Specific proteolytic cleavages limit the diversity of the pool of peptides available to MHC class I molecules in living cells

MHC class I molecules display peptides selected from a poorly characterized pool of peptides available in the endoplasmic reticulum. We analyzed the diversity of peptides available to MHC class I molecules by monitoring the generation of an OVA-derived octapeptide, OVA257-264 (SL8), and its C-terminally extended analog, SL8-I. The poorly antigenic SL8-I could be detected in cell extracts only after its conversion to the readily detectable SL8 with carboxypeptidase Y. Analysis of extracts from cells expressing the minimal precursor Met-SL8-I by this method revealed the presence of SL8/Kb and the extended SL8-I/Kb complexes, indicating that the peptide pool contained both peptides. In contrast, cells expressing full length OVA generated only the SL8/Kb complex, demonstrating that the peptide pool generated from the full length precursor contained only a subset of potential MHC-binding peptides. Deletion analysis revealed that SL8-I was generated only from precursors lacking additional C-terminal flanking residues, suggesting that the generation of the C terminus of the SL8 peptide involves a specific endopeptidase cleavage. To investigate the protease responsible for this cleavage, we tested the effect of different protease inhibitors on the generation of the SL8 and SL8-I peptides. Only the proteasome inhibitors blocked generation of SL8, but not SL8-I. These findings demonstrate that the specificities of the proteases in the Ag-processing pathway, which include but are not limited to the proteasome, limit the diversity of peptides available for binding by MHC class I molecules in the endoplasmic reticulum.     

Evidence of selective processing of immunodominant epitopes in virally infected cells

Recent advances in clarifying the molecular mechanisms involved in Ag processing and presentation have relied heavily on the use of somatic cell mutants deficient in proteasome subunits, TAP transporter, and cell surface expression of MHC class I molecules. Of particular interest currently are those mutants that lack specific protease activity involved in the generation of antigenic peptides. It is theoretically possible that deficiencies of this nature could selectively prevent the cleavage of certain peptide bonds and thus generate only a subset of antigenic peptides. Gro29/Kb cell line is derived from the wild-type murine Ltk- cell line. This cell line is one example of a mutant that lacks specific protease activities. This deficiency manifests itself in an inability to generate a subset of immunodominant peptide epitopes derived from vesicular stomatitis virus and herpes simplex virus. This in turn leads to a general inability to present these viral epitopes to cytotoxic T lymphocytes (CTL). These studies describe a unique Ag processing deficiency and provide new insight into the role of proteasome-independent proteases in MHC class I-restricted peptide generation.     

Kaposi's sarcoma-associated herpesvirus K3 utilizes the ubiquitin-proteasome system in routing class major histocompatibility complexes to late endocytic compartments

Human herpesvirus 8 (HHV8) downregulates major histocompatibility complex (MHC) class I complexes from the plasma membrane via two of its genes, K3 and K5. The N termini of K3 and K5 contain a plant homeodomain (PHD) predicted to be structurally similar to RING domains found in E3 ubiquitin ligases. In view of the importance of the ubiquitin-proteasome system in sorting within the endocytic pathway, we analyzed its role in downregulation of MHC class I complexes in cells expressing K3. Proteasome inhibitors as well as cysteine and aspartyl protease inhibitors stabilize MHC class I complexes in cells expressing K3. However, proteasome inhibitors differentially affect sorting of MHC class I complexes within the endocytic pathway and prevent their delivery to a dense endosomal compartment. In this compartment, the cytoplasmic tail of MHC class I complexes is cleaved by cysteine proteases. The complex is then cleaved within the plane of the membrane by an aspartyl protease, resulting in a soluble MHC class I fragment composed of the lumenal domain of the heavy chain, beta(2)-microglobulin (beta(2)m), and peptide. We conclude that K3 not only directs internalization, but also targets MHC class I complexes to a dense endocytic compartment on the way to lysosomes in a ubiquitin-proteasome-dependent manner.     

Thimet oligopeptidase and the stability of MHC class I epitopes in macrophage cytosol

In this study we investigated the fate of a class of proteasome-generated oligopeptides, exposing them to the crude cytosol of macrophages or to the purified recombinant thimet oligopeptidase. Among the proteasome products of known sequences are MHC class I epitopes, 13 of which were randomly chosen to be used as putative substrates. Surprisingly, our results clearly showed that the majority of the peptides were poorly or not degraded, either by the purified enzyme or by the crude macrophage cytosol. The peptides, which were resistant to hydrolysis, displayed high affinity for the thimet oligopeptidase as competitive inhibitors. Regardless of the fact that our data do not allow prediction of whether or not a specific peptide would be degraded, it seems very likely that the structural features, which rule out the stability of the MHC class I peptides in the cytosol, may have implications in an optimized repertoire selection for antigen presentation.     

