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Posts Tagged ‘antigenic peptides’


Phosphorylation-dependent interaction between antigenic peptides and MHC class I

Curator: Larry H. Bernstein, MD, FCAP

 

 

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Nat Immunol. 2008 Nov;9(11):1236-43.    http://dx.doi.org:/10.1038/ni.1660.  Epub 2008 Oct 5.
Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of ‘transformed self’. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.
Figure 1
Bioinformatic characterization of the HLA-A2–restricted phosphopeptide repertoire. (a) Distribution of phosphorylated residues among naturally processed (A2 phosphopeptide) and predicted HLA-A2 binding phosphopeptides (Phosphosite, EMBL). The frequency of phosphorylated residues at each position is displayed for naturally processed HLA-A2 associated phosphopeptides, and for peptides in EMBL and Phosphosite datasets that contain phosphorylation sites and are predicted, according to criteria described in Methods, to bind HLA-A2. (b) Representation of positively charged residues (Arg or Lys) at P1 among naturally processed HLA-A2 associated phosphopeptides, phosphopeptides from the EMBL or Phosphosite datasets that are predicted to bind HLA-A2 and contain a p-Ser residue at the P4 position, and datasets of naturally processed non-phosphorylated peptides (B-LCL) and known HLA-A2 binding peptides (Immune Epitope). Selection criteria for the latter two datasets are described in Methods. * = P<0.001, NS= not significant. (c, d) Representation of subdominant residues at the P2 anchor position (c) and the PC (P9) position (d) in naturally processed HLA-A2 associated phosphopeptides and in datasets of naturally processed non-phosphorylated peptides and known HLA-A2 binding peptides.
Changes in protein expression or metabolism due to intracellular infection or cellular transformation modify the repertoire of peptides generated and therefore displayed by class I MHC molecules, resulting in presentation of “altered self” to the immune system. T cell receptor (TCR)-mediated recognition of specific MHC-bound peptides by CD8 T lymphocytes results in cytolytic activity and release of pro-inflammatory cytokines, which are key components of anti-viral and anti-tumor immunity. Evidence suggests that peptides containing post-translational modifications (PTM), including deamidation, cysteinylation, glycosylation, and phosphorylation, contribute to the pool of MHC-bound peptides presented at the cell surface and represent potential targets for T cell recognition2. Indeed, the majority of naturally occurring PTM-bearing peptides defined to date can be discriminated from their unmodified homologs specifically by T cells2-4.  …..
Recent studies have highlighted protein phosphorylation as a process with the capacity to generate unique peptides bound to class I MHC molecules. Significant numbers of different phosphorylated peptides are presented by several HLA-A and HLA-B alleles that are prevalent in humans3,4, demonstrating their widespread potential as antigens. Moreover, CD8+ T lymphocytes recognize these phosphopeptides in a manner that is both peptide sequence-specific and phosphate-dependent3, 4. Thus, phosphopeptides can be immunologically distinguished from their non-phosphorylated counterparts. Consistent with their presentation by class I MHC molecules, most phosphorylated peptides are derived from proteins that function intracellularly, and processing of both model and naturally occurring phosphopeptides is dependent on transport into the endoplasmic reticulum (ER) by transporter associated with antigen processing (TAP)3, 5. Furthermore, rapid degradation by the proteasome, a process that regulates the activity of many transcription factors, cell growth modulators, signal transducers and cell cycle proteins6-8, is frequently dependent on target protein phosphorylation9-11. ….
Phosphopeptide antigens are of significant therapeutic interest because deregulation of protein kinase activity, normally tightly controlled, is one of the hallmarks of malignant transformation and is thought to contribute directly to oncogenic signaling pathways involved in cell growth, differentiation and survival13-15. In addition, mutation-induced deregulation of a limited number of critical kinases can often lead to activation of several signaling cascades and increases in the extent of protein phosphorylation within the cell16-18. These considerations strongly suggest that alterations in protein phosphorylation during malignancy represent a distinctive immunological signature of “transformed self”. Consistent with this notion, the phosphopeptides presented by HLA-A*0201….

