ERAP2 supports TCR recognition of three immunotherapy targeted tumor epitopes.

The therapy of cancer by adoptive T cell transfer (ACT) requires T cell receptors (TCRs) with optimal affinity for HLA class I-bound peptides (pHLA-I). But not every patient responds to ACT. Therefore, it is critical to understand the individual factors influencing the recognition of HLA class I-bound peptides (pHLA-I) by TCRs. Focusing on three immunotherapy-targeted human HLA-A* 02:01-presented T cell epitopes we investigated the contribution of the ER-resident aminopeptidases ERAP1 and ERAP2 to TCR recognition of cancer cells. We found that ERAP2 on its own, when expressed in ERAP-deficient cells, elicited a strong CTL response towards the Tyrosinase368-376 epitope. In vitro generated TAP-dependent N-terminally extended epitope precursor peptides were differently customized by ERAP1 and ERAP2 and thus may serve as potential source for the Tyrosinase368-376 epitope. ERAP2 also influenced recognition of the gp100209-217 tumor epitope and enhanced T cell recognition of the MART-126/27-35 epitope in the absence of ERAP1 expression. Our results underline the relevance of ERAP2 for tumor epitope presentation and TCR recognition and may need to be considered when designing ACT in the future.

A Conserved Cysteine Residue in Coxsackievirus B3 Protein 3A with Implication for Elevated Virulence.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

A cytosolic disulfide bridge-supported dimerization is crucial for stability and cellular distribution of Coxsackievirus B3 protein 3A.

RNA viruses in the Picornaviridae family express a large 250 kDa viral polyprotein that is processed by virus-encoded proteinases into mature functional proteins with specific functions for virus replication. One of these proteins is the highly conserved enteroviral transmembrane protein 3A that assists in reorganizing cellular membranes associated with the Golgi apparatus. Here, we studied the molecular properties of the Coxsackievirus B3 (CVB3) protein 3A with regard to its dimerization and its functional stability. By applying mutational analysis and biochemical characterization, we demonstrate that protein 3A forms DTT-sensitive disulfide-linked dimers via a conserved cytosolic cysteine residue at position 38 (Cys38). Homodimerization of CVB3 protein 3A via Cys38 leads to profound stabilization of the protein, whereas a C38A mutation promotes a rapid proteasome-dependent elimination of its monomeric form. The lysosomotropic agent chloroquine (CQ) exerted only minor stabilizing effects on the 3A monomer but resulted in enrichment of the homodimer. Our experimental data demonstrate that disulfide linkages via a highly conserved Cys-residue in enteroviral protein 3A have an important role in the dimerization of this viral protein, thereby preserving its stability and functional integrity.

Coxsackievirus B3 Exploits the Ubiquitin-Proteasome System to Facilitate Viral Replication.

Infection by RNA viruses causes extensive cellular reorganization, including hijacking of membranes to create membranous structures termed replication organelles, which support viral RNA synthesis and virion assembly. In this study, we show that infection with coxsackievirus B3 entails a profound impairment of the protein homeostasis at virus-utilized membranes, reflected by an accumulation of ubiquitinylated proteins, including K48-linked polyubiquitin conjugates, known to direct proteins to proteasomal degradation. The enrichment of membrane-bound ubiquitin conjugates is attributed to the presence of the non-structural viral proteins 2B and 3A, which are known to perturb membrane integrity and can cause an extensive rearrangement of cellular membranes. The locally increased abundance of ubiquitinylated proteins occurs without an increase of oxidatively damaged proteins. During the exponential phase of replication, the oxidative damage of membrane proteins is even diminished, an effect we attribute to the recruitment of glutathione, which is known to be required for the formation of infectious virus particles. Furthermore, we show that the proteasome contributes to the processing of viral precursor proteins. Taken together, we demonstrate how an infection with coxsackievirus B3 affects the cellular protein and redox homeostasis locally at the site of viral replication and virus assembly.

High-affinity T-cell receptor specific for MyD88 L265P mutation for adoptive T-cell therapy of B-cell malignancies.

