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Biomedical subjects

Peter Cresswell

Publications and source records attributed to Peter Cresswell.

At least 19 recordsLinked to original sources
PubMed2006

An N-linked glycan modulates the interaction between the CD1d heavy chain and beta 2-microglobulin.

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.

Amino Acid Sequence↗
PubMed2006

A role for the endoplasmic reticulum protein retrotranslocation machinery during crosspresentation by dendritic cells.

Crosspresentation of exogenous antigens (Ags) to CD8(+) T cells by dendritic cells generally requires their entry into the cytosol. Here we show that both soluble and phagocytosed extracellular Ags accessed the cytosol via molecular components required for endoplasmic reticulum (ER)-associated degradation (ERAD). Exogenous Pseudomonas aeruginosa Exotoxin A, which inhibits protein translocation from the ER to the cytosol, abrogated crosspresentation. Exotoxin A also prevented the transporter associated with antigen processing (TAP) inhibitor, ICP47, from entering the cytosol and blocking TAP-mediated peptide transport. In an in vitro model of retrotranslocation, the AAA ATPase p97, an enzyme critical for ERAD, was the only cytosolic cofactor required for protein export from isolated phagosomes. Functional p97 was also required for crosspresentation but not conventional presentation. Thus, crosspresentation appears to result from an adaptation of the retrotranslocation mechanisms involved in the degradation of misfolded ER proteins.

ADP Ribose Transferases↗
PubMed2006

Exposure of the promonocytic cell line THP-1 to Escherichia coli induces IFN-gamma-inducible lysosomal thiol reductase expression by inflammatory cytokines.

IFN-gamma-inducible lysosomal thiol reductase (GILT), which plays a role in MHC class II-restricted processing and presentation of Ags containing disulfide bonds, can be induced in various cell types by the cytokine IFN-gamma. APCs, including circulating macrophages, constitutively express high levels of GILT, although the pathways regulating its expression in these cells have not been characterized. In this study, we used the promonocytic cell line THP-1, an established model for monocyte to macrophage differentiation, to investigate the induction of GILT upon exposure to bacteria. We show that contact with LPS or intact Escherichia coli causes THP-1 cells to undergo programmed differentiation, characterized by adhesion, cytokine secretion, and up-regulation of Ag processing and presentation components, including GILT. Unlike GILT induction in response to IFN-gamma treatment, induction by bacteria is dependent on new protein synthesis, NF-kappaB signaling, and secretion of the inflammatory cytokines TNF and IL-1beta. Furthermore, we show that both cytokines are sufficient for GILT induction in the absence of a microbial stimulus. The majority of GILT synthesized by differentiated THP-1 cells is secreted as the precursor form rather than being transported to, and maturing in, lysosomes, suggesting a novel role for GILT in cells of the macrophage lineage.

Antigen-Presenting Cells↗
PubMed2006

Herpes simplex virus evades natural killer T cell recognition by suppressing CD1d recycling.

Natural killer T cells, which are stimulated by lipids presented by CD1d molecules, are crucial in antiviral host defense. How viruses evade natural killer T cell recognition remains unclear. Here we show that infection with herpes simplex virus type 1 (HSV-1) reduced CD1d surface expression on antigen-presenting cells. HSV-1 did not inhibit CD1d protein synthesis or enhance constitutive CD1d endocytosis. Instead, HSV-1 prevented the reappearance of endocytosed CD1d on the cell surface by redistributing endocytosed CD1d to the lysosome limiting membrane. HSV-1 might also inhibit the transport of newly synthesized CD1d to the cell surface. Such inhibition of CD1d surface expression impaired antigen-presenting cell-mediated stimulation of natural killer T cells, supporting the idea that this mechanism may be an important HSV-1 immune evasion strategy.

Antigen Presentation↗
PubMed2006

Enhanced and prolonged cross-presentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles.