Nα-Terminal Acetylation for T Cell Recognition: Molecular Basis of MHC Class I–Restricted Nα-Acetylpeptide Presentation

As one of the most common posttranslational modifications (PTMs) of eukaryotic proteins, Nα-terminal acetylation (Nt-acetylation) generates a class of Nα-acetylpeptides that are known to be presented by MHC class I at the cell surface. Although such PTM plays a pivotal role in adjusting proteolysis, the molecular basis for the presentation and T cell recognition of Nα-acetylpeptides remains largely unknown. In this study, we determined a high-resolution crystallographic structure of HLA (HLA)-B*3901 complexed with an Nα-acetylpeptide derived from natural cellular processing, also in comparison with the unmodified-peptide complex. Unlike the α-amino–free P1 residues of unmodified peptide, of which the α-amino group inserts into pocket A of the Ag-binding groove, the Nα-linked acetyl of the acetylated P1-Ser protrudes out of the groove for T cell recognition. Moreover, the Nt-acetylation not only alters the conformation of the peptide but also switches the residues in the α1-helix of HLA-B*3901, which may impact the T cell engagement. The thermostability measurements of complexes between Nα-acetylpeptides and a series of MHC class I molecules derived from different species reveal reduced stability. Our findings provide the insight into the mode of Nα-acetylpeptide–specific presentation by classical MHC class I molecules and shed light on the potential of acetylepitope-based immune intervene and vaccine development.

Produced by Ag processing and proteasomal degradation of intracellular proteins, polypeptides serve as CTL epitopes presented by MHC class I molecules, which play a critical role in cellular immunity (1). Eukaryotic proteins bearing various posttranslational modifications (PTMs) can generate a group of modified Ags, which contribute to a special repertoire of MHC-associated peptides presented at the cell surface as potential targets for TCR-mediated recognition. A modified peptide may become a new Ag because of the distinguished antigenicity compared with its unmodified homolog. A variety of natural peptide Ags containing modification have been observed that can be immunologically discriminated by T cells from their unmodified homologs as “altered self” (2). Thus, the significance of PTMs on epitopes and the application of modified peptides in vaccine development for immunotherapy against cancer and autoimmune diseases have been increasingly appreciated (3, 4).

The molecular bases of the presentation of peptides with several PTMs by MHC class I molecules have been successfully explicated. For instance, the formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of H2-M3 (5); both the glycan and the phosphate moieties of the central region of the glycopeptides (6, 7) and the phosphopeptides (8, 9), respectively, are exposed to enable TCR binding, and the deimination (citrullination) of arginine on a peptide presented by two HLA-B27 subtypes induces distinct peptide conformations (10).

Nα-terminal acetylation (Nt-acetylation) is one of the most common PTMs, occurring on the vast majority of eukaryotic proteins. In humans, >80% of the different varieties of intracellular proteins are irreversibly Nt-acetylated by Nα-acetyltransferases, often after the removal of the initiator methionine. Only a subset of the penultimate residues (Ala, Ser, Thr, Cys, and Val) or the retained initiator methionine can be acetylated at the α-amino (NH2) groups (11). A recent study found that acetylated N-terminal residues of eukaryotic proteins act as specific degradation signals (Ac-N-degrons) that are recognized by specific ubiquitin ligases (12). A subsequent systematic analysis demonstrated that Nt-acetylation can also represent an early determining factor in the cellular sorting for prevention of protein targeting to the secretory pathway (13). These findings suggested that Nt-acetylation–mediated inhibition of secretion could contribute to the retention of proteins in the cytosol where they may subsequently be ubiquitinylated through the specific recognition of their Ac-N-degrons and thereby generating Nt-acetylated proteasomal digestion products (14). Hence, these Nt-acetylated polypeptides in the form of MHC-associated neoantigens stand a good chance to be recognized by T cells. This has indeed been illuminated in an Nt-acetylated MHC class II–restricted peptide derived from myelin basic protein, which stimulates murine T cells to elicit experimental autoimmune encephalomyelitis, whereas the nonacetylated form does not (15). A structural study subsequently suggested that the Nt-acetylation of this peptide is essential for MHC class II binding (16).

