Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules.

In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.

Epstein-Barr virus infection is associated with the nuclear factor-kappa B p65 signaling pathway in renal cell carcinoma.

BACKGROUND: There have been few studies regarding viral involvement in the pathogenesis of renal cell carcinoma (RCC). The aim of this study was to examine the possible association of Epstein-Barr virus (EBV) infection with clinicopathological features and cellular biomarkers including p53, p16INK4a, Ki-67 and nuclear factor-kappa B (NF-κB) in RCC tumors. METHODS: In this prospective study, 122 histologically confirmed Formalin-fixed Paraffin-embedded RCC tissue specimens along with 96 specimens of their corresponding peritumoral tissues and 23 samples of blunt renal injuries were subjected to nested polymerase chain reaction (nPCR) in order to amplify EBV DNA sequences. The expression of p53, p16INK4a, Ki-67 and NF-κB was investigated by immunohistochemistry (IHC) assay. Statistical analysis was employed to demonstrate the possible associations. RESULTS: Infection with EBV was found to be significantly associated with RCC. Our results indicate that p65 NF-κB signaling pathway is probably involved in EBV-mediated RCC pathogenesis. Moreover, we found p53, Ki-67 and cytoplasmic NF-κB expression to be associated with tumor nuclear grade in RCC patients. The expression of p53 and Ki-67 was associated with primary tumor category as well. In addition, p53 overexpression was significantly more frequent among nonconventional RCC tumors than the conventional histologic type. CONCLUSIONS: Infection with EBV is likely to play an important role in the development of RCC through the constitutive and permanent activation of NF-κB p65 signaling pathway. However, more experiments and supporting data are required to reach a decisive conclusion.

Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae.

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αß heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence. [Formula: see text].

Vinylboronic Acids as Efficient Bioorthogonal Reactants for Tetrazine Labeling in Living Cells.

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present in the cell. The tetrazine ligation is very suitable as a bioorthogonal reaction because of its selectivity and high reaction rates with several alkenes and alkynes. Recently, we described vinylboronic acids (VBAs) as novel hydrophilic bioorthogonal moieties that react efficiently with dipyridyl- s-tetrazines and used them for protein modification in cell lysate. It is not clear, however, whether VBAs are suitable for labeling experiments in living cells because of the possible coordination with, for example, vicinal carbohydrate diols. Here, we evaluated VBAs as bioorthogonal reactants for labeling of proteins in living cells using an irreversible inhibitor of the proteasome and compared the reactivity to that of an inhibitor containing norbornene, a widely used reactant for the tetrazine ligation. No large differences were observed between the VBA and norbornene probes in a two-step labeling approach with a cell-penetrable fluorescent tetrazine, indicating that the VBA gives little or no side reactions with diols and can be used efficiently for protein labeling in living cells.

Proteasome-mediated degradation of tyrosine hydroxylase triggered by its phosphorylation: a new question as to the intracellular location at which the degradation occurs.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability of TH determined by the degradation remains unknown; although the TH molecule phosphorylated at its Ser19 was observed in the nucleus, and the phosphorylation suspected to trigger its proteasome-mediated degradation. Computer-assisted analysis using the cNLS Mapper program predicted that two sequences of nuclear localization signals (NLS) exist in the N-terminus of TH molecule containing the phosphorylation sites Ser19, Ser31, and Ser40 (Pro9-Arg38 and Lys12-Ile42): the NLS scores indicated that TH could become localized in both nucleus and cytoplasm. Moreover, inhibition of the importin α/ß-mediated nuclear import pathway increased the level of TH phosphorylated at its Ser19 in PC12D cells. The results suggest that TH might be imported to nucleus from cytoplasm to be degraded. Recent studies revealed that proteasomes predominantly exist in the nucleus rather than in the cytoplasm to degrade the nuclear proteins related to cell-cycle progression, gene expression, DNA damage, and DNA repair. Therefore, these studies suggest that the relationship between the phosphorylation and the nuclear localization of the TH molecule should be a matter of focus to understand the mechanism of proteasome-mediated degradation of the enzyme as a first priority.

