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Simon J Powis

Publications and source records attributed to Simon J Powis.

13 recordsLinked to original sources
PubMed2006

CLIP-region mediated interaction of Invariant chain with MHC class I molecules.

An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules.

Amino Acid Sequence↗
PubMed2006

Lack of tyrosine 320 impairs spontaneous endocytosis and enhances release of HLA-B27 molecules.

Several lines of evidence suggest that endocytosis of MHC class I molecules requires conserved motifs within the cytoplasmic domain. In this study, we show, in the C58 rat thymoma cell line transfected with HLA-B27 molecules, that replacement of the highly conserved tyrosine (Tyr320) in the cytoplasmic domain of HLA-B27 does not hamper cell surface expression of beta2-microglobulin H chain heterodimers or formation of misfolded molecules. However, Tyr320 replacement markedly impairs spontaneous endocytosis of HLA-B27. Although wild-type molecules are mostly internalized via endosomal compartments, Tyr320-mutated molecules remain at the plasma membrane in which partial colocalization with endogenous transferrin receptors can be observed, also impairing their endocytosis. Finally, we show that Tyr320 substitution enhances release of cleaved forms of HLA-B27 from the cell surface. These studies show for the first time that Tyr320 is most likely part of a cytoplasmic sorting motif involved in spontaneous endocytosis and shedding of MHC class I molecules.

Amino Acid Motifs↗
PubMed2006

Mutational analysis of the oxidoreductase ERp57 reveals the importance of the two central residues in the redox motif.

The oxidoreductase ERp57 is involved in the formation and breaking of disulfide bonds in assembling proteins within the environment of the endoplasmic reticulum. Site-directed mutants of the redox-active Cys-Gly-His-Cys motif within an isolated ERp57 sub-domain have been studied. Whereas mutation of either cysteine residue abolished reductase activity, substitution of the central residues resulted in retention of partial activity. Alkylation studies indicated that the central residue mutants retained the normal disulfide bond in the motif, whereas this disulfide bond became more resistant to reduction following addition of a third residue into the redox motif, demonstrating an optimum spacing within the redox-active motif of ERp57.

Alkylation↗
PubMed2005

Different MHC class I heavy chains compete with each other for folding independently of beta 2-microglobulin and peptide.

We reported previously that different MHC class I molecules can compete with each other for cell surface expression in F(1) hybrid and MHC class I transgenic mice. In this study, we show that the competition also occurs in transfected cell lines, and investigate the mechanism. Cell surface expression of an endogenous class I molecule in Chinese hamster ovary (CHO) cells was strongly down-regulated when the mouse K(d) class I H chain was introduced by transfection. The competition occurred only after K(d) protein translation, not at the level of RNA, and localization studies of a CHO class I-GFP fusion showed that the presence of K(d) caused retention of the hamster class I molecule in the endoplasmic reticulum. The competition was not for beta(2)-microglobulin, because a single chain version of K(d) that included mouse beta(2)-microglobulin also had a similar effect. The competition was not for association with TAP and loading with peptide, because a mutant form of the K(d) class I H chain, not able to associate with TAP, caused the same down-regulation of hamster class I expression. Moreover, K(d) expression led to a similar level of competition in TAP2-negative CHO cells. Competition for cell surface expression was also found between different mouse class I H chains in transfected mouse cells, and this competition prevented association of the H chain with beta(2)-microglobulin. These unexpected new findings show that different class I H chains compete with each other at an early stage of the intracellular assembly pathway, independently of beta(2)-microglobulin and peptide.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Misfolding of major histocompatibility complex class I molecules in activated T cells allows cis-interactions with receptors and signaling molecules and is associated with tyrosine phosphorylation.

Knowledge of the origin and biochemical status of beta(2)-microglobulin-free or misfolded major histocompatibility complex (MHC)-I molecules is essential for understanding their pleiotropic properties. Here we show that in normal human T cells, misfolding of MHC-I molecules is turned on upon activation and cell division and is proportional to the level of proliferation. Immunoprecipitation showed that a number of proteins are associated with MHC-I heavy chains at the surface of activated T cells, including the CD8alphabeta receptor and the chaperone tandem calreticulin/ERp57, associations that rely upon the existence of a pool of HC-10-reactive molecules. Biochemical analysis showed that misfolded MHC-I molecules present at the cell surface are fully glycosylated mature molecules. Importantly, misfolded MHC-I molecules are tyrosine phosphorylated and are associated with kinase activity. In vitro kinase assays followed by reprecipitation indicated that tyrosine phosphorylation of the class I heavy chain is probably mediated by a Src tyrosine kinase because Lck was found associated with HC-10 immunocomplexes. Finally, we show that inhibition of tyrosine phosphorylation by using the Src-family tyrosine kinase inhibitor PP2 resulted in enhanced release of MHC-I heavy chains from the cell surface of activated T cells and a slight down-regulation of cell surface W6/32-reactive molecules. This study provides new insights into the biology of MHC-I molecules and suggests that tyrosine phosphorylation may be involved in the regulation of MHC-I misfolding and expression.

Biotinylation↗
PubMed2004

Competition for access to the rat major histocompatibility complex class I peptide-loading complex reveals optimization of peptide cargo in the absence of transporter associated with antigen processing (TAP) association.

Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptide-loading complex (PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing (TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-Aa, when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-Au molecule from associating with TAP. However, stable RT1-Au molecules are assembled efficiently in competition with RT1-Aa, demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-Aa, which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-Aa, which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-Aa binding. Full optimization of RT1-Aa requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo.

Animals↗
PubMed2003

Formation of HLA-B27 homodimers and their relationship to assembly kinetics.

The human HLA-B27 class I molecule exhibits a strong association with the inflammatory arthritic disorder ankylosing spondylitis and other related arthropathies. Major histocompatibility complex class I heavy chains normally associate with beta(2)-microglobulin and peptide in the endoplasmic reticulum before transit to the cell surface. However, an unusual characteristic of HLA-B27 is its ability to form heavy chain homodimers through an unpaired cysteine at position 67 in the peptide groove. Homodimers have previously been detected within the ER and at the cell surface, but their mechanism of formation and role in disease remain undefined. Here we demonstrate, in the rat C58 thymoma cell line and in human HeLa cells transfected with HLA-B27, that homodimer formation involves not only cysteine at position 67 but also the conserved structural cysteine at position 164. We also show that homodimer formation can be induced in the non-disease-associated HLA class I allele HLA-A2 by slowing its assembly rate by incubation of cells at 26 degrees C, suggesting that homodimer formation in the endoplasmic reticulum may occur as a result of the slower folding kinetics of HLA-B27. Finally, we report an association between unfolded HLA-B27 molecules and immunoglobulin-binding protein at the cell surface.

Animals↗
PubMed2003

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype.

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Two highly divergent ancient allelic lineages of the transporter associated with antigen processing (TAP) gene in Xenopus: further evidence for co-evolution among MHC class I region genes.

In the frog Xenopus, MHC class I antigen presentation and processing genes (the immunoproteasome LMP2 and LMP7 and the transporter TAP1 and TAP2) seem to be closely linked in a primordial organization. Two distinct lineages of class Ia and LMP7 loci were previously identified, thus strongly suggesting co-evolution among 'class I region' genes. We now show that the Xenopus MHC 'class I region' lies between class II and class III genes and we have isolated two distinct alleles at both the TAP1 and TAP2 loci. The alleles at each locus are remarkably divergent from each other and phylogenetic tree analysis revealed in both cases that they diverged from each other 60-100 million years ago (MYA). For lineage-frequency and linkage analysis, 25 wild-caught X. laevis and 16 X. tropicalis were examined. The two lineages were present in different frequencies for X. laevis and X. tropicalis. Nevertheless, in all cases, the LMP7, TAP1, and TAP2 lineages were found in a set comprising one of the two lineages. Furthermore, like the LMP7 lineages, the TAP lineages were detected in most Xenopus species that diverged from a common ancestor 80-100 MYA, suggesting that the 'class I region' biallelic lineages are under balancing selection.

Alleles↗
PubMed2003

Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification.

Major histocompatibility complex (MHC) class I molecules bind and present short peptides to cells of the immune system. The oxidoreductase ERp57 is involved in the assembly of MHC class I molecules and is a component of the peptide loading complex, where it is found disulfide-bonded to tapasin. We have studied ERp57 and the ERp57-tapasin conjugate by rapid acidification of the intracellular environment with trichloroacetic acid (TCA), followed by thiol modification with the alkylating agent 4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). By using TCA/AMS treatment, non-tapasin-associated ERp57 is shown to exist almost exclusively in a reduced state, suggesting that both thioredoxin-like CXXC motifs are exposed and reduced. A 110-kDa product is readily detected with this TCA/AMS protocol and is confirmed as an ERp57-tapasin conjugate by its absence from the tapasin-deficient .220 cell line and by immunoblotting with both ERp57- and tapasin-specific antisera. The ERp57-tapasin conjugate can also be modified with the oxidizing agent diamide, indicating that within the pool of ERp57-tapasin complexes the free, non-tapasin-linked CXXC motif exists in both oxidized and reduced states, suggesting availability to undergo redox reactions.

Acids↗
PubMed2002

The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules.

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone-assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide-loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N-terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation.

Amino Acid Motifs↗
PubMed2002

Interactions formed by individually expressed TAP1 and TAP2 polypeptide subunits.

The transporter associated with antigen processing (TAP) supplies peptides into the lumen of the endoplasmic reticulum (ER) for binding by major histocompatibility complex (MHC) class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each a 'half-transporter' encoding a transmembrane domain and a nucleotide-binding domain. Immunoprecipitation of rat TAP1 and TAP2 expressed individually in the human TAP-deficient cell line, T2, revealed that both bound the endogenously expressed HLA-A2 and -B51 class I molecules. Using HLA-encoding recombinant vaccinia viruses HLA-A*2501, -B*2704, -B*3501 and -B*4402, alleles also associated with both TAP1 and TAP2. Thus, TAP1 and TAP2 do not appear to differ in their ability to interact with MHC class I alleles. Single TAP polypeptide subunits also formed MHC class I peptide-loading complexes, and their nucleotide-binding domains retained the ability to interact with ATP, and may permit the release of peptide-loaded MHC class I molecules in the absence of a peptide transport cycle. It is also demonstrated by chemical cross-linking that TAP2, but not TAP1, has the ability to form a homodimer complex both in whole cells and in detergent lysates. Together these data indicate that single TAP polypeptide subunits possess many of the features of the TAP heterodimer, demonstrating them to be useful models in the study of ATP-binding cassette (ABC) transporters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