Structure-based identification of MHC binding peptides: Benchmarking of prediction accuracy

Identification of MHC binding peptides is essential for understanding the molecular mechanism of immune response. However, most of the prediction methods use motifs/profiles derived from experimental peptide binding data for specific MHC alleles, thus limiting their applicability only to those alleles for which such data is available. In this work we have developed a structure-based method which does not require experimental peptide binding data for training. Our method models MHC-peptide complexes using crystal structures of 170 MHC-peptide complexes and evaluates the binding energies using two well known residue based statistical pair potentials, namely Betancourt-Thirumalai (BT) and Miyazawa-Jernigan (MJ) matrices. Extensive benchmarking of prediction accuracy on a data set of 1654 epitopes from class I and class II alleles available in the SYFPEITHI database indicate that BT pair-potential can predict more than 60% of the known binders in case of 14 MHC alleles with AUC values for ROC curves ranging from 0.6 to 0.9. Similar benchmarking on 29,522 class I and class II MHC binding peptides with known IC(50) values in the IEDB database showed AUC values higher than 0.6 for 10 class I alleles and 9 class II alleles in predictions involving classification of a peptide to be binder or non-binder. Comparison with recently available benchmarking studies indicated that, the prediction accuracy of our method for many of the class I and class II MHC alleles was comparable to the sequence based methods, even if it does not use any experimental data for training. It is also encouraging to note that the ranks of true binding peptides could further be improved, when high scoring peptides obtained from pair potential were re-ranked using all atom forcefield and MM/PBSA method.     

A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors

The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.     

The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response

The generation of antigenic peptides bound and presented to the immune system by MHC class I molecules predominantly depends on the function of the proteasome system. Stimulation of cells with interferon gamma induces the incorporation of three active site bearing beta-subunits into the 20S proteasome and the formation of the PA28 proteasome modulator complex. PA28 alters the cleavage properties of the proteasome and enhances MHC class I antigen presentation. Thus, by cytokine induced change of the proteasome system cells may alter the proteolytic properties of the 20S proteasome and may render an organism more flexible in its peptide generation capacity.     

A peptide filtering relation quantifies MHC class I peptide optimization

Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human Immunodeficiency Virus Gag-Pol polyprotein.     

Roles for asparagine endopeptidase in class II MHC-restricted antigen processing

The adaptive immune response depends on the creation of suitable peptides from foreign antigens for display on MHC molecules to T lymphocytes. Similarly, MHC-restricted display of peptides derived from self proteins results in the elimination of many potentially autoreactive T cells. Different proteolytic systems are used to generate the peptides that are displayed as T cell epitopes on class I compared with class II MHC molecules. In the case of class II MHC molecules, the proteases that reside within the endosome/lysosome system of antigen-presenting cells are responsible; surprisingly, however, there are relatively few data on which enzymes are involved. Recently we have asked whether proteolysis is required simply in a generic sense, or whether the action of particular enzymes is needed to generate specific class II MHC-associated T cell epitopes. Using the recently identified mammalian asparagine endopeptidase as an example, we review recent evidence that individual enzymes can make clear and non-redundant contributions to MHC-restricted peptide display.     

The origin of proteasome-inhibitor resistant HLA class I peptidomes: a study with HLA-A*68:01

Some HLA class I molecules bind a significant fraction of their constitutive peptidomes in the presence of proteasome inhibitors. In this study, A*68:01-bound peptides, and their parental proteins, were characterized through massive mass spectrometry sequencing to refine its binding motif, including the nearly exclusive preference for C-terminal basic residues. Stable isotope tagging was used to distinguish proteasome-inhibitor sensitive and resistant ligands. The latter accounted for less than 20% of the peptidome and, like in HLA-B27, arose predominantly from small and basic proteins. Under the conditions used for proteasome inhibition in vivo, epoxomicin and MG-132 incompletely inhibited the hydrolysis of fluorogenic substrates specific for the tryptic or for both the tryptic and chymotryptic subspecificities, respectively. This incomplete inhibition was also reflected in the cleavage of synthetic peptide precursors of A*68:01 ligands. For these substrates, the inhibition of the proteasome resulted in altered cleavage patterns. However these alterations did not upset the balance between cleavage at peptide bonds resulting in epitope destruction and those leading to their generation. The results indicate that inhibitor-resistant HLA class I ligands are not necessarily produced by non-proteasomal pathways. However, their generation is not simply explained by decreased epitope destruction upon incomplete proteasomal inhibition and may require additional proteolytic steps acting on incompletely processed proteasomal products.     

