New Insights into the Mechanisms of Proteasome-Mediated Peptide Splicing Learned from Comparing Splicing Efficiency by Different Proteasome Subtypes.

By tying peptide fragments originally distant in parental proteins, the proteasome can generate spliced peptides that are recognized by CTL. This occurs by transpeptidation involving a peptide-acyl-enzyme intermediate and another peptide fragment present in the catalytic chamber. Four main subtypes of proteasomes exist: the standard proteasome (SP), the immunoproteasome, and intermediate proteasomes ß1-ß2-ß5i (single intermediate proteasome) and ß1i-ß2-ß5i (double intermediate proteasome). In this study, we use a tandem mass tag-quantification approach to study the production of six spliced human antigenic peptides by the four proteasome subtypes. Peptides fibroblast growth factor-5172-176/217-220, tyrosinase368-373/336-340, and gp10040-42/47-52 are better produced by the SP than the other proteasome subtypes. The peptides SP110296-301/286-289, gp100195-202/191or192, and gp10047-52/40-42 are better produced by the immunoproteasome and double intermediate proteasome. The current model of proteasome-catalyzed peptide splicing suggests that the production of a spliced peptide depends on the abundance of the peptide splicing partners. Surprisingly, we found that despite the fact that reciprocal peptides RTK_QLYPEW (gp10040-42/47-52) and QLYPEW_RTK (gp10047-52/40-42) are composed of identical splicing partners, their production varies differently according to the proteasome subtype. These differences were maintained after in vitro digestions involving identical amounts of the splicing fragments. Our results indicate that the amount of splicing partner is not the only factor driving peptide splicing and suggest that peptide splicing efficiency also relies on other factors, such as the affinity of the C-terminal splice reactant for the primed binding site of the catalytic subunit.

Tryptophanemia is controlled by a tryptophan-sensing mechanism ubiquitinating tryptophan 2,3-dioxygenase.

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding noncatalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers and unmasks a four-amino acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The nonmetabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia as required for both neurological and immune homeostasis.

Influenza A Virus Infection Induces Viral and Cellular Defective Ribosomal Products Encoded by Alternative Reading Frames.

The importance of antiviral CD8+ T cell recognition of alternative reading frame (ARF)-derived peptides is uncertain. In this study, we describe an epitope (NS1-ARF21-8) present in a predicted 14-residue peptide encoded by the +1 register of NS1 mRNA in the influenza A virus (IAV). NS1-ARF21-8 elicits a robust, highly functional CD8+ T cell response in IAV-infected BALB/c mice. NS1-ARF21-8 is presented from unspliced NS mRNA, likely from downstream initiation on a Met residue that comprises the P1 position of NS1-ARF21-8 Derived from a 14-residue peptide with no apparent biological function and negligible impacts on IAV infection, infectivity, and pathogenicity, NS1-ARF21-8 provides a clear demonstration of how immunosurveillance exploits natural errors in protein translation to provide antiviral immunity. We further show that IAV infection enhances a model cellular ARF translation, which potentially has important implications for virus-induced autoimmunity.

Cytosolic Processing Governs TAP-Independent Presentation of a Critical Melanoma Antigen.

Cancer immunotherapy has been flourishing in recent years with remarkable clinical success. But as more patients are treated, a shadow is emerging that has haunted other cancer therapies: tumors develop resistance. Resistance is often caused by defects in the MHC class I Ag presentation pathway critical for CD8 T cell-mediated tumor clearance. TAP and tapasin, both key players in the pathway, are frequently downregulated in human cancers, correlating with poor patient survival. Reduced dependence on these factors may promote vaccine efficiency by limiting immune evasion. In this study, we demonstrate that PMEL209-217, a promising phase 3 trial-tested antimelanoma vaccine candidate, is robustly presented by various TAP- and/or tapasin-deficient cell lines. This striking characteristic may underlie its potency as a vaccine. Surprisingly, cytosolic proteasomes generate the peptide even for TAP-independent presentation, whereas tripeptidyl peptidase 2 (TPP2) efficiently degrades the epitope. Consequently, inhibiting TPP2 substantially boosts PMEL209-217 presentation, suggesting a possible strategy to improve the therapeutic efficacy of the vaccine.

Peptide splicing by the proteasome.

The proteasome is the major protease responsible for the production of antigenic peptides recognized by CD8+ cytolytic T cells (CTL). These peptides, generally 8-10 amino acids long, are presented at the cell surface by major histocompatibility complex (MHC) class I molecules. Originally, these peptides were believed to be solely derived from linear fragments of proteins, but this concept was challenged several years ago by the isolation of anti-tumor CTL that recognized spliced peptides, i.e. peptides composed of fragments distant in the parental protein. The splicing process was shown to occur in the proteasome through a transpeptidation reaction involving an acyl-enzyme intermediate. Here, we review the steps that led to the discovery of spliced peptides as well as the recent advances that uncover the unexpected importance of spliced peptides in the composition of the MHC class I repertoire.

