Natural proteome diversity links aneuploidy tolerance to protein turnover.

Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy-an imbalance in chromosome copy numbers-is frequent1,2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory3-5. Here we generate a proteomic resource and merge it with genomic1 and transcriptomic6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins-especially subunits of protein complexes-show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes.

Structural Visualization of the Formation and Activation of the 50S Ribosomal Subunit during In Vitro Reconstitution.

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.

ER-aminopeptidase 1 determines the processing and presentation of an immunotherapy-relevant melanoma epitope.

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 gp100209-217 epitope, which is liberated from the melanoma differentiation antigen gp100PMEL17 , 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 gp100209-217 -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.

Proteolytic dynamics of human 20S thymoproteasome.

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 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.

CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion.

The T210M Substitution in the HLA-a*02:01 gp100 Epitope Strongly Affects Overall Proteasomal Cleavage Site Usage and Antigen Processing.

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.

The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits ß5i/LMP7 (LMP, low molecular weight protein) or ß1i/LMP2 and ß5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit ß2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. ß2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.

Proteasome isoforms exhibit only quantitative differences in cleavage and epitope generation.

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard- and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage-site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage-site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard- and immunoproteasomes are due to quantitative differences in the proteasome-generated antigenic peptides.

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases.

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.

Adult human liver contains intermediate-type proteasomes with different enzymatic properties.

BACKGROUND: The 20S proteasome is the proteolytic core of the major intracellular protein degradative system, the ubiquitin-proteasome system. Since little is known about proteasomes of human liver, we have investigated the proteasome spectrum in adult human liver. MATERIAL AND METHODS: 20S proteasomes were chromatographically purified from adult human liver and from HuH7 cells. They were divided into subpopulations and subtypes and characterized with regard to their proteolytic activities using short fluorogenic oligo- and long poly-peptide substrates. Their subunit composition was studied by immunoblotting. RESULTS: Proteasomes from adult human liver tissue can be separated into three subpopulations (I, II, III), each of which is composed of several subtypes, which total to a spectrum of 14 different subtypes. Two minor subtypes contain only the immuno-subunits ß1i and ß5i but not their standard counterparts; all others are intermediate subtypes containing ß1 and ß5 standard- and ß1i and ß5i immuno-subunits in various compositions. With regard to the proteolytic activities we observed that a decreasing content of subunit ß1i in the subtypes goes along with a decreasing ratio of chymotrypsin-like/caspase-like activity, whereas the degradation rate of a 30 mer polypeptide substrate increased with decreasing ß1i content. By comparison, 20S proteasomes from HuH7 cells do not contain immuno-subunits but are pure standard proteasomes, which can be separated into three subtypes. CONCLUSION: These findings suggest that adult human liver contains a spectrum of 14 different 20S proteasome subtypes with different enzymatic properties reflecting most probably an adaptive response of liver cell functions to challenging factors during lifetime.

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo.

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8(+) T cells. Three IFN-γ-inducible catalytic proteasome (immuno)subunits as well as the IFN-γ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFN-γ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits ß5i/LMP7 and ß2i/MECL-1. Analyses of MHC class I cell-surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8(+) T-cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides; however, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen-specific CD8(+) T-cell responses.

High-throughput proteomics of nanogram-scale samples with Zeno SWATH MS.

The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.

The 20S proteasome splicing activity discovered by SpliceMet.

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.

Adhesion dynamics in the neocortex determine the start of migration and the post-migratory orientation of neurons.

The neocortex is stereotypically organized into layers of excitatory neurons arranged in a precise parallel orientation. Here we show that dynamic adhesion both preceding and following radial migration is essential for this organization. Neuronal adhesion is regulated by the Mowat-Wilson syndrome-associated transcription factor Zeb2 (Sip1/Zfhx1b) through direct repression of independent adhesion pathways controlled by Neuropilin-1 (Nrp1) and Cadherin-6 (Cdh6). We reveal that to initiate radial migration, neurons must first suppress adhesion to the extracellular matrix. Zeb2 regulates the multipolar stage by transcriptional repression of Nrp1 and thereby downstream inhibition of integrin signaling. Upon completion of migration, neurons undergo an orientation process that is independent of migration. The parallel organization of neurons within the neocortex is controlled by Cdh6 through atypical regulation of integrin signaling via its RGD motif. Our data shed light on the mechanisms that regulate initiation of radial migration and the postmigratory orientation of neurons during neocortical development.

Differential interferon responses enhance viral epitope generation by myocardial immunoproteasomes in murine enterovirus myocarditis.

