Constitutive Proteasome Subunits Research Articles

Proteomic Analysis of Human iPSC-Derived Neural Stem Cells and Motor Neurons Identifies Proteasome Structural Alterations.

Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system. We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs. The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the β5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased β5 (PSMB5) in MNs compared to HFFs. The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit β5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation.

Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging

—Proteasomes are key components of the ubiquitin–proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit β5; increased expression of genes encoding immune proteasome subunits β5i and β1i. In tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the β5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

Species-specific protein-protein interactions govern the humanization of the 20S proteasome in yeast.

Yeast and humans share thousands of genes despite a billion years of evolutionary divergence. While many human genes can functionally replace their yeast counterparts, nearly half of the tested shared genes cannot. For example, most yeast proteasome subunits are "humanizable," except subunits comprising the β-ring core, including β2c (HsPSMB7, a constitutive proteasome subunit). We developed a high-throughput pipeline to humanize yeast proteasomes by generating a large library of Hsβ2c mutants and screening them for complementation of a yeast β2 (ScPup1) knockout. Variants capable of replacing ScPup1 included (1) those impacting local protein-protein interactions (PPIs), with most affecting interactions between the β2c C-terminal tail and the adjacent β3 subunit, and (2) those affecting β2c proteolytic activity. Exchanging the full-length tail of human β2c with that of ScPup1 enabled complementation. Moreover, wild-type human β2c could replace yeast β2 if human β3 was also provided. Unexpectedly, yeast proteasomes bearing a catalytically inactive HsPSMB7-T44A variant that blocked precursor autoprocessing were viable, suggesting an intact propeptide stabilizes late assembly intermediates. In contrast, similar modifications in human β2i (HsPSMB10), an immunoproteasome subunit and the co-ortholog of yeast β2, do not enable complementation in yeast, suggesting distinct interactions are involved in human immunoproteasome core assembly. Broadly, our data reveal roles for specific PPIs governing functional replaceability across vast evolutionary distances.

Interplay between proteasome function and inflammatory responses in e-cig vapor condensate-challenged lung epithelial cells.

Exposure to cigarettes and other nicotine-based products results in persistent inflammation in the lung. In recent years, electronic cigarettes (e-cigs) have become extremely popular among adults and youth alike. E-cigarette vapor-induced oxidative stress promotes protein breakdown, DNA damage and cell death, culminating in a variety of respiratory diseases. The proteasome, a multi-catalytic protease, superintends protein degradation within the cell. When cells are stimulated with inflammatory cytokines such as IFN-γ and TNF-α, the constitutive catalytic proteasome subunits are replaced by the inducible subunits-low-molecular mass polypeptide (LMP)2 (β1i), multi-catalytic endopeptidase complex-like (MECL)1 (β2i), and LMP7 (β5i), which are required for the production of certain MHC class I-restricted T-cell epitopes. In this study, we used human alveolar epithelial cells (A549) and exposed them to filtered air or (1%) tobacco-flavored (TF) electronic cigarette vapor condensate(ECVC) ± nicotine (6mg/ml) (TF-ECVC ± N) for 24h. We observed an increase in the levels of IFN-γ, TNF-α, and inducible proteasome subunits (LMP7/PSMB8, LMP2/PSMB9, MECL1/PSMB10), and a reduced expression of constitutive proteasome subunits (β1/PSMB6 and β2/PSMB7) in challenged A549 cells. Interestingly, knockdown of the inducible proteasome subunit LMP7 reversed ECVC-induced expression of NADPH oxidase and immunoproteasome subunits in A549 cells. In addition, pre-exposure to an LMP7 inhibitor (ONX-0914) abrogated the mRNA expression of several NOX subunits and rescued the excessive production/release of inflammatory cytokines/chemokines (IL-6, IL-8, CCL2, and CCL5) in ECVC-challenged cells. Our findings suggest an important role of LMP7 in regulating the expression of inflammatory mediators during ECVC exposure. Overall, our results provide evidence for proteasome-dependent ROS-mediated inflammation in ECVC-challenged cells.

Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS.

The mammalian 20S catalytic core of the proteasome is made of 14 different subunits (α1-7 and β1-7) but exists as different subtypes depending on the cell type. In immune cells, for instance, constitutive catalytic proteasome subunits can be replaced by the so-called immuno-catalytic subunits, giving rise to the immunoproteasome. Proteasome activity is also altered by post-translational modifications (PTMs) and by genetic variants. Immunochemical methods are commonly used to investigate these PTMs whereby protein-tagging is necessary to monitor their effect on 20S assembly. Here, we present a new miniaturized workflow combining top-down and bottom-up mass spectrometry of immunopurified 20S proteasomes that analyze the proteasome assembly status as well as the full proteoform footprint, revealing PTMs, mutations, single nucleotide polymorphisms (SNPs) and induction of immune-subunits in different biological samples, including organoids, biopsies and B-lymphoblastoid cell lines derived from patients with proteasome-associated autoinflammatory syndromes (PRAAS). We emphasize the benefits of using top-down mass spectrometry in preserving the endogenous conformation of protein modifications, while enabling a rapid turnaround (1 h run) and ensuring high sensitivity (1-2 pmol) and demonstrate its capacity to semi-quantify constitutive and immune proteasome subunits.

Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the β5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the β5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit β5; increased expression of genes encoding immune proteasome subunits β5i and β1i. In tissue homogenates of aged mice, an increase in the content of immune subunits β1i and β2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the β5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the β5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

High Immunoproteasome Expression As Indicator for Sensitivity to Bortezomib-Containing Chemotherapy in Newly Diagnosed, Standard and Intermediate Risk, T-Cell Acute Lymphoblastic Leukemia Patients from Children's Oncology Group Trial AALL1231

Background: Proteasome inhibition by bortezomib (BTZ) has previously been shown to be beneficial for a subset of patients with relapsed/refractory acute leukemia. Several (pre)clinical studies showed that relatively high immunoproteasome expression (β1i and β5i) may serve as a biomarker for BTZ-sensitivity and aid to select patients that may respond well to BTZ-containing therapy (Cloos J et al. Cancer Metastasis Rev. 2017). In this study we investigated whether we could confirm these findings in newly diagnosed T-cell acute lymphoblastic leukemia patients (T-ALL) enrolled in the COG-AALL1231 phase 3 clinical trial (Teachey DT et al. J Clin Oncol 2022). In that trial, patients in treatment arm A received augmented Berlin-Frankfurt-Münster (aBFM) therapy, while for those in treatment arm B, BTZ was added to the aBFM regimen. Methods: Baseline immuno- and constitutive proteasome subunit expression was determined in bone marrow samples of 77 standard and intermediate risk T-ALL patients using Western blot. To correct for potential variation between blots, two cell lines were included as internal quantification controls, i.e. the human (BTZ-sensitive) T-ALL cell line CCRF-CEM (CEM-WT) and its subline (CEM-BTZ200) with acquired resistance to 200 nM BTZ. Protein bands were quantified and calculated relative to β-actin and the subunit expression of CEM-WT. Mann-Whitney U and log-rank tests were used for between group comparisons of proteasome subunit expressions and event free survival (EFS), respectively. Induction failure, relapse and death (induction and remission) were considered as EFS events. Results: In the bone marrow samples, a very strong correlation was observed between immunoproteasome β5i and β1i subunit expression (R=0.9, P<0.0001, Figure 1A). The correlation for constitutive subunits was R=0.7, P<0.0001. Interestingly, females had significantly higher β1i and β5i immunoproteasome levels compared to males with medians of 5.1 (range: 0.9-18.7) and 1.6 (0.3-17.9) respectively for the β1i subunit (P =0.05), and medians of 4.8 (1.9-16.1) and 1.8 (0.6-10.8) respectively for the β5i subunit (P =0.0084). Three year EFS was used as a measure for response to therapy. In patients treated with the BTZ-containing therapy (arm B), higher immunoproteasome expression levels (>0.8, based on Max-stat analysis) correlated with an improved EFS (P=0.034 for β1i and P=0.011 for β5i, Figure 1B). In contrast, in treatment arm A, higher immunoproteasome expression levels were associated with a relatively poor EFS, although this difference was not statistically significant likely due to low patient numbers in the low immunoproteasome subunit expression group in arm A (β1i N=5 and β5i N=1, for arm B β1i N=6 and β5i N=4). No other factors, including age, ethnicity, and gender, were found to be significantly associated with EFS. Discussion: These results support our hypothesis that patients with higher immunoproteasome expression levels are responsive to proteasome inhibitors such as BTZ, with better EFS after BTZ-containing therapy. When comparing the patients with high immunoproteasome expression of arm A versus arm B, the latter displays a better EFS (3 years EFS 96% for arm B vs 80% for arm A, P=0.08). Although not statistically significant, this factor may be indicative of a beneficial effect of BTZ-treatment in this subgroup of patients. The limitation of this study is the relatively low number of samples in each subgroup, in particular the patients with low expression of immunoproteasome subunits. Since most patients show a high level of immunoproteasome, only the few with low expression might not benefit from BTZ treatment and may be allocated for alternative strategies. The relevance of the immunoproteasome for BTZ sensitivity in these samples will be further examined by proteasome subunit catalytic activity measurements with subunit-specific substrates and other known factors contribution to diminished BTZ sensitivity (Niewerth et al. Drug Resist Updates 2015). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proteasomes in the Cells of the Ocular Surface in Health and Disease