Long-Peptide Cross-Presentation by Human Dendritic Cells Occurs in Vacuoles by Peptide Exchange on Nascent MHC Class I Molecules.

Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results may have implications for the development of vaccines based on long peptides.

Deciphering preferential interactions within supramolecular protein complexes: the proteasome case.

In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin-proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.

A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming.

Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work.

The capture proteasome assay: A method to measure proteasome activity in vitro.

Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes ß5i or ß1i-ß5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.

An antigenic peptide produced by reverse splicing and double asparagine deamidation.

A variety of unconventional translational and posttranslational mechanisms contribute to the production of antigenic peptides, thereby increasing the diversity of the peptide repertoire presented by MHC class I molecules. Here, we describe a class I-restricted peptide that combines several posttranslational modifications. It is derived from tyrosinase and recognized by tumor-infiltrating lymphocytes isolated from a melanoma patient. This unusual antigenic peptide is made of two noncontiguous tyrosinase fragments that are spliced together in the reverse order. In addition, it contains two aspartate residues that replace the asparagines encoded in the tyrosinase sequence. We confirmed that this peptide is naturally presented at the surface of melanoma cells, and we showed that its processing sequentially requires translation of tyrosinase into the endoplasmic reticulum and its retrotranslocation into the cytosol, where deglycosylation of the two asparagines by peptide-N-glycanase turns them into aspartates by deamidation. This process is followed by cleavage and splicing of the appropriate fragments by the standard proteasome and additional transport of the resulting peptide into the endoplasmic reticulum through the transporter associated with antigen processing (TAP).

Inefficient exogenous loading of a tapasin-dependent peptide onto HLA-B*44:02 can be improved by acid treatment or fixation of target cells.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Database of T cell-defined human tumor antigens: the 2013 update.

The plethora of tumor antigens that have been--and are still being--defined required systematization to provide a comprehensive overview of those tumor antigens that are the most relevant targets for cancer immunotherapy approaches. Here, we provide a new update of a peptide database resource that we initiated many years ago. This database compiles all human antigenic peptides described in the literature that fulfill a set of strict criteria needed to ascertain their actual "tumor antigen" nature, as we aim at guiding scientists and clinicians searching for appropriate cancer vaccine candidates (www.cancerimmunity.org/peptide). In this review, we revisit those criteria in light of recent findings related to antigen processing. We also introduce the 29 new tumor antigens that were selected for this 2013 update. Two of the new peptides show unusual features, which will be briefly discussed. The database now comprises a total of 403 tumor antigenic peptides.

Proprotein convertases process Pmel17 during secretion.

Pmel17 is a melanocyte/melanoma-specific protein that traffics to melanosomes where it forms a fibrillar matrix on which melanin gets deposited. Before being cleaved into smaller fibrillogenic fragments the protein undergoes processing by proprotein convertases, a class of serine proteases that typically recognize the canonical motif RX(R/K)R↓. The current model of Pmel17 maturation states that this processing step occurs in melanosomes, but in light of recent reports this issue has become controversial. We therefore addressed this question by thoroughly assessing the processing kinetics of either wild-type Pmel17 or a secreted soluble Pmel17 derivative. Our results demonstrate clearly that processing of Pmel17 occurs during secretion and that it does not require entry of the protein into the endocytic system. Strikingly, processing proceeds even in the presence of the secretion inhibitor monensin, suggesting that Pmel17 is an exceptionally good substrate. In line with this, we find that newly synthesized surface Pmel17 is already quantitatively cleaved. Moreover, we demonstrate that Pmel17 function is independent of the sequence identity of its unconventional proprotein convertase-cleavage motif that lacks arginine in P4 position. The data alter the current view of Pmel17 maturation and suggest that the multistep processing of Pmel17 begins with an early cleavage during secretion that primes the protein for later functional processing.

Differences in the production of spliced antigenic peptides by the standard proteasome and the immunoproteasome.

Peptide splicing allows the production of antigenic peptides composed of two fragments initially non-contiguous in the parental protein. The proposed mechanism of splicing is a transpeptidation occurring within the proteasome. Three spliced peptides, derived from FGF-5, melanoma protein gp100 and nuclear protein SP110, have been described. Here, we compared the production of these spliced peptides by the standard proteasome and the immunoproteasome. Differential isotope labelling was used to quantify (by mass spectrometry) the fragments contained in digests obtained with precursor peptides and purified proteasomes. The results show that both the standard and the immunoproteasomes can produce spliced peptides although they differ in their efficiency of production of each peptide. The FGF-5 and gp100 peptides are more efficiently produced by the standard proteasome, whereas the SP110 peptide is more efficiently produced by the immunoproteasome. This seems to result from differences in the production of the two splicing partners, which depends on a balance between cleavages liberating or destroying those fragments. By showing that splicing depends on the efficiency of production of the splicing partners, these results also support the transpeptidation model of peptide splicing. Furthermore, given the presence of immunoproteasomes in dendritic cells and cells exposed to IFN-γ, the findings may be relevant for vaccine design.

