of murine TAP by ICP47-1 and ICP47-2 could be explained by an inability of both proteins to bind to murine TAP.Previously, we showed that herpes simplex virus serotype 1 (HSV-1) ICP47 (ICP47-1) caused major histocompatibility complex (MHC) class I proteins to be retained in the endoplasmic reticulum (ER) of cells and that antigen presentation to CD8⁺ T cells was inhibited after ICP47-1 was expressed in human fibroblasts (9). ICP47-1 blocked peptide transport across the ER membrane by TAP (2, 6), so that, without peptides, class I proteins were retained in the ER. By contrast, ICP47 did not detectably inhibit MHC class I antigen presentation in mouse cells (9) and inhibited murine TAP poorly (2, 6). ICP47-1 inhibited peptide binding to TAP without affecting the binding of ATP (1, 7) and bound with high affinity, and in a stable fashion, to human TAP (7). Peptides could competitively inhibit ICP47 binding to TAP, consistent with the hypothesis that ICP47-1 binds to a site which includes the peptide binding domain of TAP (7). Others have suggested that the present data do not exclude a distortion in TAP caused by the binding of ICP47 at a site distant from the peptide binding site (3). This seems improbable given our observations that ICP47 inhibits peptide binding and that peptides competitively inhibit ICP47 binding. In order for peptides to inhibit ICP47 binding and vice versa, one would have to invoke allosteric inhibition by both ICP47 and peptides, a highly unlikely prospect.The predicted amino acid sequence of HSV type 2 ICP47 (ICP47-2) was recently described (3), and it was of some interest that ICP47-1 and ICP47-2 share only 42% amino acid identity (see Fig. ​Fig.1A).1A). Most of the homology is near the N termini and in the central regions of the molecules. A peptide including residues 2 to 35 of ICP47-1 blocked human TAP in permeabilized cells (3). This observation was somewhat surprising given that this peptide did not include residues 33 to 51, a sequence that is most homologous between ICP47-1 and ICP47-2. Presumably, this conserved domain, and even the C-terminal third of the protein, is important in virus-infected cells for stability or for functions that are not apparent in this in vitro assay involving detergent-permeabilized cells.Open in a separate windowFIG. 1Comparison of ICP47-1 and ICP47-2 protein sequences and preparation of purified proteins. (A) The predicted amino acid sequences of ICP47-1 derived from HSV-1 strain 17 (6a) and of ICP47-2 derived from HSV-2 strain HG52 (3) are shown. The boldface, underlined letters denote identical amino acids, and the italicized letters denote conserved residues. (B) ICP47-1 and ICP47-2 were produced in Escherichia coli by expressing the proteins as GST fusion proteins by fusing the ICP47 coding sequences to GST sequences in plasmid pGEX-2T as described previously (7). Lysates from bacteria were incubated with glutathione-Sepharose and washed several times, and then ICP47-1 or ICP47-2 was eluted by incubation with thrombin, which cleaves between the GST and ICP47 sequences (7). The thrombin was inactivated with phenylmethylsulfonyl fluoride, and the ICP47 preparations were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Bradford protein analysis. The positions of GST-ICP47, GST, and ICP47 protein, as well as those of molecular weight markers 104, 80, 48, 34, 24, and 18 KDa in size, are indicated.Given the differences between the primary structures of ICP47-1 and ICP47-2, we were interested in whether ICP47-2 might inhibit the murine TAP. If this were the case, it would make possible animal studies of the effects of ICP47. Here, we have produced a recombinant form of ICP47-2 and compared the effects of ICP47-2 and ICP47-1 on human and murine TAP proteins expressed in insect cell microsomes. Like ICP47-1, ICP47-2 efficiently blocked human TAP but even at high concentrations did not effectively block murine TAP. Moreover, there was little or no significant binding of either protein to insect microsomes containing mouse TAP.The HSV-2 ICP47 gene was subcloned from plasmid pBB17, which contains a KpnI-HindIII 8,477-bp fragment derived from the genome of HSV-2 strain HG52 inserted into pUC19, by using PCR to amplify ICP47-2 coding sequences. One PCR primer hybridized with the 5′ end of the ICP47-2 coding sequences and extended 5′ to generate a new BglII site just upstream of the initiation codon. The second PCR primer hybridized with 3′ sequences of the ICP47-2 gene, then diverged to produce an EcoRI site just downstream of the translation termination codon. After PCR, the DNA fragment was digested with EcoRI and inserted into the HincII (blunt) and EcoRI sites of pUC19, producing plasmid pUC47-2, which was subjected to DNA sequencing. The ICP47-2 coding sequences were excised from pUC47-2 with BglII and EcoRI and inserted into the BamHI and EcoRI sites of pGEX-2T to generate a fusion protein with glutathione S-transferase (GST). The ICP47-GST fusion protein was expressed in bacteria and purified by using glutathione-Sepharose, and then the GST sequences were removed with thrombin as described previously for ICP47-1 (7). A comparison between the predicted amino acid sequences of ICP47-2 and ICP47-1 is shown in Fig. ​Fig.1,1, with a comparative gel (Fig. ​(Fig.1B)1B) showing the purified preparations of ICP47-1 and ICP47-2 from bacteria. Microsomes purified from Sf9 insect cells infected with baculoviruses expressing human TAP1 and TAP2 have been described previously (7, 8), as were microsomes from Drosophila cells expressing murine TAP1 and TAP2 (1). We previously estimated that approximately 2% of the protein associated with the insect microsomes was human TAP (7), and the microsomes containing mouse TAP possessed similar TAP activity (see below). Peptide translocation by these microsomes was measured by using a library of ¹²⁵I-labelled peptides (5) that are glycosylated after transport into the ER. Radioactive peptides able to bind to concanavalin A were quantified as an indirect measure of peptide transport (6). Over a range of membranes from 2.5 to 20 μl, with protein concentrations of 10 to 12 mg/ml for human TAP microsomes and 5.0 to 7.0 mg/ml for mouse TAP microsomes, there was a linear increase in peptide transport (Fig. ​(Fig.2).2). Thus, peptides and ATP were not limiting. Peptide transport was specific because the transport observed with control membranes not containing TAP amounted to less than 1% of that observed when microsomes contained TAP. The levels of peptide transport associated with microsomes containing human or mouse TAP were also compared and standardized. Thus, in subsequent assays, 7.5 to 10 μl of microsomes exhibiting similar amounts of TAP activity were used. Open in a separate windowFIG. 