BACKGROUND: Adoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable side effects. Also, a substantial fraction of patients have refractory disease. Novel treatment approaches with more precise targeting may be an appealing alternative. Oncogenic somatic mutations represent ideal targets because of tumor specificity. Mutation-derived neoantigens can be recognized by T-cell receptors (TCRs) in the context of MHC-peptide presentation. METHODS: Here we have generated T-cell lines from healthy donors by autologous in vitro priming, targeting a missense mutation on the adaptor protein MyD88, changing leucine at position 265 to proline (MyD88 L265P), which is one of the most common driver mutations found in B-cell lymphomas. RESULTS: Generated T-cell lines were selectively reactive against the mutant HLA-B*07:02-restricted epitope but not against the corresponding wild-type peptide. Cloned TCRs from these cell lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with a mutation-specific TCR (TCR-T cells) recognized and killed B-cell lymphoma cell lines characterized by intrinsic MyD88 L265P mutation. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models. In addition, initial safety screening did not indicate any sign of off-target reactivity. CONCLUSION: Taken together, our data suggest that mutation-specific TCRs can be used to target the MyD88 L265P mutation, and hold promise for precision therapy in a significant subgroup of B-cell malignancies, possibly achieving the goal of absolute tumor specificity, a long sought-after dream of immunotherapy.

Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells.

Donor-specific antibodies (DSAs) have a deleterious effect on allografts and remain a major immunologic barrier in transplantation. Current therapies to eliminate DSAs are ineffective in highly HLA-sensitized patients. Proteasome inhibitors have been employed as a strategy to target bone marrow plasma cells (BMPCs), the source of long-term antibody production; however, their efficacy has been limited by poorly defined drug-resistance mechanisms. Here, we performed transcriptomic profiling of CD138+ BMPCs that survived in vivo desensitization therapy with the proteasome inhibitor carfilzomib to identify mechanisms of drug resistance. The results revealed a genomic signature that included increased expression of the immunoproteasome, a highly specialized proteasomal variant. Western blotting and functional studies demonstrated that catalytically active immunoproteasomes and the immunoproteasome activator PA28 were upregulated in carfilzomib-resistant BMPCs. Carfilzomib-resistant BMPCs displayed reduced sensitivity to the proteasome inhibitors carfilzomib, bortezomib, and ixazomib, but enhanced sensitivity to an immunoproteasome-specific inhibitor ONX-0914. Finally, in vitro carfilzomib treatment of BMPCs from HLA-sensitized patients increased levels of the immunoproteasome ß5i (PSMB8) catalytic subunit suggesting that carfilzomib therapy directly induces an adaptive immunoproteasome response. Taken together, our results indicate that carfilzomib induces structural changes in proteasomes and immunoproteasome formation.

ERAP1-Dependent Antigen Cross-Presentation Determines Efficacy of Adoptive T-cell Therapy in Mice.

Cytotoxic T lymphocytes can reject established tumors if their target peptide is efficiently presented by MHC class I molecules (pMHC-I) on the surface of cancerous cells. Therapeutic success upon adoptive T-cell transfer (ATT), however, requires additional cross-presentation of the same pMHC-I on noncancerous cells. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that customizes the N-terminus of proteasome-generated peptides so they can be loaded onto MHC-I molecules in the endoplasmic reticulum (ER). We show here that ERAP1 is critically involved in the process of tumor rejection and assumes a dual role by independently operating on both sides. Direct presentation of two MHC-I-restricted epitopes of a cancer-driving transplantation rejection antigen through ERAP1 moderately affected tumor rejection by adoptively transferred T-cell receptor gene-modified T cells in each case. ERAP1 expression by antigen cross-presenting cells of the ATT recipients was critical for expansion of therapeutic monospecific T cells and correlated with tumor rejection. Specifically, lack of ERAP1 expression in the ATT recipient's noncancerous cells enabled progression of pMHC-I-positive, IFNγ-responsive tumors, despite the presence of antigen-specific functional cytotoxic T lymphocytes. These data reveal a decisive role for ERAP1 in T-cell-mediated tumor rejection and will enhance the choice of MHC-I-restricted epitopes targeted by adoptive T-cell transfer.Significance: This study demonstrates a role of ERAP1 in the efficacy of adoptive T-cell transfer and has potential to improve personalized T-cell therapy for solid tumors. Cancer Res; 78(12); 3243-54. ©2018 AACR.

Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features.

Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.

Proteasome ß5i Subunit Deficiency Affects Opsonin Synthesis and Aggravates Pneumococcal Pneumonia.

Immunoproteasomes, harboring the active site subunits ß5i/LMP7, ß1i/LMP2, and ß2i/MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the systemic immune response in ß5i/LMP7-/- mice. Our data show that ß5i/LMP7 deficiency, which affected the subunit composition of proteasomes in murine macrophages and liver, was accompanied by reduced transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins. The diminished opsonin expression suggested an impaired humoral immune response against invading pneumococci resulting in an aggravated systemic dissemination of S. pneumoniae in ß5i/LMP7-/- mice. The impaired bacterial elimination in ß5i/LMP7-/- mice was accompanied by an aggravated course of pneumonia with early mortality as a consequence of critical illness during the late phase of disease. In summary our results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections.

Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's ß5 Catalytic Subunit.

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the ß5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly ß5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of ß5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the ß5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered.

Major Histocompatibility Complex (MHC) Class I Processing of the NY-ESO-1 Antigen Is Regulated by Rpn10 and Rpn13 Proteins and Immunoproteasomes following Non-lysine Ubiquitination.

The supply of MHC class I-restricted peptides is primarily ensured by the degradation of intracellular proteins via the ubiquitin-proteasome system. Depending on the target and the enzymes involved, ubiquitination is a process that may dramatically vary in terms of linkages, length, and attachment sites. Here we identified the unique lysine residue at position 124 of the NY-ESO-1 cancer/testis antigen as the acceptor site for the formation of canonical Lys-48-linkages. Interestingly, a lysine-less form of NY-ESO-1 was as efficient as its wild-type counterpart in supplying the HLA-A*0201-restricted NY-ESO-1157-165 antigenic peptide. In fact, we show that the regulation of NY-ESO-1 processing by the ubiquitin receptors Rpn10 and Rpn13 as a well as by the standard and immunoproteasome is governed by non-canonical ubiquitination on non-lysine sites. In summary, our data underscore the significance of atypical ubiquitination in the modulation of MHC class I antigen processing.

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.

Evidence for anti-apoptotic roles of proteasome activator 28γ via inhibiting caspase activity.

Proteasome activator PA28γ (REGγ, Ki antigen) has recently been demonstrated to display anti-apoptotic properties via enhancing Mdm2-p53 interaction, thereby facilitating ubiquitination and down-regulation of the tumor suppressor p53. In this study we demonstrate a correlation between cellular PA28γ levels and the sensitivity of cells towards apoptosis in different cellular contexts thereby confirming a role of proteasome activator PA28γ as an anti-apoptotic regulator. We investigated the anti-apoptotic role of PA28γ upon UV-C stimulation in B8 mouse fibroblasts stably overexpressing the PA28γ-encoding PSME3 gene and upon butyrate-induced apoptosis in human HT29 adenocarcinoma cells with silenced PSME3 gene. Interestingly, our results demonstrate that PA28γ has a strong influence on different apoptotic hallmarks, especially p53 phosphorylation and caspase activation. In detail, PA28γ and effector caspases mutually restrict each other. PA28γ is a caspase substrate, if PA28γ levels are low. In contrast, PA28γ overexpression reduces caspase activities, including the caspase-dependent processing of PA28γ. Furthermore, overexpression of PA28γ resulted in a nuclear accumulation of transcriptional active p53. In summary, our findings indicate that even in a p53-dominated cellular context, pro-apoptotic signaling might be overcome by PA28γ-mediated caspase inhibition.

α-Keto phenylamides as P1'-extended proteasome inhibitors.

The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off-target-related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA-approved proteasome inhibitors bortezomib (1) or carfilzomib (2). A systematic comparison of electrophilic headgroups recently identified the class of α-keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure-activity relationship (SAR)-based approach for rational ligand design using an electronic parameter (Hammett's σ) and in silico molecular modeling. This resulted in the tripeptidic α-keto phenylamide BSc4999 [(S)-3-(benzyloxycarbonyl-(S)-leucyl-(S)-leucylamino)-5-methyl-2-oxo-N-(2,4-dimethylphenyl)hexanamide, 6 a], a highly potent (IC50 = 38 nM), cell-permeable, and slowly reversible covalent inhibitor which targets both the primed and non-primed sites of the proteasome's substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α-keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies.