For MHC class I, the first Nt-acetylated natural ligand was identified more than a decade ago (17). However, the mode of interaction of this acetylated peptide with class I molecules remained largely enigmatic. To understand this, we determined the crystal structures of a naturally occurring Nt-acetylated self-peptide (NAc-SL9) and two nonmodified variants (SL9 and HL8), respectively, in complex with HLA-B*3901. Taken together with the thermostability analyses of Nα-acetylpeptides complexed with a series of class I molecules of human and murine origin, we elucidated that Nt-acetylation exerts a destabilizing effect on peptide–MHC (pMHC) complex, thereby influencing TCR recognition.

……

Our results here provide the structural and thermodynamic insights into the presentation of Nt-acetylated peptides by MHC class I molecules. The structure of the Nα-acetylpeptide in complex with HLA-B*3901 outlines a molecular interpretation of the reduced stability of MHC class I–bound Nt-acetylated peptides and also highlights a potential influence of Nt-acetylation on antigenic identity and T cell recognition. In addition, the structure elucidation of HLA-B*3901, the predominant B39 subtype, also is valuable in studying immune diseases associated with this MHC allele.

In a previous report, the Nt-formyl group on an Nt-formylated peptide binds to the bottom of the peptide-binding groove of the murine MHC class I H2-M3 playing an anchoring role for MHC class I binding (Supplemental Fig. 2A) (5). In our study, the methyl and carbonyl groups of the acetyl are rotated upwards like two arms that push the peptide-binding groove open (Fig. 2G, Supplemental Fig. 2B), thereby altering its immunogenicity at the expense of the pMHC stability. The thermostability we tested from seven human and one murine complexes indicates a general feature of Nα-acetylpeptide in weakening the binding affinity to MHC class I, which could be revealed by the gel-filtration chromatography of pMHC refolding assays as well (Supplemental Fig. 3). Their instability would partially explain why, as yet, such epitopes are rarely found. Within N-terminal residues of eukaryotic proteins, Ser is the most frequently acetylated in vivo (11). The Ala, Thr, Cys, and Val residues can also be Nt-acetylated and have small side chains like Ser. Thus, the rotation of P1 residues observed in the pHLA-B*3901 complex with an acetylated P1-Ser could very well be a general mode in Nα-acetylpeptide binding. In contrast, the long side chain of Met precludes it from being rotated into pocket A, but a certain reorientation is presumed to take place in the acetylated P1-Met based on the thermal instability (Fig. 6H). Besides the accommodation of the acetyl moiety, Nt-acetylation is presumed to decrease the stability of the pHLA-B*3901 complex as a result of the conformational switch of the Arg62. Arg62 in the α1-helix is largely conserved in almost all HLA-B and -C allotypes (Table V). For other HLA class I (Table V, Fig. 8), the long charged side chains of the residues in position 62 (Glu62 of A24 and Gln62 of A11 and so on) also may interact with the acetyl. Hence, the residue in position 62 plays a key role in the interaction between acetyl group and the H chain, which may influence not only the Nα-acetylpeptide binding to HLA molecules but also the TCR docking.