CD8(+) T-cell responses are critical in the immunological control of tumours and infectious diseases. To prime CD8(+) T cells against these cell-associated antigens, exogenous antigens must be cross-presented by professional antigen-presenting cells (APCs). While cross-presentation of soluble antigens by dendritic cells is detectable in vivo, the efficiency is low, limiting the clinical utility of protein-based vaccinations. To enhance the efficiency of presentation, we generated nanoparticles from a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), to deliver antigen into the major histocompatibility complex (MHC) class I antigen presentation pathway. In primary mouse bone marrow-derived dendritic cells (BMDCs), the MHC class I presentation of PLGA-encapsulated ovalbumin (OVA) stimulated T cell interleukin-2 secretion at 1000-fold lower concentration than soluble antigen and 10-fold lower than antigen-coated latex beads. The microparticles also served as an intracellular antigen reservoir, leading to sustained MHC class I presentation of OVA for 72 hr, decreasing by only 20% after 96 hr, a time at which the presentation of soluble and latex bead-associated antigens was undetectable. Cytosol extraction demonstrated that antigen delivery via PLGA particles increased the amount of protein that escaped from endosomes into the cytoplasm, thereby increasing the access of exogenous antigen to the classic MHC class I loading pathway. These data indicate that the unique properties of PLGA particle-mediated antigen delivery dramatically enhance and sustain exogenous antigen presentation by MHC class I, potentially facilitating the clinical use of these particles in vaccination.

Animals↗
PubMed2005

Tapasin and ERp57 form a stable disulfide-linked dimer within the MHC class I peptide-loading complex.

We previously showed that the major histocompatibility complex (MHC) class I chaperone tapasin can be detected as a mixed disulfide with the thiol-oxidoreductase ERp57. Here we show that tapasin is a unique and preferred substrate, a substantial majority of which is disulfide-linked to ERp57 within the cell. Tapasin upregulation by interferon-gamma induces sequestration of the vast majority of ERp57 into the MHC class I peptide-loading complex. The rate of tapasin-ERp57 conjugate formation is unaffected by the absence of beta2-microglubulin (beta2m), and is independent of calnexin or calreticulin interactions with monoglucosylated N-linked glycans. The heterodimer forms spontaneously in vitro upon mixing recombinant ERp57 and tapasin. Noncovalent interactions between the native proteins inhibit the reductase activity of the thioredoxin CXXC motif within the N-terminal a domain of ERp57 to maintain its interaction with tapasin. Disruption of these interactions by denaturation allows reduction to proceed. Thus, tapasin association specifically inhibits the escape pathway required for disulfide-bond isomerization within conventional protein substrates, suggesting a specific structural role for ERp57 within the MHC class I peptide-loading complex.

Antiporters↗
PubMed2005

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor.

Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-gamma-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-gamma. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.

Alkyl and Aryl Transferases↗
PubMed2005

Mechanisms of MHC class I-restricted antigen processing and cross-presentation.

In this review, we discuss recent data from our laboratory that address two aspects of major histocompatibility complex (MHC) class I-restricted antigen processing. First, we consider the nature of the peptide-loading complex, which is the assembly of proteins in the endoplasmic reticulum (ER) into which newly synthesized MHC class I-beta(2) microglobulin (beta(2)m) heterodimers are incorporated, and the mechanisms involved in MHC class I assembly and peptide loading that are facilitated by the peptide-loading complex. Second, we discuss mechanisms of cross-presentation, the phenomenon whereby extracellular and luminal protein antigens can be processed by antigen-presenting cells, particularly dendritic cells, and presented by MHC class I molecules to CD8(+) T cells. The focus of the discussion is mainly on the human MHC class I system.

Animals↗
PubMed2005

Stoichiometric tapasin interactions in the catalysis of major histocompatibility complex class I molecule assembly.