Ubiquitin-proteasome system and ER stress in the retina of diabetic rats.

PURPOSE: Diabetic retinopathy (DR) is the most frequently occurring complication of diabetes. Alterations in ubiquitin-proteasome system (UPS) have been associated with several degenerative disorders. Hence, in this study, we investigated the status and role of UPS and ER stress in the retina of diabetic rats. METHODS: Diabetes was induced in rats by streptozotocin. Retinal markers, ER stress markers, components of UPS, ERAD, and autophagy were analyzed after 2- and 4-months of diabetes. Apoptosis was analyzed by TUNEL Assay. RESULTS: There were increased acellular capillaries and pericyte loss in diabetic rat retina. Decreased protein expression of UPS components - ubiquitin activating enzyme (E1), deubiquitinating enzymes (UCHL1 and UCHL5), SIAH1 (E3 ligase) and free ubiquitin were observed in the diabetic rats. Increased ER stress markers (ATF6, XBP1, and CHOP), decreased expression of HRD1, declined autophagy (LC3B) and increased apoptosis were observed in diabetic rats. Interestingly, treatment of diabetic rats with a chemical chaperone (4-PBA) restored the levels of DUBs and ameliorated ER stress-induced retinal cell death in type 1 diabetic rats. CONCLUSION: The declined UPS components: E1 and HRD1 in the retina of diabetic rats could elicit ER stress, and the prolonged ER stress may trigger CHOP-mediated neuronal apoptosis.

Site-specific labeling of proteins for electron microscopy.

Electron microscopy is commonly employed to determine the subunit organization of large macromolecular assemblies. However, the field lacks a robust molecular labeling methodology for unambiguous identification of constituent subunits. We present a strategy that exploits the unique properties of an unnatural amino acid in order to enable site-specific attachment of a single, readily identifiable protein label at any solvent-exposed position on the macromolecular surface. Using this method, we show clear labeling of a subunit within the 26S proteasome lid subcomplex that has not been amenable to labeling by traditional approaches.

Development and function of cortical thymic epithelial cells.

The thymic cortex provides a microenvironment that supports the generation and T cell antigen receptor (TCR)-mediated selection of CD4(+)CD8(+)TCRαß(+) thymocytes. Cortical thymic epithelial cells (cTECs) are the essential component that forms the architecture of the thymic cortex and induces the generation as well as the selection of newly generated T cells. Here we summarize current knowledge on the development, function, and heterogeneity of cTECs, focusing on the expression and function of ß5t, a cTEC-specific subunit of the thymoproteasome.

Top-down protein identification of proteasome proteins with nanoLC-FT-ICR-MS employing data-independent fragmentation methods.

A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (continuous accumulation of selected ions)-CAD. The N-termini for 9 of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass-measurement accuracy with the LC-FT-ICR system for the 20- to 30-kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100-kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact-protein fragmentation and is an effective addition to the growing inventory of dissociation methods that are compatible with online protein separation coupled to FT-ICR-MS.

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue.

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.

Human ASPL/TUG interacts with p97 and complements the proteasome mislocalization of a yeast ubx4 mutant, but not the ER-associated degradation defect.

BACKGROUND: In mammalian cells, ASPL is involved in insulin-stimulated redistribution of the glucose transporter GLUT4 and assembly of the Golgi apparatus. Its putative yeast orthologue, Ubx4, is important for proteasome localization, endoplasmic reticulum-associated protein degradation (ERAD), and UV-induced degradation of RNA polymerase. RESULTS: Here, we show that ASPL is a cofactor of the hexameric ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast. In addition, ASPL interacts in vitro with NSF, another hexameric ATPase complex. ASPL localizes to the ER membrane. The central area in ASPL, containing both a SHP box and a UBX domain, is required for binding to the p97 N-domain. Knock-down of ASPL does not impair degradation of misfolded secretory proteins via the ERAD pathway. Deletion of UBX4 in yeast causes cycloheximide sensitivity, while ubx4 cdc48-3 double mutations cause proteasome mislocalization. ASPL alleviates these defects, but not the impaired ERAD. CONCLUSIONS: In conclusion, ASPL and Ubx4 are homologous proteins with only partially overlapping functions. Both interact with p97/Cdc48, but while Ubx4 is important for ERAD, ASPL appears not to share this function.

