Proteasome Catalytic Subunits Research Articles

Immunoproteasome activation expands the MHC class I immunopeptidome, unmasks neoantigens and enhances T-cell antimyeloma activity.

Proteasomes generate antigenic peptides that are presented on the tumor surface to cytotoxic T-lymphocytes (CTLs). Immunoproteasomes are highly-specialized proteasome variants that are expressed at higher levels in antigen-presenting cells and contain replacements of the three constitutive proteasome catalytic subunits to generate peptides with a hydrophobic C-terminus that fit within the groove of MHC class I (MHC-I) molecules. A hallmark of cancer is the ability to evade immunosurveillance by disrupting the antigen presentation machinery and downregulating MHC-I antigen presentation. High-throughput screening was performed to identify Compound A, a novel molecule that selectively increased immunoproteasome activity and expanded the number and diversity of MHC-I-bound peptides presented on multiple myeloma (MM) cells. Compound A increased the presentation of individual MHC-I-bound peptides >100-fold and unmasked tumor-specific neoantigens on myeloma cells. Global proteomic integral stability assays determined that Compound A binds the proteasome structural subunit PSMA1 and promotes association of the proteasome activator PA28α/β (PSME1/PSME2) with immunoproteasomes. CRISPR/Cas9 silencing of PSMA1, PSME1, or PSME2 as well as treatment with immunoproteasome-specific suicide inhibitors abolished the effects of Compound A on antigen presentation. Treatment of MM cell lines and patient bone marrow-derived CD138+ cells with Compound A increased the antimyeloma activity of allogenic and autologous T-cells. Compound A was well-tolerated in vivo and co-treatment with allogeneic T-cells reduced the growth of myeloma xenotransplants in NSG mice. Taken together, our results demonstrate the paradigm-shifting impact of immunoproteasome activators to diversify the antigenic landscape, expand the immunopeptidome, potentiate T-cell-directed therapy, and reveal actionable neoantigens for personalized T-cell immunotherapy.

Clickable Probes for Pathogen Proteasomes: Synthesis and Applications.

The 20S proteasome is a multimeric protease complex that is essential for proteostasis in the cell. Small molecule proteasome inhibitors are approved drugs for various cancers and are advancing clinically as antiparasitics. Although tools and technologies to study the 20S proteasome have advanced, only one probe is commercially available to image proteasome activity. This probe consists of a fluorescently labeled, peptidyl vinyl sulfone that binds to one or more of the catalytic proteasome subunits. Here, we synthesized two, active site-directed epoxyketone probes, LJL-1 and LJL-2, that were based on the peptidyl backbones of the anticancer drugs, carfilzomib and bortezomib, respectively. Each probe was conjugated, via click chemistry, to a bifunctional group comprising 5-carboxytetramethylrhodamine (TAMRA) and biotin to, respectively, visualize and enrich the 20S proteasome from protein extracts of two eukaryotic pathogens, Leishmania donovani and Trichomonas vaginalis. Depending on species, each probe generated a different subunit-binding profile by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the biotin tag enabled the enrichment of the bound subunits which were then formally identified by proteomics. Species differences in the order of electrophoretic migration by the β subunits were also noted. Finally, both probes reacted specifically with the 20S subunits in contrast to the commercial vinyl sulfone probe that cross reacted with cysteine proteases. LJL-1 and LJL-2 should find general utility in the identification and characterization of pathogen proteasomes, and serve as reagents to evaluate the specificity and mechanism of binding of new antiparasitic proteasome inhibitors.

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.

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.

Allantopyrone A interferes with the degradation of hypoxia-inducible factor 1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Allantopyrone A is an α-pyrone metabolite that was originally isolated from the endophytic fungus Allantophomopsis lycopodina KS-97. We previously demonstrated that allantopyrone A exhibits anti-cancer, anti-inflammatory, and neuroprotective activities. In the present study, we showed that allantopyrone A up-regulated the protein expression of hypoxia-inducible factor (HIF)-1α in human fibrosarcoma HT-1080 cells. It also up-regulated the mRNA expression of BNIP3 and ENO1, but not other HIF target genes or HIF1A. Allantopyrone A did not inhibit the prolyl hydroxylation of HIF-1α, but enhanced the ubiquitination of cellular proteins. Consistent with this result, chymotrypsin-like and trypsin-like proteasome activities were reduced, but not completely inactivated by allantopyrone A. Allantopyrone A decreased the amount of proteasome catalytic subunits. Therefore, the present results showed that allantopyrone A interfered with the degradation of HIF-1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome

The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic β2 and β5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the β5 P1 binding pocket, compromising compound binding. Dual targeting of P.falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.