The discoveries that intracellular proteins with Ac-N-degrons are inhibited from being secreted (13) and then are degraded via ubiquitylation (12) raise many questions on the biological significance of acetylation-mediated proteolysis (14). The Nt-acetylated peptides with the size of MHC class I ligands (8–11 aa) as neoepitopes for CD8+ T cells, represent one of the possible roles of the Nt-acetylated digestion products. The vast armory of intracellular proteins that are frequently Nt-acetylated can create a large pool of Nα-acetylpeptides for Ag presentation and T cell surveying. The Nt-acetylation potentially impacts the TCR-MHC interaction in three different aspects: 1) the direct interaction of the solvent-exposed acetyl moiety; 2) the altered conformation of the central region of the peptide main chain; and 3) the conformational switches of the MHC residues. The Nt-acetylation creation of a distinctive pMHC landscape and participation in a potential binding element for TCR engagement described in our results highlights needs for further investigation into the Nα-acetylpeptide–specific TCR repertoires.  ……

see…J Immunol 2014; 192:5509-5519   http://dx.doi.org:/10.4049/jimmunol.1400199   http://www.jimmunol.org/content/192/12/5509

Supplementary http://www.jimmunol.org/content/suppl/2014/05/14/jimmunol.1400199.DCSupplemental.html
References http://www.jimmunol.org/content/192/12/5509.full#ref-list-1

 

The Cellular Redox Environment Alters Antigen Presentation*

Jonathan A. Trujillo,§12Nathan P. Croft,1Nadine L. Dudek,1Rudragouda ChannappanavarAlex TheodossisAndrew I. Webb,…., Jamie Rossjohn,‡‡,§§5Stanley Perlman,§6 and Anthony W. Purcell,7
The Journal of Biological Chemistry 289; 27979-27991.
http://dx.doi.org:/10.1074/jbc.M114.573402

Capsule

Background: Modification of cysteine residues, including glutathionylation, commonly occurs in peptides bound to and presented by MHC molecules.

Results: Glutathionylation of a coronavirus-specific T cell epitope results in diminished CD8 T cell recognition.

Conclusion: Cysteine modification of a T cell epitope negatively impacts the host immune response.

Significance: Cross-talk between virus-induced oxidative stress and the T cell response probably occurs, diminishing host cell recognition of infected cells.

Cysteine-containing peptides represent an important class of T cell epitopes, yet their prevalence remains underestimated. We have established and interrogated a database of around 70,000 naturally processed MHC-bound peptides and demonstrate that cysteine-containing peptides are presented on the surface of cells in an MHC allomorph-dependent manner and comprise on average 5–10% of the immunopeptidome. A significant proportion of these peptides are oxidatively modified, most commonly through covalent linkage with the antioxidant glutathione. Unlike some of the previously reported cysteine-based modifications, this represents a true physiological alteration of cysteine residues. Furthermore, our results suggest that alterations in the cellular redox state induced by viral infection are communicated to the immune system through the presentation of S-glutathionylated viral peptides, resulting in altered T cell recognition. Our data provide a structural basis for how the glutathione modification alters recognition by virus-specific T cells. Collectively, these results suggest that oxidative stress represents a mechanism for modulating the virus-specific T cell response.

Antigen Presentation     Antigen Processing     Glutathionylation     Mass Spectrometry (MS)     Oxidation-Reduction (Redox)     Redox Regulation     T-cell     Viral Immunology