Splicing of distant peptide fragments occurs in the proteasome by transpeptidation and produces the spliced antigenic peptide derived from fibroblast growth factor-5.

Peptide splicing is a newly described mode of production of antigenic peptides presented by MHC class I molecules, whereby two noncontiguous fragments of the parental protein are joined together after excision of the intervening segment. Three spliced peptides have been described. In two cases, splicing involved the excision of a short intervening segment of 4 or 6 aa and was shown to occur in the proteasome by transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by the N terminus of the other peptide fragment. For the third peptide, which is derived from fibroblast growth factor-5 (FGF-5), the splicing mechanism remains unknown. In this case, the intervening segment is 40 aa long. This much greater length made the transpeptidation model more difficult to envision. Therefore, we evaluated the role of the proteasome in the splicing of this peptide. We observed that the spliced FGF-5 peptide was produced in vitro after incubation of proteasomes with a 49-aa-long precursor peptide. We evaluated the catalytic mechanism by incubating proteasomes with various precursor peptides. The results confirmed the transpeptidation model of splicing. By transfecting a series of mutant FGF-5 constructs, we observed that reducing the length of the intervening segment increased the production of the spliced peptide, as predicted by the transpeptidation model. Finally, we observed that trans-splicing (i.e., splicing of fragments from two distinct proteins) can occur in the cell, but with a much lower efficacy than splicing of fragments from the same protein.

Insights into the processing of MHC class I ligands gained from the study of human tumor epitopes.

The molecular definition of tumor antigens recognized by cytolytic T lymphocytes (CTL) started in the late 1980s, at a time when the MHC class I antigen processing field was in its infancy. Born together, these two fields of science evolved together and provided each other with critical insights. Over the years, stimulated by the potential interest of tumor antigens for cancer immunotherapy, scientists have identified and characterized numerous antigens recognized by CTL on human tumors. These studies have provided a wealth of information relevant to the mode of production of antigenic peptides presented by MHC class I molecules. A number of tumor antigenic peptides were found to result from unusual mechanisms occurring at the level of transcription, translation or processing. Although many of these mechanisms occur in the cell at very low level, they are relevant to the immune system as they determine the killing of tumor cells by CTL, which are sensitive to low levels of peptide/MHC complexes. Moreover, these unusual mechanisms were found to occur not only in tumor cells but also in normal cells. Thereby, the study of tumor antigens has illuminated many aspects of MHC class I processing. We review here those insights into the MHC I antigen processing pathway that result from the characterization of human tumor antigens recognized by CTL.

Functional Differences between Proteasome Subtypes.

Four proteasome subtypes are commonly present in mammalian tissues: standard proteasomes, which contain the standard catalytic subunits ß1, ß2 and ß5; immunoproteasomes containing the immuno-subunits ß1i, ß2i and ß5i; and two intermediate proteasomes, containing a mix of standard and immuno-subunits. Recent studies revealed the expression of two tissue-specific proteasome subtypes in cortical thymic epithelial cells and in testes: thymoproteasomes and spermatoproteasomes. In this review, we describe the mechanisms that enable the ATP- and ubiquitin-dependent as well as the ATP- and ubiquitin-independent degradation of proteins by the proteasome. We focus on understanding the role of the different proteasome subtypes in maintaining protein homeostasis in normal physiological conditions through the ATP- and ubiquitin-dependent degradation of proteins. Additionally, we discuss the role of each proteasome subtype in the ATP- and ubiquitin-independent degradation of disordered proteins. We also discuss the role of the proteasome in the generation of peptides presented by MHC class I molecules and the implication of having different proteasome subtypes for the peptide repertoire presented at the cell surface. Finally, we discuss the role of the immunoproteasome in immune cells and its modulation as a potential therapy for autoimmune diseases.

Navigating Critical Challenges Associated with Immunopeptidomics-Based Detection of Proteasomal Spliced Peptide Candidates.

Within the tumor immunology community, the topic of proteasomal spliced peptides (PSP) has generated a great deal of controversy. In the earliest reports, careful biological validation led to the conclusion that proteasome-catalyzed peptide splicing was a rare event. To date, six PSPs have been validated biologically. However, the advent of algorithms to identify candidate PSPs in mass spectrometry data challenged this notion, with several studies concluding that the frequency of spliced peptides binding to MHC class I was quite high. Since this time, much debate has centered around the methodologies used in these studies. Several reanalyses of data from these studies have led to questions about the validity of the conclusions. Furthermore, the biological and technical validation that should be necessary for verifying PSP assignments was often lacking. It has been suggested therefore that the research community should unite around a common set of standards for validating candidate PSPs. In this review, we propose and highlight the necessary steps for validation of proteasomal splicing at both the mass spectrometry and biological levels. We hope that these guidelines will serve as a foundation for critical assessment of results from proteasomal splicing studies.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction.

Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.