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.

Investigation of the P1' and P2' sites of IQF substrates and their selectivity toward 20S proteasome subunits.

High levels of expression and activity of the 20S proteasome have been linked to many types of pathologies, including neoplasia, autoimmune disorders, neurodegenerative diseases and many more. Moreover, distinguishing between 20S proteasome catalytic subunits is neglected, although it may provide further insight into the development of pathologies. Several approaches have been developed to detect 20S proteasome activity, one of which is internally quenched fluorescent (IQF) substrates, which currently suffer from low efficiency and sensitivity. Previous reports focused on peptides including natural amino acids; therefore, in this report, we synthesized and analyzed IQF substrates with both natural and unnatural amino acids in the P1' and P2' positions to investigate their influences on selectivity toward 20S proteasome subunits. We found that elongation of the substrate by the P1'and P2' positions increased specificity in comparison to tetrapeptides. Moreover, we were able to obtain IQF substrates for the Ch-L subunit, which was characterized by higher selectivity than formerly used tetrapeptides. These findings may further contribute to the development of novel diagnostic tools for 20S proteasome-dependent disorders.

Susceptibilities of Ugandan Plasmodium falciparum Isolates to Proteasome Inhibitors.

The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC50 values <100 nM. TDI8304, a macrocylic peptide lead compound with drug-like properties, had a median IC50 of 16 nM. Sequencing genes encoding the β2 and β5 catalytic proteasome subunits, the predicted targets of the inhibitors, and five additional proteasome subunits, identified two mutations in β2 (I204T, S214F), three mutations in β5 (V2I, A142S, D150E), and three mutations in other subunits. The β2 S214F mutation was associated with decreased susceptibility to two peptide boronates, with IC50s of 181 nM and 2635 nM against mutant versus 62 nM and 477 nM against wild type parasites for MMV1579506 and MMV1794229, respectively, although significance could not be formally assessed due to the small number of mutant parasites with available data. The other β2 and β5 mutations and mutations in other subunits were not associated with susceptibility to tested compounds. Against culture-adapted Ugandan isolates, two asparagine ethylenediamines and the peptide proteasome inhibitors WLW-vinyl sulfone and WLL-vinyl sulfone (which were not studied ex vivo) demonstrated low nM activity, without decreased activity against β2 S214F mutant parasites. Overall, proteasome inhibitors had potent activity against P. falciparum isolates circulating in Uganda, and genetic variation in proteasome targets was uncommon.

Exploring the Anticancer Effects of Brominated Plastoquinone Analogs with Promising Cytotoxic Activity in MCF-7 Breast Cancer Cells via Cell Cycle Arrest and Oxidative Stress Induction.

Plastoquinone analogs are privileged structures among the known antiproliferative natural product-based compound families. Exploiting one of these analogs as a lead structure, we report the investigation of the brominated PQ analogs (BrPQ) in collaboration with the National Cancer Institute of Bethesda within the Developmental Therapeutics Program (DTP). These analogs exhibited growth inhibition in the micromolar range across leukemia, non-small cell lung cancer (EKVX, HOP-92, and NCI-H522), colon cancer (HCT-116, HOP-92), melanoma (LOX IMVI), and ovarian cancer (OVCAR-4) cell lines. One brominated PQ analog (BrPQ5) was selected for a full panel five-dose in vitro assay by the NCI’s Development Therapeutic Program (DTP) division to determine GI50, TGI, and LC50 parameters. The brominated PQ analog (BrPQ5) displayed remarkable activity against most tested cell lines, with GI50 values ranging from 1.55 to 4.41 µM. The designed molecules (BrPQ analogs) obeyed drug-likeness rules, displayed a favorable predictive Absorption, Distribution, Metabolism, and Excretion (ADME) profile, and an in silico simulation predicted a possible BrPQ5 interaction with proteasome catalytic subunits. Furthermore, the in vitro cytotoxic activity of BrPQ5 was assessed, and IC50 values for U-251 glioma, MCF-7 and MDA-MB-231 breast cancers, DU145 prostate cancer, HCT-116 colon cancer, and VHF93 fibroblast cell lines were evaluated using an MTT assay. MCF-7 was the most affected cell line, and the effects of BrPQ5 on cell proliferation, cell cycle, oxidative stress, apoptosis/necrosis induction, and proteasome activity were further investigated in MCF-7 cells. The in vitro assay results showed that BrPQ5 caused cytotoxicity in MCF-7 breast cancer cells via cell cycle arrest and oxidative stress induction. However, BrPQ5 did not inhibit the catalytic activity of the proteasome. These results provide valuable insights for further discovery of novel antiproliferative agents.