2Peptide transport by insect microsomes containing human or murine TAP. Microsomes were derived from insect Sf9 cells coinfected with BacTAP1 and BacTAP2 (Human TAP) (7) or from Sf9 cells infected with a control baculovirus, BacgH (Human control). Alternatively, microsomes were derived from Drosophila cells induced to express mouse TAP (Murine TAP) (1) or from Drosophila cells which were not induced to express mouse TAP (Murine control). Various concentrations of each microsome preparation were incubated with ¹²⁵I-labelled peptides and 5 mM ATP in a volume of 150 μl for 10 min at 23°C. The microsomes were washed, pelleted, and disrupted in detergent as described previously (7). Peptides able to bind to concanavalin A-Sepharose were eluted with alpha-methylmannoside and quantified (7).ICP47-2 inhibited peptide transport by human TAP, and the inhibition was similar to that of ICP47-1; the 50% inhibitory concentration (IC₅₀) for ICP47-2 was 0.24 μM and for ICP47-1 was 0.27 μM (Fig. ​(Fig.3A).3A). In other experiments the IC₅₀ values for ICP47-1 and ICP47-2 varied from 0.15 to 0.35 μM, and there were no experiments in which there was a significant difference in the abilities of the two proteins to inhibit human TAP. Moreover, the binding properties of ICP47-2 to human TAP were similar to those of ICP47-1. Binding experiments were performed as described previously for ICP47-1 (7) by using membranes containing human TAP and ¹²⁵I-labelled ICP47-2. Specific binding of ICP47-2 was calculated by subtracting the binding to control microsomes derived from insect cells infected with a baculovirus expressing HSV gH (7). The binding of ICP47-2 was saturable, so that at a protein concentration of 1 μM approximately 16 ng of protein bound to human TAP (Fig. ​(Fig.4A).4A). In previous experiments with a similar preparation of insect microsomes containing human TAP, the binding of ICP47-1 also saturated at 15 to 16 ng (7). The ICP47-2 binding data were analyzed in a standard Scatchard plot, and the K_D was calculated to be 4.8 × 10⁻⁸ M (Fig. ​(Fig.4B),4B), compared with 5.2 × 10⁻⁸ M for ICP47-1 (7). These values are greater than those of high-affinity peptides that bind to human TAP with affinities reaching 4 × 10⁻⁷ M, though the vast majority of peptides bind to TAP with much lower affinities (8). Open in a separate windowFIG. 3Inhibition of human and murine TAP-mediated peptide transport by ICP47-1 and ICP47-2. TAP assays were performed as described in the legend for Fig. ​Fig.22 by using insect microsomes containing human TAP (10 μl of membranes containing 12 mg of membrane protein per ml) (A) or murine TAP (7.5 μl of membranes containing 4.8 mg of membrane protein per ml but with equivalent levels of TAP activity compared with microsomes containing human TAP) (B) and various concentrations of ICP47-1 and ICP47-2. The results shown are combined from two separate experiments, each involving human and murine TAP.Open in a separate windowFIG. 4Binding of ICP47-2 to human TAP. (A) Microsomes (15 μl of membranes with a 7.5-mg/ml concentration of membrane protein) derived from Sf9 cells expressing TAP1 and TAP2 or expressing HSV-1 gH (control membranes not containing TAP) were incubated with various amounts of ¹²⁵I-labelled ICP47-2 for 60 min at 4°C as described previously (7). Binding to control membranes was subtracted from binding to microsomes containing TAP at each point. (B) Scatchard analysis of the data in panel A. The K_D for ICP47-2 binding to TAP was calculated to be 4.8 × 10⁻⁸ M.To determine whether ICP47-2 could inhibit the murine TAP, microsomes from insect cells expressing mouse TAP were incubated with various concentrations of ICP47-1 and ICP47-2 and TAP assays were performed. Inhibition of the mouse TAP was observed with both ICP47-1 and ICP47-2, but relatively high concentrations of both proteins were required (Fig. ​(Fig.3B).3B). The IC₅₀ values for ICP47-1 and ICP47-2 in this experiment were 10.8 and 16.2 μM, respectively. However, we were unable to reduce TAP activity beyond approximately 40% with ICP47-1 or ICP47-2 concentrations reaching 30 μM. This was 100 times the concentration required to inhibit human TAP by 50%. We attempted to measure the specific binding of radiolabelled ICP47-1 and ICP47-2 to microsomes containing mouse TAP in experiments similar to those shown in Fig. ​Fig.4.4. However, there was little specific binding of ICP47-1 and ICP47-2, and it was difficult to measure binding at lower protein concentrations. We therefore measured binding at a single, higher protein concentration (2.75 μM), one sufficient to inhibit 10 to 20% of the mouse TAP activity and all of the human TAP activity. In this experiment, specific binding to microsomes containing murine TAP was determined by subtracting the binding to microsomes from insect cells that were not induced to express murine TAP (1). The binding of ICP47-1 and ICP47-2 to human TAP was easily measured (Fig. ​(Fig.5),5), although under these conditions it is important to note that ICP47-1 and ICP47-2 were present at concentrations beyond those required to saturate the TAP (Fig. ​(Fig.4A).4A). By contrast, it was found that there was little or no significant binding of ICP47-1 or ICP47-2 to microsomes containing murine TAP when background binding to control membranes was subtracted. In the experiment shown, specific ICP47-2 binding was greater than zero, but in other experiments this binding was less than zero, and thus we concluded that there was no detectable binding overall. In every experiment, it was clear that the level of binding of ICP47-1 and ICP47-2 to murine TAP was at least 25-fold lower than to human TAP. However, the human TAP present in these microsomes was limiting in these experiments, and thus it is very likely that the 25-fold difference between the levels of binding to human and mouse TAP is an underestimate. More likely this difference is 50- to 100-fold. On the basis of the inhibitory concentrations required to block murine TAP and the binding studies described above, estimates of the binding affinities of ICP47-1 and ICP47-2 for murine TAP may fall in the range of 5 × 10⁻⁶ M. Therefore, ICP47-1 and ICP47-2 bind poorly to the murine TAP, and this largely accounts for their inability to block mouse TAP peptide transport. Open in a separate windowFIG. 5Binding of ICP47-1 and ICP47-2 to microsomes containing murine TAP. Microsomes containing human TAP or control membranes without human TAP (100 μg of membrane protein per 150-μl assay) or microsomes containing mouse TAP or control membranes without mouse TAP (50 μg of membrane protein with the same TAP activity as with the human microsomes) were incubated with ¹²⁵I-labelled ICP47-1 or ICP47-2 at 2.75 μM for 60 min at 4°C. The microsomes were washed twice, pelleted, and disrupted with detergents as described previously (7). Radioactivity associated with the microsomes was quantified by gamma counting. “ICP47 bound” refers to specific binding, calculated by subtracting the binding to control membranes (without TAP) from that observed with microsomes containing human or murine TAP.In summary, ICP47-2 and ICP47-1 could block human TAP and bound to TAP with similar high affinities. It was interesting that these two proteins, whose primary structures are only about 40% identical, inhibit human TAP with indistinguishable profiles and bind to human TAP with virtually identical affinities. Moreover, both proteins blocked murine TAP poorly and only at high protein concentrations and could not bind to murine TAP. These results, at face value, would suggest that mice will not be an appropriate model in which to test the effects of ICP47 on HSV replication or as a selective inhibitor of CD8⁺ T-cell responses in other systems. However, we recently found that an HSV-1 ICP47 mutant showed dramatically reduced neurovirulence in mice, without altering the course of disease in the cornea (4). Therefore, ICP47 may attain sufficient concentrations in certain cells in the nervous systems of mice to inhibit TAP. This may be related to the fact that TAP and class I proteins are expressed at low levels in the nervous system. Alternatively, ICP47 may have other functions in the nervous system.     