A role for cathepsin L and cathepsin S in peptide generation for MHC class II presentation

The enzymes that degrade proteins to peptides for presentation on MHC class II molecules are poorly understood. The cysteinal lysosomal proteases, cathepsin L (CL) and cathepsin S (CS), have been shown to process invariant chain, thereby facilitating MHC class II maturation. However, their role in Ag processing is not established. To examine this issue, we generated embryonic fibroblast lines that express CL, CS, or neither. Expression of CL or CS mediates efficient degradation of invariant chain as expected. Ag presentation was evaluated using T cell hybridoma assays as well as mass spectroscopic analysis of peptides eluted from MHC class II molecules. Interestingly, we found that the majority of peptides are presented regardless of CL or CS expression, although these proteases often alter the relative levels of the peptides. However, for a subset of Ags, epitope generation is critically regulated by CL or CS. This result suggests that these cysteinal proteases participate in Ag processing and generate qualitative and quantitative differences in the peptide repertoires displayed by MHC class II molecules.     

Function of the transport complex TAP in cellular immune recognition

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility complex (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. The MHC-encoded ABC transporter works in concert with the proteasome and MHC class I molecules for the antigen presentation on the cell surface for T cell recognition. TAP forms a heterodimer where each subunit consists of a hydrophilic nucleotide binding domain and a hydrophobic transmembrane domain. The transport mechanism is a multistep process composed of an ATP-independent peptide association step which induces a structural reorganization of the transport complex that may trigger the ATP-driven transport of the peptide into the endoplasmic reticulum lumen. By using combinatorial peptide libraries, the substrate selectivity and the recognition principle of TAP have been elucidated. TAP maximizes the degree of substrate diversity in combination with high substrate affinity. This ABC transporter is also unique as it is closely associated with chaperone-like proteins involved in bonding of the substrate onto MHC molecules. Most interestingly, virus-infected and malignant cells have developed strategies to escape immune surveillance by affecting TAP expression or function.     

Effect of decreasing the affinity of the class II-associated invariant chain peptide on the MHC class II peptide repertoire in the presence or absence of H-2M

The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-A(b), this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates.     

Molecular cloning of cDNA that encode MHC class I molecules from a New World primate (Saguinus oedipus). Natural selection acts at positions that may affect peptide presentation to T cells

To investigate the evolutionary pressures that drive the generation of polymorphism in primate MHC class I molecules, three cDNA that encode MHC class I alleles from a New World monkey, the cotton-top tamarin (Saguinus oedipus), were cloned and sequenced. These tamarin MHC class I alleles contained amino acid substitutions not found in any of the previously sequenced human MHC class I alleles. Moreover, the majority of these unique amino acid substitutions was located in the Ag recognition site at positions that have been shown to be critical in the presentation of viral peptides to T cells in mice and humans. These data suggest that selective pressures on MHC class I molecules preferentially act on the Ag recognition site and that the peptide binding or presenting functions of these molecules may drive the generation of MHC class I polymorphism. The novel Ag recognition sites of the tamarin MHC class I molecules, in addition to their restricted polymorphism, might account for the unusual susceptibility of the cotton-top tamarin to human pathogens.     

Quantifying recruitment of cytosolic peptides for HLA class I presentation: impact of TAP transport

MHC class I ligands are recruited from the cytosolic peptide pool, whose size is likely to depend on the balance between peptide generation by the proteasome and peptide degradation by downstream peptidases. We asked what fraction of this pool is available for presentation, and how the size of this fraction is modulated by peptide affinity for the TAP transporters. A model epitope restricted by HLA-A2 and a series of epitope precursors with N-terminal extensions by single residues modifying TAP affinity were expressed in a system that allowed us to monitor and modulate cytosolic peptide copy numbers. We show that presentation varies strongly according to TAP affinities of the epitope precursors. The fraction of cytosolic peptides recruited for MHC presentation does not exceed 1% and is more than two logs lower for peptides with very low TAP affinities. Therefore, TAP affinity has a substantial impact on MHC class I Ag presentation.     