The ATP/Mg2+ Balance Affects the Degradation of Short Fluorogenic Substrates by the 20S Proteasome

Proteasomes hydrolyze most cellular proteins. The standard reaction to determine proteasome activity in cellular lysate or elsewhere contains AMC-conjugated peptide substrate, ATP, Mg2+, and DTT. ATP and Mg2+ are included to maintain 26S proteasome functionality. However, most cellular proteasomes are 20S proteasomes, and the effects of ATP on the turnover of fluorogenic substrates by 20S complexes are largely unknown. Here, we evaluated the effect of ATP alone or in combination with Mg2+ on the degradation of AMC-conjugated fluorogenic substrates by purified 20S proteasomes. Degradation of substrates used to determine chymotrypsin-, caspase- and trypsin-like proteasome activities was gradually decreased with the rise of ATP concentration from 0.25 to 10 mM. These effects were not associated with the blockage of the proteasome catalytic subunit active sites or unspecific alterations of AMC fluorescence by the ATP. However, ATP-induced peptide degradation slowdown was rescued by the addition of Mg2+. Moreover, the substrate degradation efficacy was proportional to the Mg2+/ATP ratio, being equal to control values when equimolar concentrations of the molecules were used. The obtained results indicate that when proteasome activity is assessed, the reciprocal effects of ATP and Mg2+ on the hydrolysis of AMC-conjugated fluorogenic substrates by the 20S proteasomes should be considered.

Nitazoxanide and related thiazolides induce cell death in cancer cells by targeting the 20S proteasome with novel binding modes

Nitazoxanide and related thiazolides are a novel class of anti-infectious agents against protozoan parasites, bacteria and viruses. In recent years, it is demonstrated that thiazolides can also induce cell cycle arrest and apoptotic cell death in cancer cells. Due to their fast proliferating nature, cancer cells highly depend on the proteasome system to remove aberrant proteins. Many of these aberrant proteins are regulators of cell cycle progression and apoptosis, such as the cyclins, BCL2 family members and nuclear factor of κB (NF-κB). Here, we demonstrate at both molecular and cellular levels that the 20S proteasome is a direct target of NTZ and related thiazolides. By concurrently inhibiting the multiple catalytic subunits of 20S proteasome, NTZ promotes cell cycle arrest and triggers cell death in colon cancer cells, either directly or as a sensitizer to other anti-tumor agents, especially doxorubicin. We further show that the binding mode of NTZ in the β5 subunit of the 20S proteasome is different from that of bortezomib and other existing proteasome inhibitors. These findings provide new insights in the design of novel small molecular proteasome inhibitors as anti-tumor agents suitable for solid tumor treatment in an oral dosing form.

Comparative analysis of proteins associated with 26S and 20S proteasomes isolated from rabbit brain and liver

We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.

Comparative analysis of proteins associated with 26S and 20S proteasomes isolated from rabbit brain and liver

