Prediction of intracellular targets of a small compound by analyzing peptides presented on MHC class I

In chemical biology, the elucidation of chemical target is crucial for successful drug development. Because MHC class I molecules present peptides from intracellular damaged proteins, it might be possible to identify targets of a chemical by analyzing peptide sequences on MHC class I. Therefore, we treated cells with the autophagy-inducing chemical TMD-457 and identified the peptides presented on MHC class I. Many of the peptides were derived from molecules involved in ER trafficking and ER stress, which were confirmed by morphological and biochemical analyses. Therefore, our results demonstrate that analyzing MHC class I peptides is useful for the detection of chemical targets.     

Assembly and transport of MHC class II molecules.

At the surface of antigen-presenting cells MHC class I and class II molecules present peptides to respectively CD8+ and CD4+ T cells. MHC class I molecules acquire peptides right after synthesis in the endoplasmic reticulum. MHC class II molecules do not acquire peptides in the endoplasmic reticulum but instead associate with a third chain, the invariant chain which impedes peptide binding. Subsequently the invariant chain takes MHC class II molecules to the endosomal/lysosomal compartment thanks to a targeting signal retained in its cytoplasmic tail. It then dissociates from the MHC class II dimer to allow it to bind peptides.     

Regulation of MHC class I and II gene transcription: differences and similarities

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8⁺ (cytotoxic) and CD4⁺ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes. Received: 12 January 1998     

Processing of MHC class I presented antigens

The immune defences of our organism against pathogens and malignant transformation rely to a large extent on surveillance by cytotoxic T lymphocytes. This surveillance in turn depends on the antigen processing system, which provides peptide samples of the cellular protein composition to MHC (major histocompatibility complex) class I molecules displayed on the cell surface. To continuously and almost in real time provide a representative sample of the array of proteins synthesized by the cell, this system exploits some fundamental pathways of the cellular metabolism, with the help of several dedicated players acting exclusively in antigen processing. Thus, a key element in the turnover of cellular proteins, protein degradation by cytosolic proteasome complexes, is exploited as source of peptides, by recruiting a minor fraction of the produced peptides as ligands for MHC class I molecules. These peptides can be further processed and adapted to the precise binding requirements of allelic MHC class I molecules by enzymes in the cytosol and endoplasmic reticulum. The latter compartment is equipped with several dedicated players helping peptide assembly with class I molecules. These include the TAP (transporter associated with antigen processing) membrane transporter pumping peptides into the ER, and tapasin, a chaperone with a structure similar to MHC molecules that tethers class I molecules awaiting peptide loading to the TAP transporter, and mediates optimization of MHC class I ligand by a still somewhat mysterious mechanism. Additional "house-keeping" chaperones that are known to act in concert in ER quality control, assist and control correct folding, oxidation and assembly of MHC class I molecules. While this processing system handles exclusively endogenous cellular proteins in most cells, dendritic cells employ one or several special pathways to shuttle exogenous, internalized proteins into the system, in a process referred to as cross-presentation. Deciphering the cell biological mechanism creating the link between the endosomal and secretory pathways that enables cross-presentation is one of the challenges faced by contemporary research in the field of MHC class I antigen processing.     

MHC class Ib molecules bridge innate and acquired immunity

Our understanding of the classical MHC class I molecules (MHC class Ia molecules) has long focused on their extreme polymorphism. These molecules present peptides to T cells and are central to discrimination between self and non-self. By contrast, the functions of the non-polymorphic MHC class I molecules (MHC class Ib molecules) have been elusive, but emerging evidence reveals that, in addition to antigen presentation, MHC class Ib molecules are involved in immunoregulation. As we discuss here, the subset of MHC class Ib molecules that presents peptides to T cells bridges innate and acquired immunity, and this provides insights into the origins of acquired immunity.     

MHC Class I/peptide interactions: Binding specificity and kinetics

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (k_d) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured k_d values for antigenic peptide/class I interactions at 25°C are in the range of ca 10^?4–10^?6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.     

Anti-peptide antibody blocks peptide binding to MHC class I molecules in the endoplasmic reticulum

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.     

Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members

The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)–encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family–associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.     

Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells.

Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.     

Leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for MHC class I antigen presentation

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases.     

Mechanisms of Function of Tapasin,a Critical Major Histocompatibility Complex Class I Assembly Factor

For their efficient assembly in the endoplasmic reticulum (ER), major histocompatibility complex (MHC) class I molecules require the specific assembly factors transporter associated with antigen processing (TAP) and tapasin, as well as generic ER folding factors, including the oxidoreductases ERp57 and protein disulfide isomerase (PDI), and the chaperone calreticulin. TAP transports peptides from the cytosol into the ER. Tapasin promotes the assembly of MHC class I molecules with peptides. The formation of disulfide‐linked conjugates of tapasin with ERp57 is suggested to be crucial for tapasin function. Important functional roles are also suggested for the tapasin transmembrane and cytoplasmic domains, sites of tapasin interaction with TAP. We show that interactions of tapasin with both TAP and ERp57 are correlated with strong MHC class I recruitment and assembly enhancement. The presence of the transmembrane/cytosolic regions of tapasin is critical for efficient tapasin–MHC class I binding in interferon‐γ‐treated cells, and contributes to an ERp57‐independent mode of MHC class I assembly enhancement. A second ERp57‐dependent mode of tapasin function correlates with enhanced MHC class I binding to tapasin and calreticulin. We also show that PDI binds to TAP in a tapasin‐independent manner, but forms disulfide‐linked conjugates with soluble tapasin. Thus, full‐length tapasin is important for enhancing recruitment of MHC class I molecules and increasing specificity of tapasin–ERp57 conjugation. Furthermore, tapasin or the TAP/tapasin complex has an intrinsic ability to recruit MHC class I molecules and promote assembly, but also uses generic folding factors to enhance MHC class I recruitment and assembly.     