Two inhibitors of the ubiquitin proteasome system enhance long-term memory formation upon olfactory conditioning in the honeybee (Apis mellifera).

In honeybees (Apis mellifera), the proteasome inhibitor Z-Leu-Leu-Leu-CHO (MG132) enhances long-term memory (LTM) formation. Studies in vertebrates using different inhibitors of the proteasome demonstrate the opposite, namely an inhibition of memory formation. The reason for this contradiction remains unclear. MG132 is an inhibitor of the proteasome, but also blocks other proteases. Accordingly, one possible explanation might be that other proteases affected by MG132 are responsible for the enhancement of LTM formation. We test this hypothesis by comparing the effect of MG132 and the more specific proteasome inhibitor clasto-lactacystin beta-lactone (ß-lactone). We show that these two inhibitors block the activity of the proteasome in honeybee brains to a similar extent, do not affect the animals' survival but do enhance LTM retention upon olfactory conditioning. Thus, the enhancement of LTM formation is not due to MG132-specific side effects, but to inhibition of a protease targeted by MG132 and ß-lactone, i.e. the proteasome.

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.

Trypanosoma cruzi infection down-modulates the immunoproteasome biosynthesis and the MHC class I cell surface expression in HeLa cells.

Generally, Trypanosoma cruzi infection in human is persistent and tends to chronicity, suggesting that the parasite evade the immune surveillance by down regulating the intracellular antigen processing routes. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome. However, upon IFN-γ stimulation, the catalytic constitutive subunits of the proteasome are replaced by the subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 to form the immunoproteasome. In this scenario, we analyzed whether the expression and activity of the constitutive and the immunoproteasome as well as the expression of other components of the MHC class I pathway are altered during the infection of HeLa cells with T. cruzi. By RT-PCR and two-dimensional gel electrophoresis analysis, we showed that the expression and composition of the constitutive proteasome is not affected by the parasite. In contrast, the biosynthesis of the ß1i, ß2i, ß5i immunosubunits, PA28ß, TAP1 and the MHC class I molecule as well as the proteasomal proteolytic activities were down-regulated in infected-IFN-γ-treated cell cultures. Taken together, our results provide evidence that the protozoan T. cruzi specifically modulates its infection through an unknown posttranscriptional mechanism that inhibits the expression of the MHC class I pathway components.

Cross-presentation of synthetic long peptides by human dendritic cells: a process dependent on ERAD component p97/VCP but Not sec61 and/or Derlin-1.

Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8(+) CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as "cross-presentation". Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16-40 containing the HLA-A2-restricted epitope26-35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16-40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16-40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26-35 peptide from the SLP16-40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.

Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities.

Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits ß1i and ß5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only ß5i beside standard-subunits. In favour of a relative increase of the major subpopulation Ι, subpopulation ΙΙ and ΙΙΙ were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.

T lymphocytes export proteasomes by way of microparticles: a possible mechanism for generation of extracellular proteasomes.

The 20S proteasome is almost exclusively localized within cells. High levels of extracellular proteasomes are also found circulating in the blood plasma of patients suffering from a variety of inflammatory, autoimmune and neoplastic diseases. However, the origin of these proteasomes remained enigmatic. Since the proteome of microparticles, small membrane enclosed vesicles released from cells, was shown to contain proteasomal subunits, we studied whether intact proteasomes are actively released into the extracellular space. Using human primary T lymphocytes stimulated with CaCl2 and the calcium ionophore A23187 to induce membrane blebbing we demonstrate that microparticles contain proteolytically active 20S proteasomes as well as the proteasome activator PA28 and subunits of the 19S proteasome regulator. Furthermore, our experiments reveal that incubation of in vitro generated T lymphocyte-microparticles with sphingomyelinase results in the hydrolysis of the microparticle membranes and subsequent release of proteasomes from the vesicles. Thus, we here show for the first time that functional proteasomes can be exported from activated immune cells by way of microparticles, the dissolution of which may finally lead to the generation of extracellular proteasomes.