Polyubiquitinated proteins, proteasome, and glycogen characterize the particle-rich cytoplasmic structure (PaCS) of neoplastic and fetal cells.

A particle-rich cytoplasmic structure (PaCS) concentrating ubiquitin-proteasome system (UPS) components and barrel-like particles in clear, cytoskeleton- and organelle-free areas has recently been described in some neoplasms and in genetic or infectious diseases at risk of neoplasia. Ultrastructurally similar particulate cytoplasmic structures, interpreted as glycogen deposits, have previously been reported in clear-cell neoplasms and some fetal tissues. It remains to be investigated whether the two structures are the same, colocalize UPS components and polysaccharides, and have a role in highly proliferative cells such as fetal and neoplastic cells. We used immunogold electron microscopy and confocal immunofluorescence microscopy to examine human and mouse fetal tissues and human neoplasms. Fetal and neoplastic cells both showed colocalization of polyubiquitinated proteins, 19S and 20S proteasomes, and polysaccharides, both glycogen and chondroitin sulfate, inside cytoplasmic structures showing all distinctive features of PaCSs. Poorly demarcated and/or hybrid (ribosomes admixed) UPS- and glycogen-enriched areas, likely stages in PaCS development, were also seen in some fetal cells, with special reference to those, like primary alveolar pulmonary cells or pancreatic centroacinar cells, having a crucial role in organogenesis. UPS- and glycogen-rich PaCSs developed extensively in clear-cell neoplasms of the kidney, ovary, pancreas, and other organs, as well as, in infantile, development-related tumors replicating fetal patterns, such as choroid plexus papilloma. UPS-mediated, ATP-dependent proteolysis and its potential energy source, glycogen metabolism, may have a crucial, synergic role in embryo-/organogenesis and carcinogenesis.

Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity.

Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity.

Mapping the structural topology of the yeast 19S proteasomal regulatory particle using chemical cross-linking and probabilistic modeling.

Structural characterization of proteasome complexes is an essential step toward understanding the ubiquitin-proteasome system. Currently, high resolution structures are not available for the 26S proteasome holocomplex as well as its subcomplex, the 19S regulatory particle (RP). Here we have employed a novel integrated strategy combining chemical cross-linking with multistage tandem mass spectrometry to define the proximity of subunits within the yeast 19S RP to elucidate its topology. This has resulted in the identification of 174 cross-linked peptides of the yeast 19S RP, representing 43 unique lysine-lysine linkages within 24 nonredundant pair-wise subunit interactions. To map the spatial organization of the 19S RP, we have developed and utilized a rigorous probabilistic framework to derive maximum likelihood (ML) topologies based on cross-linked peptides determined from our analysis. Probabilistic modeling of the yeast 19S AAA-ATPase ring (i.e., Rpt1-6) has produced an ML topology that is in excellent agreement with known topologies of its orthologs. In addition, similar analysis was carried out on the 19S lid subcomplex, whose predicted ML topology corroborates recently reported electron microscopy studies. Together, we have demonstrated the effectiveness and potential of probabilistic modeling for unraveling topologies of protein complexes using cross-linking data. This report describes the first study of the 19S RP topology using a new integrated strategy combining chemical cross-linking, mass spectrometry, and probabilistic modeling. Our results have provided a solid foundation to advance our understanding of the 19S RP architecture at peptide level resolution. Furthermore, our methodology developed here is a valuable proteomic tool that can be generalized for elucidating the structures of protein complexes.