PurposeLimbal stem cell deficiency (LSCD) is associated with corneal conjunctivalization that compromises corneal transparency and causes visual impairment. Previously, we found marked changes in proteasome population in corneas of rabbit with LSCD, and showed immunoproteasome (IPR) presence in central corneas during conjunctivalization compared to healthy rabbit cornea. In the present study, we further map the distribution of proteasomes population in the ocular surface.Methodsgene expression intensity obtained by microarray analysis of human healthy corneas and conjunctiva previously reported by the group of Dr. Wolosin (Turner H.C. et al 2007) was used to conduct this study. Constitutive proteasome (CPR) subunits (B5, B2, and B1) were compared to the inducible immunoproteasome (IPR) subunits (B5i, B2i, and B1i). The ratio between CPR and IPR subunits was calculated for cornea and for conjunctiva.ResultsThe comparison of the ratio B5/B5i, B2/B2i and B1/B1i between cornea and conjunctiva showed a significant high expression of CPR in corneas and a high expression of IPR in conjunctivae. In addition, PA28, the regulatory complex of IPR, and the major histocompatibility complex, class I A and B, were found significantly expressed in conjunctiva. Keratins K3/K12 were specifically expressed in cornea and keratins K4/K13 were specifically expressed in conjunctiva, which could be used to normalize the data analysis.ConclusionThe results indicated that conjunctivae contain high levels of IPR and that during conjunctivalization, corneas lose CPR to express IPR. The ration CPR/IPR could be used as an indication of conjunctivalization level in case of corneal injuries.

Omega-3 PUFAs Suppress IL-1β-Induced Hyperactivity of Immunoproteasomes in Astrocytes