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection, with host-specific outcomes ranging from complete recovery in resistant mice to chronic disease in susceptible hosts. To identify susceptibility factors that modulate the course of viral myocarditis, we show that type-I interferon (IFN) responses are considerably impaired in acute CVB3-induced myocarditis in susceptible mice, which have been linked to immunoproteasome (IP) formation. Here we report that in concurrence with distinctive type-I IFN kinetics, myocardial IP formation peaked early after infection in resistant mice and was postponed with maximum IP expression concomitant to massive inflammation and predominant type-II IFN responses in susceptible mice. IP activity is linked to a strong enhancement of antigenic viral peptide presentation. To investigate the impact of myocardial IPs in CVB3-induced myocarditis, we identified two novel CVB3 T cell epitopes, virus capsid protein 2 [285-293] and polymerase 3D [2170-2177]. Analysis of myocardial IPs in CVB3-induced myocarditis revealed that myocardial IP expression resulted in efficient epitope generation. As opposed to the susceptible host, myocardial IP expression at early stages of disease corresponded to enhanced CVB3 epitope generation in the hearts of resistant mice. We propose that this process may precondition the infected heart for adaptive immune responses. In conclusion, type-I IFN-induced myocardial IP activity at early stages coincides with less severe disease manifestation in CVB3-induced myocarditis.

Generation of in silico predicted coxsackievirus B3-derived MHC class I epitopes by proteasomes.

Proteasomes are known to be the main suppliers of MHC class I (MHC-I) ligands. In an attempt to identify coxsackievirus B3 (CVB3)-MHC-I epitopes, a combined approach of in silico MHC-I/transporters associated with antigen processing (TAP)-binding and proteasomal cleavage prediction was applied. Accordingly, 13 potential epitopes originating from the structural and non-structural protein region of CVB3 were selected for further in vitro processing analysis by proteasomes. Mass spectrometry demonstrated the generation of seven of the 13 predicted MHC-I ligands or respective ligand precursors by proteasomes. Detailed processing analysis of three adjacent MHC-I ligands with partially overlapping sequences, i.e. VP2(273-281), VP2(284-292) and VP2(285-293), revealed the preferential generation predominantly of the VP2(285-293) epitope by immunoproteasomes due to altered cleavage site preferences. The VP2(285-293) peptide was identified to be a high affinity binder, rendering VP2(285-293) a likely candidate for CD8 T cell immunity in CVB3 infection. In conclusion, the concerted usage of different in silico prediction methods and in vitro epitope processing/presentation studies was supportive in the identification of CVB3 MHC-I epitopes.

Large database for the analysis and prediction of spliced and non-spliced peptide generation by proteasomes.

Proteasomes are the main producers of antigenic peptides presented to CD8+ T cells. They can cut proteins and release their fragments or recombine non-contiguous fragments thereby generating novel sequences, i.e. spliced peptides. Understanding which are the driving forces and the sequence preferences of both reactions can streamline target discovery in immunotherapies against cancer, infection and autoimmunity. Here, we present a large database of spliced and non-spliced peptides generated by proteasomes in vitro, which is available as simple CSV file and as a MySQL database. To generate the database, we performed in vitro digestions of 55 unique synthetic polypeptide substrates with different proteasome isoforms and experimental conditions. We measured the samples using three mass spectrometers, filtered and validated putative peptides, identified 22,333 peptide product sequences (15,028 spliced and 7,305 non-spliced product sequences). Our database and datasets have been deposited to the Mendeley (doi:10.17632/nr7cs764rc.1) and PRIDE (PXD016782) repositories. We anticipate that this unique database can be a valuable source for predictors of proteasome-catalyzed peptide hydrolysis and splicing, with various future translational applications.

Parkin contributes to synaptic vesicle autophagy in Bassoon-deficient mice.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5. Here, we show that increased autophagy in Bassoon knockout neurons depends on poly-ubiquitination and that the loss of Bassoon leads to elevated levels of ubiquitinated synaptic proteins per se. Our data show that Bassoon knockout neurons have a smaller SV pool size and a higher turnover rate as indicated by a younger pool of SV2. The E3 ligase Parkin is required for increased autophagy in Bassoon-deficient neurons as the knockdown of Parkin normalized autophagy and SV protein levels and rescued impaired SV recycling. These data indicate that Bassoon is a key regulator of SV proteostasis and that Parkin is a key E3 ligase in the autophagy-mediated clearance of SV proteins.

Ultra-High-Throughput Clinical Proteomics Reveals Classifiers of COVID-19 Infection.

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum and plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up to 180 samples per day, enables high precision quantification, and reduces batch effects for large-scale and longitudinal studies. We use our platform on samples collected from a cohort of early hospitalized cases of the SARS-CoV-2 pandemic and identify 27 potential biomarkers that are differentially expressed depending on the WHO severity grade of COVID-19. They include complement factors, the coagulation system, inflammation modulators, and pro-inflammatory factors upstream and downstream of interleukin 6. All protocols and software for implementing our approach are freely available. In total, this work supports the development of routine proteomic assays to aid clinical decision making and generate hypotheses about potential COVID-19 therapeutic targets.

Analysis of Proteasome-Generated Antigenic Peptides by Mass Spectrometry.

Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by purified 20S proteasomes in in vitro experiments. This approach also provides a convenient and sensitive way to monitor the different processing characteristics of proteasome isoforms. The combination of high performance liquid chromatography (HPLC) with ESI-MS allows for the analysis of complex samples with separation in their specific constituents by LC and their subsequent detection by MS.