Regulation of MHC class I heterodimer stability and interaction with TAP by tapasin

 Major histocompatibility complex (MHC) class I molecules are heterodimers of a class I heavy chain and β₂-microglobulin that bind peptides supplied by the MHC region-encoded transporters associated with antigen processing (TAP). Peptide binding by class I heterodimers is necessary for their maturation into stable complexes and is dependent on their physical association with TAP. In human mutant 721.220 cells, however, a novel genetic defect causes the failure of class I heterodimers to associate with TAP. This deficiency correlates with lack of expression of a glycoprotein, tapasin (TAP-associated glycoprotein), which has been found in association with class I heterodimers and TAP. Employing a transcomplementation analysis, we obtained evidence co-localizing the genetic defect of mutant 220 cells and the structural or a regulatory gene controlling the expression of tapasin on the short arm of chromosome 6, which includes the MHC. Expression of tapasin and the normal interaction of class I heterodimers with TAP are concomitantly restored, indicating the probable function of tapasin as a physical link between these complexes. In further support of this model, the absence of tapasin in mutant 220 cells correlates with reduced class I heterodimer stability, suggesting that tapasin may stabilize class I heterodimers and thereby enhance their association with TAP. These results further implicate tapasin in a mechanism that promotes peptide binding by class I heterodimers through their interaction with TAP. Received: 20 March 1997 / Revised: 2 June 1997     