Expression of the oncoprotein gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumor.

OBJECTIVE: The oncoprotein, gankyrin, is known to facilitate cell proliferation through phosphorylation and degradation of retinoblastoma protein. In the present study, we evaluated the expression of gankyrin and phosphorylated retinoblastoma protein in human testis and testicular germ cell tumors. METHODS: The effects of suppression of gankyrin by locked nucleic acid on phosphorylation status of retinoblastoma and cell proliferation were analyzed using western blot analysis and testicular tumor cell line NEC8. The expressions of gankyrin, retinoblastoma and retinoblastoma protein were analyzed in 93 testicular germ cell tumor samples and five normal human testis by immunohistochemistry. The retinoblastoma protein expression was determined using an antibody to retinoblastoma protein, Ser795. RESULTS: Gankyrin was expressed in NEC8 cells as well as a normal human testis and testicular tumors. Suppression of gankyrin by locked nucleic acid led to suppression of retinoblastoma protein and cell proliferation in NEC8 cells. Immunohistochemistry of normal testis showed that gankyrin is expressed dominantly in spermatocytes. In testicular germ cell tumors, high expressions of gankyrin and phosphorylated-retinoblastoma protein were observed in seminoma and embryonal carcinoma, whereas the expressions of both proteins were weak in histological subtypes of non-seminoma. Growing teratoma and testicular malignant transformation tissues expressed phosphorylated-retinoblastoma protein strongly, but gankyrin faintly. CONCLUSION: Gankyrin is dominantly expressed in normal spermatocytes and seminoma/embryonal carcinoma, and its expression correlates well with retinoblastoma protein expression except in the growing teratoma and testicular malignant transformation cases. These data provide new insights into the molecular mechanisms of normal spermatogenesis and pathogenesis of testicular germ cell tumors.

Quantitative proteomic analysis revealed 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone-induced up-regulation of 20S proteasome in cultured human fibroblast cells.

The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone (NNK), is a well-known carcinogen. Although the ability of the metabolically activated form of NNK to generate DNA adducts is well established, little is known about the cellular pathways perturbed by NNK in its native state. In this study, we utilized stable isotope labeling by amino acid in cell culture (SILAC), together with mass spectrometry, to assess the perturbation of protein expression in GM00637 human skin fibroblast cells upon NNK exposure. With this approach, we were able to quantify 1412 proteins and 137 of them were with significantly altered expression following NNK exposure, including the up-regulation of all subunits of the 20S proteasome core complex. The up-regulation of the 20S core complex was also reflected by a significant increase in 20S proteasome activities in GM00637, IMR90, and MCF-7 cells upon NNK treatment. Furthermore, the ß-adrenergic receptor (ß-AR) antagonist propranolol could attenuate significantly the NNK-induced increase in proteasome activity in all the three cell lines, suggesting that up-regulation of the 20S proteasome may be mediated through the ß-AR. Additionally, we found that NNK treatment altered the expression levels of other important proteins including mitochondrial proteins, cytoskeleton-associated proteins, and proteins involved in glycolysis and gluconeogenesis. Results from the present study provided novel insights into the cellular mechanisms targeted by NNK.

Activity-based near-infrared fluorescent probe for LMP7: a chemical proteomics tool for the immunoproteasome in living cells.

Probing the unknown: The immunoproteasome, an alternative form of the constitutive proteasome, has been implicated in a number of pathological states such as cancer and autoimmune diseases. In an effort to understand the role of the immunoproteasome in cells, the first immunoproteasome-specific near-infrared fluorescent probe has been developed.

The C-terminal fragment of the immunoproteasome PA28S (Reg alpha) as an early diagnosis and tumor-relapse biomarker: evidence from mass spectrometry profiling.

