Search

Throughout its evolution, the human immune system has developed a plethora of strategies to diversify the antigenic peptide sequences that can be targeted by the CD8+ T cell response against pathogens and aberrations of self. Here we provide a general overview of the mechanisms that lead to the diversity of antigens presented by MHC class I complexes and their recognition by CD8+ T cells, together with a more detailed analysis of recent progress in two important areas that are highly controversial: the prevalence and immunological relevance of unconventional antigen peptides; and cross-recognition of antigenic peptides by the T cell receptors of CD8+ T cells.

Anticancer immunotherapies demand optimal epitope targets, which could include proteasome-generated spliced peptides if tumor cells were to present them. Here, we show that spliced peptides are widely presented by MHC class I molecules of colon and breast carcinoma cell lines. The peptides derive from hot spots within antigens and enlarge the antigen coverage. Spliced peptides also represent a large number of antigens that would otherwise be neglected by patrolling T cells. These antigens tend to be long, hydrophobic, and basic. Thus, spliced peptides can be a key to identifying targets in an enlarged pool of antigens associated with cancer.

The human immune system relies on the capability of CD8+ T cells to patrol body cells, spot infected cells and eliminate them. This cytotoxic response is supposed to be limited to infected cells to avoid killing of healthy cells. To enable this, CD8+ T cells have T Cell Receptors (TCRs) which should discriminate between self and non-self through the recognition of antigenic peptides bound to Human Leukocyte Antigen class I (HLA-I) complexes—i.e., HLA-I immunopeptidomes—of patrolled cells. The majority of these antigenic peptides are produced by proteasomes through either peptide hydrolysis or peptide splicing. Proteasome-generated cis-spliced peptides derive from a given antigen, are immunogenic and frequently presented by HLA-I complexes. Theoretically, they also have a very large sequence variability, which might impinge upon our model of self/non-self discrimination and central and peripheral CD8+ T cell tolerance. Indeed, a large variety of cis-spliced epitopes might enlarge the pool of viral-human zwitter epitopes, i.e., peptides that may be generated with the exact same sequence from both self (human) and non-self (viral) antigens. Antigenic viral-human zwitter peptides may be recognized by CD8+ thymocytes and T cells, induce clonal deletion or other tolerance processes, thereby restraining CD8+ T cell response against viruses. To test this hypothesis, we computed in silico the theoretical frequency of zwitter non-spliced and cis-spliced epitope candidates derived from human proteome (self) and from the proteomes of a large pool of viruses (non-self). We considered their binding affinity to the representative HLA-A*02:01 complex, self-antigen expression in Medullary Thymic Epithelial cells (mTECs) and the relative frequency of non-spliced and cis-spliced peptides in HLA-I immunopeptidomes. Based on the present knowledge of proteasome-catalyzed peptide splicing and neglecting CD8+ TCR degeneracy, our study suggests that, despite their frequency, the portion of the cis-spliced peptides we investigated could only marginally impinge upon the variety of functional CD8+ cytotoxic T cells (CTLs) involved in anti-viral response.

Dissecting the different steps of the processing and presentation of tumor-associated antigens is a key aspect of immunotherapies enabling to tackle the immune response evasion attempts of cancer cells. The immunodominant glycoprotein gp100 209-217 epitope, which is liberated from the melanoma differentiation antigen gp100 PMEL17, is part of immunotherapy trials. By analyzing different human melanoma cell lines, we here demonstrate that a pool of N-terminal extended peptides sharing the common minimal epitope is generated by melanoma proteasome subtypes. In vitro and in cellulo experiments indicate that ER-resident aminopeptidase 1 (ERAP1)—but not ERAP2—defines the processing of this peptide pool thereby modulating the T-cell recognition of melanoma cells. By combining the outcomes of our studies and others, we can sketch the complex processing and endogenous presentation pathway of the gp100 209-217-containing epitope/peptides, which are produced by proteasomes and are translocated to the vesicular compartment through different pathways, where the precursor peptides that reach the endoplasmic reticulum are further processed by ERAP1. The latter step enhances the activation of epitope-specific T lymphocytes, which might be a target to improve the efficiency of anti-melanoma immunotherapy.