We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.Iz pecheni i mozga krolika vydeleny fraktsii 26S i 20S proteasom. Po dannym mass-spektrometricheskogo (MS) analiza, fraktsii 26S proteasom iz étikh organov soderzhali kataliticheskie i reguliatornye sub"edinitsy, kharakternye dlia korovoĭ chasti proteasom i dlia reguliatornykh subchastits. Fraktsii 20S proteasom mozga i pecheni soderzhali tol'ko kataliticheskie sub"edinitsy proteasom. Pomimo sub"edinits proteasom, v poluchennykh fraktsiiakh obnaruzheny komponenty ubikvitin-proteasomnoĭ sistemy, ubikvitinirovannye belki, fermenty, igraiushchie vazhnuiu rol' v metabolicheskikh protsessakh, komponenty tsitoskeleta, signal'nye, reguliatornye i zashchitnye belki, a takzhe belki reguliatsii ékspressii genov, kletochnogo deleniia i differentsirovki. Predstavlennost' riada assotsiirovannykh s proteasomami belkov byla sopostavima ili prevyshala predstavlennost' sobstvennykh komponentov proteasom. Okolo treti belkov, obshchikh dlia vsekh issledovannykh fraktsiĭ (26S i 20S proteasom mozga i pecheni), otnosiatsia k gruppe mul'tifunktsional'nykh belkov. Vyborochnaia biosensornaia validatsiia podtverdila affinnoe sviazyvanie identifitsirovannykh v khode MS analiza belkov (al'dolaza, fosfoglitseratkinaza) s 20S proteasomoĭ mozga. Sopostavlenie subproteomov 26S i 20S proteasom mozga pokazalo, chto pri udalenii komponentov reguliatornykh (19S) subchastits kolichestvo individual'nykh belkov, assotsiirovannykh s korovoĭ chast'iu proteasomy (20S), prakticheski udvaivalos'. V pecheni kolichestvo belkov, assotsiirovannykh s korovoĭ chast'iu proteasomy, posle udaleniia komponentov reguliatornykh subchastits prakticheski ne izmenialos'. Éto svidetel'stvuet o tom, chto v mozge i, vozmozhno, v drugikh organakh belki reguliatornoĭ (19S) subchastitsy igraiut vazhnuiu rol' v formirovanii proteasomnogo interaktoma.

Modulation of proteasome activity by curcumin and didemethylcurcumin

Modulation of proteasome function by pharmacological interventions and molecular biology tools is an active area of research in cancer biology and neurodegenerative diseases. Curcumin (diferuloylmethane) is a naturally occurring polyphenol that affects multiple signaling pathways. Curcumin shows anti-inflammatory, antioxidant, anti-angiogenic, or anti-apoptotic properties. Recent research suggests that the therapeutic efficacy of curcumin may be due to its activity as a potent inhibitor of the proteasome. Using in vitro cell culture and molecular docking methods, here we show that both curcumin and its synthetic polyphenolic derivative (didemethylcurcumin, CUIII) modulated proteasome activity in a biphasic manner. Curcumin and CUIII increased proteasome activity at nanomolar concentrations, but inhibited proteasome activity at micromolar concentrations. Curcumin was more effective than CUIII in increasing relative proteasome activity at nanomolar concentrations. Also, curcumin was more effective than CUIII in inhibiting relative proteasome activity at micromolar concentrations. Docking simulations of curcumin and didemethylcurcumin binding to the 20S proteasome catalytic subunit estimated K d values of 0.0054 µM and 1.3167 µM, respectively. These values correlate well with the results of the effectiveness of modulation by curcumin compared to CUIII. The small size of CUIII allows it to dock to the narrow cavity of the active site, but the binding interaction is not strong compared to curcumin. These results indicate that curcumin and its didemethyl derivative can be used to modulate proteasome activity and suggest that curcumin and its didemethyl derivative may be useful in treating two different disease classes: neurodegeneration and cancer. Communicated by Ramaswamy H. Sarma

FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity.

Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic β5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

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.

Immunosubunit β5i Knockout Suppresses Neovascularization and Restores Autophagy in Retinal Neovascularization by Targeting ATG5 for Degradation.

PurposeTo investigate the functional role of immunoproteasome subunit β5i in pathologic retinal neovascularization (RNV) and its ability to link the immunoproteasome and autophagy.MethodsOxygen-induced retinopathy (OIR) was induced in wild-type (WT) and β5i knockout (KO) mouse pups on a C57BL/6J background. Proteasome catalytic subunit expression and proteasome activity were evaluated by quantitative real-time PCR (qPCR) and proteasome activity. Retinal vascular anatomy and neovascularization were characterized and quantified by retinal vascular flat-mount staining, fluorescence angiography, platelet endothelial cell adhesion molecule (PECAM) immunostaining, and hematoxylin and eosin staining. Correlation factors, including VEGF and ICAM-1, were detected by qPCR. Autophagy was examined by transmission electron microscopy (TEM). Autophagy biomarkers, including LC3, P62, ATG5, and ATG7, were measured by immunostaining and immunoblotting. The protein interaction between β5i and ATG5 was detected by immunoprecipitation.ResultsWe observed that β5i had the greatest effect in WT OIR mice. Fundus fluorescence angiography, retinal flat-mount staining, and PECAM staining revealed that pathologic RNV decreased in β5i KO OIR mice compared with WT OIR mice. Concurrently, TEM, immunostaining, and immunoblotting showed that autophagy was induced in β5i KO OIR mice compared to WT OIR mice through increases in autophagosome and LC3 expression and a decrease in P62. Mechanistically, β5i interacted with ATG5 and promoted its degradation, leading to autophagy inhibition and pathogenic RNV.ConclusionsThis study identifies a functional role for β5i in RNV regulation. β5i deletion ameliorates RNV and restores autophagy by stabilizing ATG5. These results demonstrate the potential of β5i to serve as a bridge linking the immunoproteasome and autophagy.