Control of MHC class I traffic from the endoplasmic reticulum by cellular chaperones and viral anti-chaperones

MHC class I molecules assemble with peptides in the endoplasmic reticulum (ER). To ensure that only peptide-loaded MHC molecules leave the ER, empty molecules are retained by ER-resident chaperones, most notably the MHC-specific tapasin. ER exit of class I MHC is also controlled by viruses, but for the opposite purpose of preventing peptide presentation to T cells. Interestingly, some viral proteins are able to retain MHC class I molecules in the ER despite being transported. By contrast, other viral proteins exit the ER only upon binding to class I MHC, thereby rerouting newly synthesized class I molecules to intracellular sites of proteolysis. Thus, immune escape can be achieved by reversing, inhibiting or redirecting the chaperone-assisted MHC class I folding, assembly and intracellular transport.     

Traffic of proteins and peptides across membranes for immunosurveillance by CD8(+) T lymphocytes: a topological challenge

Cytotoxic CD8(+) T lymphocytes kill infected cells that display major histocompatibility complex (MHC) class I molecules presenting peptides processed from pathogen proteins. In general, the peptides are proteolytically processed from newly made endogenous antigens in the cytosol and require translocation to the endoplasmic reticulum (ER) for MHC class I loading. This last task is performed by the transporters associated with antigen processing (TAP). Sampling of suspicious pathogen-derived proteins reaches beyond the cytosol, and MHC class I loading can occur in other secretory or endosomal compartments besides the ER. Peptides processed from exogenous antigens can also be presented by MHC class I molecules to CD8(+) T lymphocytes, in this case requiring delivery from the extracellular medium to the processing and MHC class I loading compartments. The endogenous or exogenous antigen can be processed before or after its transport to the site of MHC class I loading. Therefore, mechanisms that allow the full-length protein or processed peptides to cross several subcellular membranes are essential. This review deals with the different intracellular pathways that allow the traffic of antigens to compartments proficient in processing and loading of MHC class I molecules for presentation to CD8(+) T lymphocytes and highlights the need to molecularly identify the transporters involved.     

Cross-presentation in viral immunity and self-tolerance

T lymphocytes recognize peptide antigens presented by class I and class II molecules encoded by the major histocompatibility complex (MHC). Classical antigen-presentation studies showed that MHC class I molecules present peptides derived from proteins synthesized within the cell, whereas MHC class II molecules present exogenous proteins captured from the environment. Emerging evidence indicates, however, that dendritic cells have a specialized capacity to process exogenous antigens into the MHC class I pathway. This function, known as cross-presentation, provides the immune system with an important mechanism for generating immunity to viruses and tolerance to self.     

Testing the role of gp96 as peptide chaperone in antigen processing

gp96 is a 96-kDa glycoprotein of the endoplasmic reticulum that is believed to be involved in antigen processing as an intermediate carrier of peptides for presentation by major histocompatibility complex (MHC) class I molecules. This function implies that gp96 carries a large array of different peptides that represent the antigenicity of the cell and can serve all MHC class I molecules. So far, the evidence regarding these peptides is largely indirect and based on experiments where mice immunized with gp96 from tumor or virus-infected cells developed T cellular immune responses with the corresponding specificities. We analyzed by mass spectrometry peptides isolated from gp96 and found a number of different peptides derived from the proteins of different cellular compartments but mostly cytoplasm and nucleus. The sequences of these peptides provide information on the specificity of antigen processing and reveal structural requirements for binding to gp96 that only partially correspond to those of peptides presented by MHC class I molecules. The yield of peptides extracted from gp96 was far substoichiometric with an estimated occupancy of this chaperone of between 0.1% and 0.4%. These results strongly argue against a regular role for gp96 as a peptide chaperone in antigen processing.     

Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.     

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.     

Chaperones and folding of MHC class I molecules in the endoplasmic reticulum

In this review we discuss the influence of chaperones on the general phenomena of folding as well as on the specific folding of an individual protein, MHC class I. MHC class I maturation is a highly sophisticated process in which the folding machinery of the endoplasmic reticulum (ER) is heavily involved. Understanding the MHC class I maturation per se is important since peptides loaded onto MHC class I molecules are the base for antigen presentation generating immune responses against virus, intracellular bacteria as well as tumours. This review discusses the early stages of MHC class I maturation regarding BiP and calnexin association, and differences in MHC class I heavy chain (HC) interaction with calnexin and calreticulin are highlighted. Late stage MHC class I maturation with focus on the dedicated chaperone tapasin is also discussed.