An efficient immunosurveillance of CD8+ T cells in the periphery depends on positive/negative selection of thymocytes and thus on the dynamics of antigen degradation and epitope production by thymoproteasome and immunoproteasome in the thymus. Although studies in mouse systems have shown how thymoproteasome activity differs from that of immunoproteasome and strongly impacts on the T cell repertoire, the proteolytic dynamics and the regulation of human thymoproteasome are unknown. By combining biochemical and computational modeling approaches, we show here that human 20S thymoproteasome and immunoproteasome differ not only in the proteolytic activity of the catalytic sites but also in the peptide transport. These differences impinge upon the quantity of peptide products rather than where the substrates are cleaved. The comparison of the two human 20S proteasome isoforms depicts different processing of antigens that are associated to tumors and autoimmune diseases.

Dysfunction of parvalbumin (PV)-expressing interneurons is thought to underlie the alterations of gamma-band oscillations observed in schizophrenia. Although the pathomechanisms of this disease remain unclear, oxidative stress induced by NMDA receptor (NMDAR) hypofunction and decreased glutathione (GSH) synthesizing capacity have been shown to lead to PV-loss and aberrant oscillatory activity. However, the individual contributions of NMDAR-inhibition and GSH-depletion to the developmental alterations observed in schizophrenia are largely unknown. We therefore investigated each condition in isolation using hippocampal slice cultures wherein interneuron maturation occurs entirely in vitro. Although both treatments caused oxidative stress, NMDAR-inhibition led to an immediate reduction in gamma oscillation frequency and a delayed loss of PV. In contrast, GSH-depletion immediately decreased PV expression and increased power, without affecting frequency. Hence, although disturbances of PV-expression and gamma oscillations coexist in schizophrenia, they can arise from separate pathological processes.

Summary: Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design. : Proteasomes both degrade proteins and ligate generated products, creating “spliced peptides” composed of distant protein parts. Platteel et al. now describe a multi-level strategy for identifying proteasome-generated spliced T cell epitopes. This work suggests ways of defining spliced epitopes within any antigen of interest and to determine their immunological relevance. Keywords: proteasome, peptide splicing, Listeria monocytogenes, antigen presentation, intracelllular bacteria, in silico analysis

Osteopontin is a pleiotropic cytokine that is involved in several diseases including multiple sclerosis. Secreted osteopontin is cleaved by few known proteases, modulating its pro-inflammatory activities. Here we show by in vitro experiments that secreted osteopontin can be processed by extracellular proteasomes, thereby producing fragments with novel chemotactic activity. Furthermore, osteopontin reduces the release of proteasomes in the extracellular space. The latter phenomenon seems to occur in vivo in multiple sclerosis, where it reflects the remission/relapse alternation. The extracellular proteasome-mediated inflammatory pathway may represent a general mechanism to control inflammation in inflammatory diseases.

MHC class I-restricted epitopes, which carry a tumor-specific mutation resulting in improved MHC binding affinity, are preferred T cell receptor targets in innovative adoptive T cell therapies. However, T cell therapy requires efficient generation of the selected epitope. How such mutations may affect proteasome-mediated antigen processing has so far not been studied. Therefore, we analyzed by in vitro experiments the effect on antigen processing and recognition of a T210M exchange, which previously had been introduced into the melanoma gp100209-217 tumor epitope to improve the HLA-A*02:01 binding and its immunogenicity. A quantitative analysis of the main steps of antigen processing shows that the T210M exchange affects proteasomal cleavage site usage within the mutgp100201-230 polypeptide, leading to the generation of an unique set of cleavage products. The T210M substitution qualitatively affects the proteasome-catalyzed generation of spliced and non-spliced peptides predicted to bind HLA-A or -B complexes. The T210M substitution also induces an enhanced production of the mutgp100209-217 epitope and its N-terminally extended peptides. The T210M exchange revealed no effect on ERAP1-mediated N-terminal trimming of the precursor peptides. However, mutant N-terminally extended peptides exhibited significantly increased HLA-A*02:01 binding affinity and elicited CD8(+) T cell stimulation in vitro similar to the wtgp100209-217 epitope. Thus, our experiments demonstrate that amino acid exchanges within an epitope can result in the generation of an altered peptide pool with new antigenic peptides and in a wider CD8(+) T cell response also towards N-terminally extended versions of the minimal epitope.

Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage. DOI: http://dx.doi.org/10.7554/eLife.07545.001, eLife digest Cells have to be able to reliably destroy or remove molecules from their interior that they no longer need. Structures called proteasomes play a central part in this complex process by cutting up and digesting proteins. Mammals have several different types of proteasomes, each made up of several protein ‘subunits’. For example, when a cell experiences inflammation some proteasomes change some of their subunits and form an immuno-proteasome. These immuno-proteasomes tend to break down proteins more quickly than ‘standard’ proteasomes, but it was not clear how they are able to do so. Liepe et al. have now combined experiments and mathematical modelling to construct a detailed model of proteasome activity. The model shows that protein transport into and out of the proteasome chamber are the steps that limit how quickly the proteasomes can break down proteins. Furthermore, these transport processes are also to a large extent responsible for the different rates at which standard and immuno-proteasomes process proteins. Liepe et al. were also able to confirm the existence of regulatory sites within the proteasome, and describe how these are arranged. Problems that alter the rate at which proteasomes break down proteins have been linked to tumors and neurological and autoimmune diseases. Liepe et al.'s model opens up the ability to study how the proteasome's activity is affected by drugs and therefore makes it easier to investigate ways of interfering with this activity for therapeutic purposes. DOI: http://dx.doi.org/10.7554/eLife.07545.002

Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function and duration of such transplanted livers are apparently comparable to those from young donors, suggesting that, despite some morphological and structural age-related changes, no major functional changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of proteasomes composition showed an age-related increased of β5i/α4 ratio, suggesting a shift toward proteasomes containing inducible subunits and a decreased content of PA28α subunit, mainly in the cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are emerging and they deserve further investigations owing to the different aging trajectories between men and women. Finally, our data support the safe use of livers from old donors for transplantation. Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function and duration of such transplanted livers are apparently comparable to those from young donors, suggesting that, despite some morphological and structural age-related changes, no major functional changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of proteasomes composition showed an age-related increased of β5i/α4 ratio, suggesting a shift toward proteasomes containing inducible subunits and a decreased content of PA28α subunit, mainly in the cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are emerging and they deserve further investigations owing to the different aging trajectories between men and women. Finally, our data support the safe use of livers from old donors for transplantation.

The ubiquitin-proteasome system is the major intracellular molecular machinery for protein degradation and maintenance of protein homeostasis in most human cells. As ubiquitin-proteasome system plays a critical role in the regulation of the immune system, it might also influence the development and progression of multiple sclerosis (MS). Bothex vivoanalyses and animal models suggest that activity and composition of ubiquitin-proteasome system are altered in MS. Proteasome isoforms endowed of immunosubunits may affect the functionality of different cell types such as CD8+and CD4+T cells and B cells as well as neurons during MS development. Furthermore, the study of proteasome-related biomarkers, such as proteasome antibodies and circulating proteasomes, may represent a field of interest in MS. Proteasome inhibitors are already used as treatment for cancer and the recent development of inhibitors selective for immunoproteasome subunits may soon represent novel therapeutic approaches to the different forms of MS. In this review we describe the current knowledge on the potential role of proteasomes in MS and discuss thepro et contraof possible therapies for MS targeting proteasome isoforms.

Immunoproteasome has been associated to neurodegenerative and autoimmune diseases as a marker and regulator of inflammatory mechanisms. Its expression in the brain may occur upon neuroinflammation in different cell types and affect a variety of homeostatic and inflammatory pathways including the oxidized protein clearance and the self-antigen presentation. In the present study we investigated the immunoproteasome expression in hippocampi and cortex of patients affected by different histopathological forms of pharmaco-resistent mesial temporal lobe epilepsy. We identified a pathology-specific pattern of immunoproteasome expression, which could provide insight into the complex neuroinflammatory pathogenic components of this disease.

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive (alpha4 and beta1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28alphabeta regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical data have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28alphabeta. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention.