Small fragments of proteins (peptides) derived from both intracellular and extracellular sources are displayed on the surface of cells by molecules encoded within the major histocompatibility complex (MHC). These peptides are recognized by T lymphocytes and provide the immune system with a surveillance mechanism for the detection of pathogens and cancer cells. The fidelity with which antigen presentation communicates changes in the intracellular proteome is critical for immune surveillance. Not only do antigens expressed at vastly different abundances need to be represented within the array of peptides selected and presented at the cell surface (collectively termed the immunopeptidome (1, 2)), but changes in their post-translational state also need to be conveyed within this complex mixture of peptides. For example, changes in antigen phosphorylation have been linked to cancer, and the presentation of phosphorylated peptides has been shown to communicate the cancerous state of cells to the immune system (36). Other types of post-translational modification play a central role in the pathogenesis of autoimmune diseases (7), such as arginine citrullination in arthritis (810), deamidation of glutamine residues in wheat proteins in celiac disease (1115), and cysteine oxidation in type 1 diabetes (16, 17). Cysteine is predicted to be present in up to 14% of potential T cell epitopes based on its prevalence in various pathogen and host proteomes (18). However, reports of cysteine-containing epitopes are much less frequent due to technical difficulties associated with synthesis and handling of cysteine-containing peptides and their subsequent avoidance in many epitope mapping studies (19). Cysteine can be modified in numerous ways, including cysteinylation (the disulfide linkage of free cysteine to peptide or protein cysteine residues), oxidation to cysteine sulfenic (oxidation), sulfinic (dioxidation) and sulfonic acids (trioxidation), S-nitrosylation, and S-glutathionylation. Such modifications may occur prior to or during antigen processing; however, the role of cysteine modification in T-cell-mediated immunity has not been systematically addressed.

In addition to constitutive presentation of a subset of oxidatively modified peptides, it is anticipated that changes in the proportion of these ligands will occur upon infection because oxidative stress, triggering of the unfolded protein response, and modulation of host cell synthesis by the virus are hallmarks of this process (2027). For example, host cell stress responses modulate expression, localization, and function of Toll-like receptors, a key event in the initiation of the immune response (28). Oxidative stress would also be predicted to affect protein function through post-translational modification of amino acids, such as cysteine. Indeed, because of the reactive nature of cysteine and the requirements for cells to regulate the redox state of proteins to maintain function, a number of scavenging systems for redox-reactive intermediates exist. The tripeptide glutathione (GSH) is one of the key intracellular antioxidants, acting as a scavenger for reactive oxygen species. Reduced GSH is equilibrated with its oxidized form, GSSG, with normal cytosolic conditions being that of the reduced state in a ratio of ∼50:1 (GSH/GSSG) (29). Modification of proteins and peptides with GSH (termed S-glutathionylation) occurs following reaction of GSSG with the thiol group of cysteine in a reaction catalyzed by the detoxifying enzyme, glutathione S-transferase (GST). A variety of cellular processes and signaling pathways, such as the induction of innate immunity, apoptosis, redox homeostasis, and cytokine production, are modulated by this GST-catalyzed post-translational modification (3032). S-Glutathionylation can eventuate via oxidative stress, whereby the intracellular levels of GSSG increase.

Given that viruses are known to induce oxidative stress (3335), the intracellular environment of viral infection may lead to an increase inS-glutathionylated cellular proteins and viral antigens. For instance, HSV infection induces an early burst of reactive oxygen species, resulting in S-glutathionylation of TRAF family members, which in turn is linked to downstream signaling and interferon production (36). The potential for modification of viral antigens subsequent to reactive oxygen species production is highlighted by S-glutathionylation of several retroviral proteases, leading to host modulation of protease function (37). Indeed large scale changes in protein S-glutathionylation are observed in HIV-infected T cell blasts (38), suggesting that functional modulation of both host and viral proteins occurs via this mechanism. Whether these S-glutathionylated proteins inhibit or enhance immune responses to the unmodified epitope or generate novel T-cell epitopes that are subsequently recognized by the adaptive immune system is unclear.

Here, we investigate the frequency of modification of cysteine-containing MHC-bound peptides by interrogating a large database of naturally processed self-peptides derived from B-lymphoblastoid cells, murine tissues, and cytokine-treated cells. In addition, the functional consequences of Cys modification of T cell epitopes was investigated using an established model of infection that involves an immunodominant cysteine-containing epitope derived from a neurotropic strain of mouse hepatitis virus, strain JHM (JHMV)8(3941). We describe S-glutathionylation of this viral T cell epitope and the functional and structural implications of redox-modulated antigen presentation. Collectively our studies suggest that S-glutathionylation plays a key, previously unappreciated role in adaptive immune recognition.

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