This study reports on the C-terminal fragment of the 11S proteasome activator complex (PA28 or Reg alpha), a novel ovarian-specific biomarker of early and late stages of ovarian cancer (OVC) relapse, in patient biopsies after chemotherapy. A total of 179 tissue samples were analyzed: 8 stage I, 55 stage III-IV, 10 relapsed serous carcinomas, 25 mucinous carcinomas and 12 borderline and 68 benign ovarian tissue samples. This fragment was detected by MALDI mass spectrometry profiling in conjunction with a novel extraction method using hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-propanol; HFIP) solvents for protein solubilization and by immunohistochemistry using a specific antibody directed against the C-terminal fragment of PA28. Due to its specific cellular localization, this fragment is a suitable candidate for early OVC diagnosis, patient prognosis and follow-up during therapy and discriminating borderline cancers. Statistical analyses performed for this marker at different OVC stages reflect a prevalence of 77.66 ± 8.77 % (with a correlation coefficient value p < 0.001 of 0.601 between OVC and benign tissue). This marker presents a prevalence of 88 % in the case of tumor relapse and is detected at 80.5 % in stage I and 81.25 % ± 1.06 in stage III-IV of OVC. The correlation value for the different OVC stages is p < 0.001 of 0.998. Taken together, this report constitutes the first evidence of a novel OVC-specific marker.

Usefulness of material recovered from distal embolic protection devices after carotid angioplasty for proteomic studies.

PURPOSE: To demonstrate the usefulness of biologic material obtained from distal embolic protection devices (DEPDs) used in carotid angioplasty for the study of atherosclerosis protein markers and to establish the effect of systemic inflammation on the protein expression of carotid atheromatous plaques. MATERIALS AND METHODS: Two-dimensional gel electrophoresis and mass spectrometry were used to study proteins obtained from debris captured in DEPDs from patients who underwent carotid angioplasty. In addition, protein expression obtained from angioplasty samples in patients with different types of systemic inflammation (measured by serum levels of high-sensitivity C-reactive protein [CRP] with a cutoff value of 3 mg/L) was compared. Finally, immunohistochemistry of atherosclerotic plaques obtained by endarterectomy was used to validate the results obtained using DEPDs. RESULTS: Proteomic studies were successfully performed using debris from DEPDs. Protein expression differences were found in debris from patients with high systemic inflammation compared with debris from patients with low systemic inflammation. Annexin A5 (ANXA5), haptoglobin precursor, purine nucleoside phosphorylase, transgelin-2 (TAGLN2), and bisphosphoglycerate mutase were upregulated in debris from patients with high systemic inflammation, and proteasome subunit 8 beta type and glutathione-S-transferase kappa 1 (GSTK1) levels were higher in debris from patients with low levels of systemic inflammation. CONCLUSIONS: Atherosclerotic plaque debris captured in DEPDs is a suitable and valid source of material for proteomic studies of atherosclerosis. Protein expression in DEPD debris is affected by systemic inflammation.

Exclusive expression of proteasome subunit {beta}5t in the human thymic cortex.

The ubiquitin-proteasome pathway, which degrades intracellular proteins, is involved in numerous cellular processes, including the supply of immunocompetent peptides to the antigen presenting machinery. Proteolysis by proteasomes is conducted by three beta subunits, beta1, beta2, and beta5, of the 20S proteasome. Recently, a novel beta subunit expressed exclusively in cortical thymic epithelial cells was discovered in mice. This subunit, designated beta5t, is a component of the thymoproteasome, a specialized type of proteasomes implicated in thymic positive selection. In this study, we show that, like its mouse counterpart, human beta5t is expressed exclusively in the thymic cortex. Human beta5t was expressed in approximately 80% of cortical thymic epithelial cells and some cortical dendritic cells. Human beta5t was incorporated into proteasomes with two other catalytically active beta subunits beta1i and beta2i, forming 20S proteasomes with subunit compositions characteristic of thymoproteasomes. The present study demonstrates, for the first time, the existence of thymoproteasomes in the human thymic cortex, indicating that thymoproteasome function is likely conserved between humans and mice.