In this study, we investigated proteasome composition and activity in the brain of Macaca fascicularis, in order to test whether this nonhuman primate species might be a suitable animal model for anti-aging therapies in the central nervous system, addressed to the ubiquitin-proteasome system. We detected the catalytic beta subunits of constitutive proteasome, as well as the PA28 regulator and a subunit of immunoproteasome (i.e., beta1i [LMP2]), in seven adult, six old, and one young nonhuman primate brains. Subunit expression and proteasome activity were not influenced by the age of the animal in any of the brain regions (temporal and frontal cortex and cerebellum) we studied. However, an area-specific susceptibility to aged-related oxidative stress emerged. On the whole, the results suggest that, compared to humans, Macaca fascicularis primates may have a different age-dependent regulation of the ubiquitin-proteasome system and, possibly, of neuroinflammation in the brain. An in silico model of the 20S immunoproteasome containing the Macaca fascicularis alpha and beta subunits, present in database or identified by our group (i.e., LMP2), has been developed. Additional information was obtained by de novo sequencing of the beta1 (delta) subunit of Macaca fascicularis. A comparison with humans suggests that in multiprotein complexes some functional subunits, such as alpha subunits, appear to be preferentially conserved during evolution.

The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-α-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, anin silicomodel of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in anin vitrocellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between α and β rings.

A common arginine to proline polymorphism is harboured at codon 72 of the human p53 gene. In this investigation, we found that fibroblasts and lymphocytes isolated from arginine allele homozygote centenarians and sexagenarians (Arg+) undergo an oxidative-stress-induced apoptosis at a higher extent than cells obtained from proline allele carriers (Pro+). At variance, the difference in apoptosis susceptibility between Arg+ and Pro+ is not significant when cells from 30-year-old people are studied. Further, we found that Arg+ and Pro+ cells from centenarians differ in the constitutive levels of p53 protein and p53/MDM2 complex, as well as in the levels of oxidative stress-induced p53/Bcl-xL complex and mitochondria-localised p53. Consistently, all these differences are less evident in cells from 30-year-old people. Finally, we investigated the in vivo functional relevance of the p53 codon 72 genotype in a group of old patients (66-99 years of age) affected by acute myocardial ischaemia, a clinical condition in which in vivo cell death occurs. We found that Arg+ patients show increased levels of Troponin I and CK-MB, two serum markers that correlate with the extent of the ischaemic damage in comparison to Pro+ patients. In conclusion, these data suggest that p53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.

As a consequence of inflammatory stimuli (such as TNFalpha and IFNgamma), some constitutive subunits of the proteasome, the principal mediator of nonlysosomal protein degradation, are replaced with other subunits, the large multifunctional proteases LMP2 and LMP7, thus originating the immunoproteasome. An age-related alteration of proteasome activity and susceptibility to TNFalpha-induced apoptosis, in which LMP2 and the nuclear factor (NF)-kappaB activation play an important role has been recently reported. In this paper, we investigated the possible influence of two LMP2 and LMP7 polymorphisms on susceptibility to TNFalpha-induced apoptosis. Our data show that an increase in susceptibility to TNFalpha-induced apoptosis is evident in long-lived people (aged88 years) in comparison to young individuals. Moreover, the modulation of LMP2 codon 60 polymorphism on TNFalpha-induced apoptosis is evident in long-lived subjects. Genotyping of 311 young people and 157 nonagenarians and centenarians revealed no changes in LMP2 codon 60 genotype frequency distribution. No correlation with TNFalpha-induced apoptosis and no difference in frequency between young people and nonagenarians/centenarians was observed when the LMP7 nucleotide 145 polymorphism was studied.

A wide difference in the susceptibility to undergo in vitro apoptosis exists among individuals, and this fact has potential implications in predicting the in vivo response to apoptotic agents, such as those employed in chemotherapy. In this report, we addressed the question whether the natural variability at p53 locus (the proline-arginine substitution at codon 72) affects the capacity of peripheral-blood mononuclear cells from healthy subjects to undergo in vitro apoptosis in response to the cytotoxic drug cytosine arabinoside. We found that cells from subjects carrying the arginine/arginine genotype undergo in vitro apoptosis at a higher extent in comparison to those from arginine/proline subjects. This finding suggests that naturally occurring genetic variability at p53 gene explains part of the inter-individual difference in the in vitro susceptibility to a chemotherapeutic drug, thus resulting as an eligible predictor marker of in vivo response to chemotherapy and its adverse effects.