The assembly of major histocompatibility complex (MHC) class I molecules with their peptide ligands in the endoplasmic reticulum (ER) requires the assistance of many proteins that form a multimolecular assemblage termed the 'peptide-loading complex'. Tapasin is the central stabilizer of this complex, which also includes the transporter associated with antigen processing (TAP), MHC class I molecules, the ER chaperone, calreticulin, and the thiol-oxidoreductase ERp57. In the present report, we investigated the requirements of these interactions for tapasin protein stability and MHC class I dissociation from the peptide-loading complex. We established that tapasin is stable in the absence of either TAP or MHC class I interaction. In the absence of TAP, tapasin interaction with MHC class I molecules is long-lived and results in the sequestration of existing tapasin molecules. In contrast, in TAP-sufficient cells, tapasin is re-utilized to interact with and facilitate the assembly of many MHC class I molecules sequentially. Furthermore, chemical cross-linking has been utilized to characterize the interactions within this complex. We demonstrate that tapasin and MHC class I molecules exist in a 1 : 1 complex without evidence of higher-order tapasin multimers. Together these studies shed light on the tapasin protein life cycle and how it functions in MHC class I assembly with peptide for presentation to CD8(+) T cells.

ATP-Binding Cassette Transporters↗
PubMed2004

Access of soluble antigens to the endoplasmic reticulum can explain cross-presentation by dendritic cells.

In dendritic cells (DCs), peptides derived from internalized particulate substrates are efficiently cross-presented by major histocompatibility complex (MHC) class I molecules. Exogenous soluble antigens are also presented by DCs but with substantially lower efficiency. Here we show that particulate and soluble antigens use different transport pathways. Particulate antigens have been shown to access peripheral endoplasmic reticulum (ER)-like phagosomes that are competent for cross-presentation, whereas we show here that soluble proteins that escape proteolysis enter the lumen of the ER. From there, they may be translocated into the cytosol by the pathway established for ER-associated degradation and their derived peptides may be transported back into the ER for binding by MHC class I molecules. MHC class I presentation involving the constitutive retrograde transport of soluble proteins to the ER by DCs may facilitate DC tolerance to components of their extracellular environment.

Animals↗
PubMed2004

Differential requirements for endosomal reduction in the presentation of two H2-E(d)-restricted epitopes from influenza hemagglutinin.

We examined the role of reduction in the presentation of two H2-E(d)-restricted epitopes (site 1 epitope (S1) and site 3 epitope (S3)) occupying distinct domains of the influenza hemagglutinin major subunit that contains four intrachain disulfide bonds and is connected to the virion by one interchain bond. S3 is situated within the stalk region that unfolds in response to mild acidification, and loads onto recycling H2-E(d) in the early endosome, while S1, located in the structurally constrained globular domain, loads onto nascent H2-E(d) in the late endosome. Predicting dependence upon reduction for either epitope seemed plausible but the results from several approaches were clear: presentation of S1 but not S3 is reduction dependent. Surprisingly, IFN-gamma-inducible lysosomal thiol reductase (GILT), the only reductase thus far known to be involved in MHC class II-restricted processing, is not necessary for the generation of S1. However, GILT is necessary for presentation of either epitope when the virus is pretreated with a reducible cross-linker. The results suggest that unfolding of the Ag, perhaps a prerequisite for proteolytic processing in many cases, proceeds either spontaneously in the early endosome or via reduction in a later endosome. They further imply mechanisms for GILT-independent reduction in the late endosome, with GILT perhaps being reserved for more intractable Ags.

Animals↗
PubMed2004

Major histocompatibility complex class I molecules expressed with monoglucosylated N-linked glycans bind calreticulin independently of their assembly status.