Nucleotide interactions with membrane-bound transporter associated with antigen processing proteins

The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4 degrees C using the photo-cross-linkable nucleotide analogs 8-azido-[gamma-(32)P]ATP and 8-azido-[alpha-(32)P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.     

Catalytic site modifications of TAP1 and TAP2 and their functional consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.     

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6

The human cytomegalovirus (HCMV) protein US6 inhibits the transporter associated with antigen processing (TAP). Since TAP transports antigenic peptides into the endoplasmic reticulum for binding to major histocompatibility class I molecules, inhibition of the transporter by HCMV US6 impairs the presentation of viral antigens to cytotoxic T lymphocytes. HCMV US6 inhibits ATP binding by TAP, hence depriving TAP of the energy source it requires for peptide translocation, yet the molecular basis for the interaction between US6 and TAP is poorly understood. In this study we demonstrate that residues 89 to 108 of the HCMV US6 luminal domain are required for TAP inhibition, whereas sequences that flank this region stabilize the binding of the viral protein to TAP. In parallel, we demonstrate that chimpanzee cytomegalovirus (CCMV) US6 binds, but does not inhibit, human TAP. The sequence of CCMV US6 differs from that of HCMV US6 in the region corresponding to residues 89 to 108 of the HCMV protein. The substitution of this region of CCMV US6 with the corresponding residues from HCMV US6 generates a chimeric protein that inhibits human TAP and provides further evidence for the pivotal role of residues 89 to 108 of HCMV US6 in the inhibition of TAP. On the basis of these observations, we propose that there is a hierarchy of interactions between HCMV US6 and TAP, in which residues 89 to 108 of HCMV US6 interact with and inhibit TAP, whereas other parts of the viral protein also bind to TAP and stabilize this inhibitory interaction.     

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis

The effects of estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10(-10) M E2 stimulated and 10(-8) M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E2. E2 was without effect in the HTC and estrogen-resistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tam-resistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E2 occurred via a different pathway that appears to be related to the presence of ER in the cells.     

Temperature-sensitive synthesis of a metalloproteinase in ts110-MSV-M-transformed NRK cells

Previously, we reported that transformation associated protein (TAP) was over-expressed in the 6m2 line, but not in their normal counterparts (1,2). 6m2 is a culture of NRK cells transformed by the ts-110 mutant of MSV-M. The synthesis of TAP and the expression of transformation properties in the 6m2 cells are all temperature-sensitive (2; 3; 4). TAP is secreted as two polypeptides of 64 kD and 68 kD (P64 and P68) (2). Experiments were carried out to determine whether any metalloproteinase (MP) activity was associated with TAP. Results of zymograms indicated that the two forms of purified TAP (P64 and P68) had MP activity, using gelatin or collagen type IV as substrates. Serum-free medium (SFM) of 6m2 cells incubated at 33 degrees C also showed two bands of MP activity, while the corresponding SFM from 6m2 cells at 39 degrees C lacked such MP activity, indicating that the synthesis of MP was temperature-sensitive. The association of MP activity with the P64 and P68 bands of TAP (purified or in SFM) was confirmed by simultaneous Western blot analysis, which showed the reactivity of the two MP bands with monoclonal or polyclonal antibodies to TAP. Accordingly, what we previously designated as TAP is apparently one form of MP, which are known to be involved in tumor cell metastasis.