The role of immunoproteasome (iP) in astroglia, the cellular component of innate immunity, has not been clarified. The results so far indicate that neuroinflammation, a prominent hallmark of Alzheimer’s disease, strongly activates the iP subunits expression. Since omega-3 PUFAs possess anti-inflammatory and pro-resolving activity in the brain, we investigated the effect of DHA and EPA on the gene expression of constitutive (β1 and β5) and inducible (iβ1/LMP2 and iβ5/LMP7) proteasome subunits and proteasomal activity in IL-1β-stimulated astrocytes. We found that both PUFAs downregulated the expression of IL-1β-induced the iP subunits, but not the constitutive proteasome subunits. The chymotrypsin-like activity was inhibited in a dose-dependent manner by DHA, and much strongly in the lower concentration by EPA. Furthermore, we established that C/EBPα and C/EBPβ transcription factors, being the cis-regulatory element of the transcription complex, frequently activated by inflammatory mediators, participate in a reduction in the iP subunits’ expression. Moreover, the expression of connexin 43 the major gap junction protein in astrocytes, negatively regulated by IL-1β was markedly increased in PUFA-treated cells. These findings indicate that omega-3 PUFAs attenuate inflammation-induced hyperactivity of iPs in astrocytes and have a beneficial effect on preservation of interastrocytic communication by gap junctions.

Synthesis of macrocyclic α-ketoamide as a selective and reversible immunoproteasome inhibitor

In recent years, the human immunoproteasome has emerged as an attractive therapeutic target for various diseases, leading to a growing interest in the discovery of immunoproteasome inhibitors that selectively target specific subunits. Herein we report the design, synthesis, and evaluation of a new immunoproteasome inhibitor that feature a macrocyclic ring containing an internal α-ketoamide warhead. This compound is a selective and reversible inhibitor of immunoproteasome subunits β1i and β5i and shows essentially no inhibition of constitutive proteasome subunits.

Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically β1, β2, and β5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits β1, β2, and β5 can be substituted by the immune βli, β2i, and β5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

Reduced Proteasome Activity and Enhanced Autophagy in Blood Cells of Psoriatic Patients.

Psoriasis is a skin disease that is accompanied by oxidative stress resulting in modification of cell components, including proteins. Therefore, we investigated the relationship between the intensity of oxidative stress and the expression and activity of the proteasomal system as well as autophagy, responsible for the degradation of oxidatively modified proteins in the blood cells of patients with psoriasis. Our results showed that the caspase-like, trypsin-like, and chymotrypsin-like activity of the 20S proteasome in lymphocytes, erythrocytes, and granulocytes was lower, while the expression of constitutive proteasome and immunoproteasome subunits in lymphocytes was increased cells of psoriatic patients compared to healthy subjects. Conversely, the expression of constitutive subunits in erythrocytes, and both constitutive and immunoproteasomal subunits in granulocytes were reduced. However, a significant increase in the autophagy flux (assessed using LC3BII/LC3BI ratio) independent of the AKT pathway was observed. The levels of 4-HNE, 4-HNE-protein adducts, and proteins carbonyl groups were significantly higher in the blood cells of psoriatic patients. The decreased activity of the 20S proteasome together with the increased autophagy and the significantly increased level of proteins carbonyl groups and 4-HNE-protein adducts indicate a proteostatic imbalance in the blood cells of patients with psoriasis.

Evaluating the immunoproteasome as a potential therapeutic target in cisplatin-resistant small cell and non-small cell lung cancer.

We evaluated the expression of proteasome subunits to assess whether the proteasome could be a therapeutic target in cisplatin-resistant lung cancer cells. Cisplatin-resistant (CR) variants were established from three non-small cell lung cancer (NSCLC) cell lines (A549, H1299, and H1975) and two small cell lung cancer (SCLC) cell lines (SBC3 and SBC5). The expression of proteasome subunits, the sensitivity to immunoproteasome inhibitors, and 20S proteasomal proteolytic activity were examined in the CR variants of the lung cancer cell lines. All five CR cell lines highly expressed one or both of the immunoproteasome subunit genes, PSMB8 and PSMB9, while no clear trend was observed in the expression of constitutive proteasome subunits. The CR cells expressed significantly higher levels of PSMB8 and PSMB9 proteins, as well. The CR variants of the H1299 and SBC3 cell lines were more sensitive to immunoproteasome inhibitors, and had significantly more proteasomal proteolytic activity than their parental counterparts. The immunoproteasome may be an effective therapeutic target in a subset of CR lung cancers. Proteasomal proteolytic activity may be a predictive marker for the efficacy of immunoproteasome inhibitors in cisplatin-resistant SCLC and NSCLC.