Celecoxib-Dependent Neuroprotection in a Rat Model of Transient Middle Cerebral Artery Occlusion (tMCAO) Involves Modifications in Unfolded Protein Response (UPR) and Proteasome.

Stroke is one of the main causes of death and disability worldwide. Ischemic stroke results in unfolded/misfolded protein accumulation in endoplasmic reticulum (ER), a condition known as ER stress. We hypothesized that previously reported neuroprotection of celecoxib, a selective inhibitor of cyclooxygenase-2, in transient middle cerebral artery occlusion (tMCAO) model, relies on the ER stress decrease. To probe this hypothesis, Sprague-Dawley rats were subjected to 1h of tMCAO and treated with celecoxib or vehicle 1 and 24h after ischemia. Protein and mRNA levels of the main hallmarks of ER stress, unfolded protein response (UPR) activation, UPR-induced cell death, and ubiquitin proteasome system (UPS) and autophagy, the main protein degradation pathways, were measured at 12 and 48h of reperfusion. Celecoxib treatment decreased polyubiquitinated protein load and ER stress marker expression such as glucose-related protein 78 (GRP78), C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP), and caspase 12 after 48h of reperfusion. Regarding the UPR activation, celecoxib promoted inositol-requiring enzyme 1 (IRE1) pathway instead of double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway. Furthermore, celecoxib treatment increased proteasome catalytic subunits transcript levels and decreased p62 protein levels, while the microtubule-associated protein 1 light chain 3 (LC3B) II/I ratio remained unchanged. Thus, the ability of celecoxib treatment on reducing the ER stress correlates with the enhancement of IRE1-UPR pathway and UPS degradation. These data support the ability of anti-inflammatory therapy in modulating ER stress and reveal the IRE1 pathway as a promising therapeutic target in stroke therapy.Graphical abstract.

Timing of Proteasome Inhibition for Prevention of Muscle Contractures in Neonatal Brachial Plexus Injury

Background: Neonatal brachial plexus injury (NBPI) causes disabling and incurable contractures, or limb stiffness, which result from proteasome-mediated protein degradation impairing the longitudinal growth of neonatally denervated muscles. We recently showed in a mouse model that the 20S proteasome inhibitor, bortezomib, prevents contractures after NBPI. Given that contractures uniquely follow neonatal denervation, the current study tests the hypothesis that proteasome inhibition during a finite window of neonatal development can prevent long-term contracture development. Methods: We used a mouse model of NBPI in which neonatal forelimb denervation at P5 causes shoulder and elbow contractures 4 weeks post-denervation. We first outlined the minimum period for proteasome inhibition to prevent contractures 4 weeks post-NBPI by treating mice with saline or bortezomib for varying durations between P8 and P32. We then compared the ability of varying durations of longer-term proteasome inhibition to prevent contractures at 8 and 12 weeks post-NBPI. Elbow and shoulder contractures, elbow flexor muscle length, and proteasome catalytic subunit activity were assessed. Findings: Proteasome inhibition can be delayed 3-4 days after denervation but is required throughout skeletal growth to prevent contractures long-term. Proteasome inhibition becomes less effective in preventing contractures beyond the neonatal period. The therapeutic effect of bortezomib is associated with attenuation of 20S proteasome β1 subunit activity. Interpretation: Temporary neonatal proteasome inhibition does not prevent contractures long-term. Instead, neonatal denervation causes a permanent longitudinal growth deficiency that must be continuously ameliorated during skeletal growth. Additional mechanisms must be explored to reduce the risk of toxicity from long-term proteasome inhibition. Funding Statement: This work was supported by grants to RC from the National Institutes of Health (NIH) (R01HD098280-01) and Pediatric Orthopaedic Society of North America (POSNA), as well as funding from the Cincinnati Children’s Hospital Division of Orthopaedic Surgery and Junior Cooperative Society. Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mice were handled according to approved institutional animal care and use committee (IACUC) protocols (#2017-0084) of the Cincinnati Children’s Hospital Medical Center. All surgeries were performed under isoflurane anesthesia, and every effort was made to minimize suffering.