Trafficking of exogenous peptides into proteasome-dependent major histocompatibility complex class I pathway following enterotoxin B subunit-mediated delivery

The B-subunit component of Escherichia coli heat-labile enterotoxin (EtxB), which binds to cell surface GM1 ganglioside receptors, was recently shown to be a highly effective vehicle for delivery of conjugated peptides into the major histocompatibility complex (MHC) class I pathway. In this study we have investigated the pathway of epitope delivery. The peptides used contained the epitope either located at the C terminus or with a C-terminal extension. Pretreatment of cells with cholesterol-disrupting agents blocked transport of EtxB conjugates to the Golgi/endoplasmic reticulum, but did not affect EtxB-mediated MHC class I presentation. Under these conditions, EtxB conjugates entered EEA1-positive early endosomes where peptides were cleaved and translocated into the cytosol. Endosome acidification was required for epitope presentation. Purified 20 S immunoproteasomes were able to generate the epitope from peptides in vitro, but 26 S proteasomes were not. Only presentation from the C-terminal extended peptide was proteasome-dependent in cells, and this was found to be significantly slower than presentation from peptides with the epitope at the C terminus. These results implicate the proteasome in the generation of the correct C terminus of the epitope and are consistent with proteasome-independent N-terminal trimming. Epitope presentation was blocked in a TAP-deficient cell line, providing further evidence that conjugated peptides enter the cytosol as well as demonstrating a requirement for the peptide transporter. Our findings demonstrate the utility of EtxB-mediated peptide delivery for rapid and efficient loading of MHC class I epitopes in several different cell types. Conjugated peptides are released from early endosomes into the cytosol where they gain access to proteasomes and TAP in the "classical" pathway of class I presentation.     

The role of the ubiquitin-proteasome pathway in MHC class I antigen processing: implications for vaccine design

Proteasomes are multisubunit enzyme complexes that reside in the cytoplasm and nucleus of eukaryotic cells. By selective protein degradation, proteasomes regulate many cellular processes including MHC class I antigen processing. Three constitutively expressed catalytic subunits are responsible for proteasome mediated proteolysis. These subunits are exchanged for three homologous subunits, the immunosubunits, in IFNgamma-exposed cells and in cells with specialized antigen presenting function. Both constitutive and immunoproteasomes degrade endogenous proteins into small peptide fragments that can bind to MHC class I molecules for presentation on the cell surface to cytotoxic T lymphocytes. However, immunoproteasomes seem to fulfill this function more efficiently. IFNgamma further induces the expression of a proteasome activator, PA28, which can also enhance antigenic peptide production by proteasomes. In this review, we will introduce the ubiquitin-proteasome system and summarize recent findings regarding the role of the IFNgamma-inducible proteasome subunits and proteasome regulators in antigen processing. We review the different ways by which tumors and viruses have been found to target the proteasome system to avoid MHC class I presentation of their antigens, and discuss recent progressions in the development of computer assisted approaches to predict CTL epitopes within larger protein sequences, based on proteasome cleavage specificity. The availability of such programs as well as a general insight into the proteasome mediated steps in MHC class I antigen processing provides us with a rational basis for the design of new antiviral and anticancer T cell vaccines.     

Host immune system strikes back

Autophagy-mediated major histocompatibility complex (MHC) class I presentation can follow either the conventional MHC class I pathway or a recently described vacuolar pathway. In the vacuolar pathway, protein degradation is effected by lysosomal proteases, peptide exchange takes place with recirculating MHC complexes and the newly formed peptide-MHC complexes reach the cell surface by the endocytic pathway. This pathway is independent of the proteasome and the transporter associated with antigen processing (TAP) complex, but generates the same, or a similar, epitope as that from the conventional MHC class I pathway. Here, we discuss different mechanisms by which autophagy mediates MHC class I-restricted antigen presentation, which is crucial to its role in the control of intracellular pathogens.     

Dynamics of Major Histocompatibility Complex Class I Association with the Human Peptide-loading Complex

Although the human peptide-loading complex (PLC) is required for optimal major histocompatibility complex class I (MHC I) antigen presentation, its composition is still incompletely understood. The ratio of the transporter associated with antigen processing (TAP) and MHC I to tapasin, which is responsible for MHC I recruitment and peptide binding optimization, is particularly critical for modeling of the PLC. Here, we characterized the stoichiometry of the human PLC using both biophysical and biochemical approaches. By means of single-molecule pulldown (SiMPull), we determined a TAP/tapasin ratio of 1:2, consistent with previous studies of insect-cell microsomes, rat-human chimeric cells, and HeLa cells expressing truncated TAP subunits. We also report that the tapasin/MHC I ratio varies, with the PLC population comprising both 2:1 and 2:2 complexes, based on mutational and co-precipitation studies. The MHC I-saturated PLC may be particularly prevalent among peptide-selective alleles, such as HLA-C4. Additionally, MHC I association with the PLC increases when its peptide supply is reduced by inhibiting the proteasome or by blocking TAP-mediated peptide transport using viral inhibitors. Taken together, our results indicate that the composition of the human PLC varies under normal conditions and dynamically adapts to alterations in peptide supply that may arise during viral infection. These findings improve our understanding of the quality control of MHC I peptide loading and may aid the structural and functional modeling of the human PLC.