The Contribution of Proteasome Subunits to Myeloma Cell Viability and Proteasome Inhibitor Sensitivity

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Proteasomal degradation within endocytic organelles mediates antigen cross-presentation.

During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.

Selective Substrates and Activity-Based Probes for Imaging of the Human Constitutive 20S Proteasome in Cells and Blood Samples.

The proteasome is an enzyme complex critical for maintaining protein homeostasis. Perturbed proteasome function leads to pathologies including cancer and autoimmune and neurodegenerative disease. Therefore, the proteasome constitutes an excellent molecular target for pharmaceutical development. Here, using the HyCoSuL approach, we designed and synthesized novel and selective fluorogenic substrates for each of these three constitutive 20S proteasome activities and applied them to assess inhibition of proteasome subunits by MG-132 and a clinically used inhibitor bortezomib. Our results confirm the utility of designed substrates in biochemical assays. Furthermore, selective peptide sequences obtained in this manner were used to construct fluorophore-labeled activity-based probes and then utilized to detect each constitutive 20S proteasome subunit simultaneously in lysates of HEK-293F cells and red blood cells. Overall, we describe a simple and rapid method useful to measure constitutive 20S proteasome activity in whole human blood samples that could enable early diagnosis of pathological states associated with aberrantly upregulated proteasome activity.

(Immuno)proteasomes as therapeutic target in acute leukemia

The clinical efficacy of proteasome inhibitors in the treatment of multiple myeloma has encouraged application of proteasome inhibitor containing therapeutic interventions in (pediatric) acute leukemia. Here, we summarize the positioning of bortezomib, as first-generation proteasome inhibitor, and second-generation proteasome inhibitors in leukemia treatment from a preclinical and clinical perspective. Potential markers for proteasome inhibitor sensitivity and/or resistance emerging from leukemia cell line models and clinical sample studies will be discussed focusing on the role of immunoproteasome and constitutive proteasome (subunit) expression, PSMB5 mutations, and alternative mechanisms of overcoming proteolytic stress.

The Immunoproteasome in Human Lens Epithelial Cells During Oxidative Stress.

The immunoproteasome is known to generate peptides for antigen presentation. However, it has also been proposed to have additional functions such as stress response. The propensity of the immunoproteasome for degradation of oxidatively damaged proteins and peptides makes it interesting in the context of cataract formation and prevention. This study hypothesized that the immunoproteasome is present in human cataractous lenses and that oxidative stress will induce its expression, affect its proteolytic activity and its intracellular location in native cultured human lens epithelial cells (HLECs). The expression of the immunoproteasome and the constitutive proteasome subunits β1i/β1, β2i/β2, and β5i/β5 were studied by using Western blotting. The chymotrypsin-like activity was investigated for possible oxidative stress response. Inhibitors specific for the immuno- and constitutive proteasome, ONX-0914 and MG-132, respectively, were used to study their relative contributions to total proteasome activity. The intracellular location of the proteasomal subunits β5i and β5 was studied by immunocytochemistry. Immunoproteasome subunits were detected both in the lens epithelium and in the lens fibers derived from cataract surgery. Oxidative stress to cultured HLECs upregulated the immunoproteasome but not the constitutive proteasome. The chymotrypsin-like activity decreased with increased oxidative stress and the two proteasome types contributed equally to total proteasome activity. Immunocytochemical labeling of subunit β5i showed mainly cytosolic localization, whereas subunit β5 was localized predominantly in the nucleus. H2O2-induced challenge increased the expression of the immunoproteasome. The present findings indicate a role for the immunoproteasome in oxidative stress management in the lens.