The assembly of major histocompatibility complex (MHC) class I molecules with peptides in the endoplasmic reticulum (ER) is a critical step in the presentation of viral antigens to CD8+ T cells. This process is subject to quality control restrictions that prevent free class I heavy chains (HCs) and peptide-free HC-beta(2)-microglobulin (beta(2)m) dimers from exiting the ER. The lectin-like chaperone calreticulin associates with HC-beta(2)m heterodimers prior to peptide binding, but its precise role in regulating the subsequent events of peptide association and ER to Golgi transport remains undefined. In vitro analysis of the assembly process has been limited by the specificity of calreticulin for monoglucosylated N-linked glycans, which are transient biosynthetic intermediates. To address this problem, we developed a novel expression system using Saccharomyces cerevisiae glycosylation mutants to produce class I HC bearing N-linked oligosaccharides with the specific structure Glc(1)Man(9)GlcNAc(2). The monoglucosylated glycan proved to be both necessary and sufficient for in vitro binding of calreticulin to MHC class I molecules. Calreticulin bound as efficiently to peptide-loaded MHC class I complexes as it did to folding intermediates created in vitro, namely free class I HC and empty HC-beta(2)m heterodimers. Thus, calreticulin is unable to discriminate between native and non-native MHC class I conformations and therefore unlikely to play a role in the recognition and release of peptide-loaded complexes from the ER. Furthermore, the recombinant expression system developed in this study can be used to produce a broad range of calreticulin substrates to elucidate its general mechanism of activity in vitro.

Calnexin↗
PubMed2004

Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.

Animals↗
PubMed2004

Lipid-protein interactions: biosynthetic assembly of CD1 with lipids in the endoplasmic reticulum is evolutionarily conserved.

The CD1 family consists of lipid antigen-presenting molecules, which include group I CD1a, CD1b, and CD1c and group II CD1d proteins. Topologically, they resemble the classical peptide antigen-presenting MHC molecules except that the large, exclusively nonpolar and hydrophobic, antigen-binding groove of CD1 has evolved to present cellular and pathogen-derived lipid antigens to specific T lymphocytes. As an approach to understanding the biochemical basis of lipid antigen presentation by CD1 molecules, we have characterized the natural ligands associated with mouse CD1d1 as well as human CD1b and CD1d molecules. We found that both group I and II CD1 molecules assemble with cellular phosphatidylinositol (PI), which contains heterogeneous fatty acyl chains. Further, this assembly occurs within the endoplasmic reticulum. Because the structures of the antigen-binding grooves of CD1a and CD1c closely resemble those of CD1b and CD1d, we conclude that the assembly of CD1 molecules with PI in the endoplasmic reticulum is evolutionarily conserved. These findings suggest that PI plays a chaperone-like role in CD1 assembly, possibly to preserve the integrity of the antigen-binding groove until CD1 binds antigenic lipids in the endocytic pathway.

Antigens, CD1↗
PubMed2004

Saposins facilitate CD1d-restricted presentation of an exogenous lipid antigen to T cells.

Members of the CD1 family present antigenic lipids to T lymphocytes. CD1 molecules survey endocytic compartments for lipid antigens that are sorted into these vesicles after incorporation into the membrane bilayer, and extraction from the bilayer is likely to be a critical step for lipid association. We hypothesized that lysosomal saposins, which are cofactors required for sphingolipid degradation, might be involved in this process. Here we show that saposins, although not required for the autoreactive recognition of CD1d by natural killer T cells, are indispensable for the binding of an exogenous lipid antigen, alpha-galactosylceramide, to CD1d in the endocytic pathway. We suggest that saposins mobilize monomeric lipids from lysosomal membranes and facilitate their association with CD1d.

Animals↗
PubMed2004

Recent developments in MHC-class-I-mediated antigen presentation.

Defective ribosomal products provide an important supply of endogenous peptides for entry into the classic MHC class I antigen presentation pathway. The recruitment of endoplasmic reticulum membrane during phagosome biogenesis allows exogenous antigens to be translocated into the cytosol as well as providing access to the class I peptide transport and loading machinery. This combination of features provides a mechanism for cross-presentation by specialised antigen presenting cells. Recent studies have shed new light on these pathways and have also described the emerging K3 family of viral ubiquitin E3 ligases, which constitutively ubiquitinate and degrade MHC class I molecules and other immunoreceptors.

Animals↗