Clinical activity of carfilzomib correlates with inhibition of multiple proteasome subunits: application of a novel pharmacodynamic assay

SummaryWhile proteasome inhibition is a validated therapeutic approach for multiple myeloma (MM), inhibition of individual constitutive proteasome (c20S) and immunoproteasome (i20S) subunits has not been fully explored owing to a lack of effective tools. We utilized the novel proteasome constitutive/immunoproteasome subunit enzyme‐linked immunosorbent (ProCISE) assay to quantify proteasome subunit occupancy in samples from five phase I/II and II trials before and after treatment with the proteasome inhibitor carfilzomib. Following the first carfilzomib dose (15–56 mg/m2), dose‐dependent inhibition of c20S and i20S chymotrypsin‐like active sites was observed [whole blood: ≥67%; peripheral blood mononuclear cells (PBMCs): ≥75%]. A similar inhibition profile was observed in bone marrow–derived CD138+ tumour cells. Carfilzomib‐induced proteasome inhibition was durable, with minimal recovery in PBMCs after 24 h but near‐complete recovery between cycles. Importantly, the ProCISE assay can be used to quantify occupancy of individual c20S and i20S subunits. We observed a relationship between MM patient response (n = 29), carfilzomib dose and occupancy of multiple i20S subunits, where greater occupancy was associated with an increased likelihood of achieving a clinical response at higher doses. ProCISE represents a new tool for measuring proteasome inhibitor activity in clinical trials and relating drug action to patient outcomes.

P13: Features of proteasome system functioning in the tumor and microenvironment cells of breast cancer

Background The tumor microenvironment plays an important role in the progression of cancer and may be regulated by proteolysis, including the proteasomes. The proteasomes modify the biologically important molecules involved in the pathogenesis and progression of a variety of malignancies, including breast cancer (BC). The aim of this study was to investigate the features of the subunit composition of proteasomes in the tumor and its microenvironment of breast cancer. Material and Methods The material for investigation was samples of tumor tissue of invasive ductal breast cancer. The study was conducted to estimate the distribution of the total pool of proteasome, proteasomes activator PA700, immune proteasome forms containing LMP7 and/or LMP2 subunit in tumor cells and stromal component using immunofluorescence. Furthermore, there was evaluated the distribution of the proteasome in the tumor. The expression of immune proteasomes and proteasomes activators in the cells was studied by immunofluorescence labeling of the cells by antibodies to immune proteasome subunits and cell markers. Fluorescence was analyzed by using a fluorescence microscope DM RXA2 (“Leica”, Germany) and confocal microscope TCS SP (“Leica”, Germany). The specificity of the primary antibodies was confirmed by check samples, at which the reaction was carried out only with the second antibody. No cross reaction between the first and second antibodies was tested by incubation of each primary antibodies with the opposing second antibodies. In addition, there was carried out labeling the cell nuclei by reagent Hoechst 33,342. Results It was found that the tumor cells comprise immune proteasomes, also PA700 and PA28αb activators. Activator PA28αb and immune proteasomes are localized in the cytoplasm of tumor cells, whereas α1, 2, 3, 5, 6, 7 subunits and PA700 activator detected in the cytoplasm and in the nuclei of tumor cells. Availability of α1, 2, 3, 5, 6, 7 subunits in the nuclei of tumor cells shows the expression of constitutive proteasome subunits. Stromal cells are characterized by a high ratio of the α1, 2, 3, 5, 6, 7 subunits to the immune LMP2 subunit as compared to cells of invasive ductal carcinoma. This means that the pool of proteasome in tumor cells of invasive ductal cancer is enriched by the immune proteasomes as compared to stromal cells. Thus, samples of invasive ductal breast cancer contain predominantly tumor cells enriched by immune proteasomes, activators PA700 and PA28αb. The presence of proteasomes in stromal component indicates that the tumor microenvironment also has active processes of proteolysis, with involving proteasome system. Probably the processes occurring in the stromal component contribute to the output of the proteasome into the extracellular space, which is confirmed by other researchers about the existence of circulating proteasome pools and their further participation in dissemination of cancer. This work was supported by the Russian Foundation for Basic Research (Grant numbers 13-04-00169 ).