scholarly journals Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

2013 ◽  
Vol 87 (23) ◽  
pp. 13035-13041 ◽  
Author(s):  
Angela M. Mitchell ◽  
R. Jude Samulski
Keyword(s):  
Cysteine Protease ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Protease Inhibition ◽  
Adeno Associated Virus

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.

In Vitro and In Vivo Proteasome Activity Profiles of Bortezomib and a Novel Proteasome Inhibitor NPI-0052.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3363-3363 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ta-Hsiang Chao ◽  
Laurence Catley ◽  
Benjamin Nicholson ◽  
Mugdha Velanker ◽  
...  
Keyword(s):  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Catalytic Activities ◽  
Proteasome Subunits ◽  
20 Nm ◽  
Whole Blood Cells

Abstract Proteasome inhibition is an effective anti-cancer therapy. Proteasome function is mediated by three catalytic activities: chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L). Kinetics of inhibition of catalytic activities may define the pharmacologic utility of proteasome inhibitors. Here we utilized two structurally distinct proteasome inhibitors Bortezomib, a dipeptide boronic acid; and a non-peptide proteasome inhibitor NPI-0052 to determine their effect on proteasome activities in vitro and in animal model. Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates shows that NPI-0052 and Bortezomib inhibit all three proteasome activities, albeit at different concentrations: NPI-0052 inhibits CT-L and T-L activities at lower concentrations than Bortezomib (NPI-0052: EC50 = 3.5 ± 0.3 nM versus Bortezomib: 7.9 ± 0.5 nM for CT-L activity; and NPI-0052: EC50 = 28 ± 2 nM versus Bortezomib: EC50 = 590 ± 67 nM for T-L activity); in contrast, higher concentrations of NPI-0052 than Bortezomib are required to inhibit C-L activity (NPI-0052 EC50 = 430 ± 34 nM versus Bortezomib: EC50 = 53 ± 10 nM for C-L activity). We next compared the effects of NPI-0052 and Bortezomib on all three proteasome activities in vivo. Mice were treated with a single MTD dose of NPI-0052 (0.15 mg/kg i.v) or Bortezomib (1 mg/kg i.v); blood samples were collected at 90 mins, 24h, 48h, 72h, or 168h; and whole blood cells were then analyzed for proteasome activity. NPI-0052 completely inhibited CT-L activity by 90 mins, which was recoverable by 168h; whereas Bortezomib-inhibited CT-L activity is recoverable at 24h. T-L activity is significantly inhibited by NPI-0052 at 90 mins, 24h, 48h, and 72h; and is recoverable by 168h; in contrast, Bortezomib enhances T-L activity. Finally, NPI-0052 inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and this activity recovered at 168h, whereas Bortezomib significantly inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and is similarly recoverable at 168h. We next utilized a novel methodology to measure proteasome activity by immunoblotting using dansylAhx3L3VS as a probe (Berkers et al., Nature Methods, 2005), which also allow for determining subunit specificity of a proteasome inhibitor. Multiple myeloma (MM) cells were cultured in the presence or absence of various concentrations of either NPI-0052 (2 nM; 7 nM: IC50; or 20 nM) or Bortezomib (2 nM; 5 nM: IC50; or 20 nM). Competition experiments between either NPI-0052 or Bortezomib and dansylAhx3L3VS revealed that NPI-0052 (7 nM) markedly inhibits the CT-L activity represented by beta-5 subunit of the proteasome and decreased the dansylAhx3L3VS-labeling of the beta-1 (C-L activity) and -2 (T-L activity) subunits. Slightly higher concentrations of Bortezomib are necessary to markedly inhibit beta-5 and -1 subunits, whereas beta-2 subunits are not inhibited. Importantly, both agents trigger apoptosis in MM cells; however, NPI-0052 is remarkably less toxic to normal lymphocytes than Bortezomib. Our data show that NPI-0052, like Bortezomib, targets the proteasome, but triggers a proteasome activity profile distinct from Bortezomib. The mechanistic insights gained from these studies will allow for improved drug design based on targeting specific proteasome subunits.

scholarly journals Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation

2018 ◽  
Vol 217 (5) ◽  
pp. 1757-1776 ◽  
Author(s):  
Zhe Sha ◽  
Helena M. Schnell ◽  
Kerstin Ruoff ◽  
Alfred Goldberg
Keyword(s):  
Proteasome Inhibitor ◽  
Neuroblastoma Cells ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Nuclear Factor ◽  
Ubiquitinated Proteins ◽  
Rapid Induction ◽  
Independent Mechanism ◽  
Transcription Factor Nuclear Factor ◽  
Inhibitor Treatment

Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.

Neurodegeneration Induced by Bortezomib Exposure in Vitro Occurs Via Proteasome Independent Mechanisms.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2859-2859
Author(s):  
Shirin Arastu-Kapur ◽  
Andrew J. Ball ◽  
Janet L. Anderl ◽  
Mark K Bennett ◽  
Christopher J Kirk
Keyword(s):  
Peripheral Nerve ◽  
Proteasome Inhibitor ◽  
Serine Proteases ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Nerve Degeneration ◽  
Differentiated Cells ◽  
Favorable Safety ◽  
Neurite Degeneration

Abstract Abstract 2859 Poster Board II-835 BACKGROUND: The dipeptide boronate proteasome inhibitor bortezomib (BTZ; Velcade®) is approved for the treatment of multiple myeloma and non-Hodgkin's lymphoma. Bortezomib-induced peripheral neuropathy (BIPN, Blood (2008)112:1593-1599) is seen in ∼30% of BTZ-treated patients and can result in dose reductions and discontinuations that may result in suboptimal levels of proteasome inhibition. Carfilzomib (CFZ), a tetrapeptide epoxyketone, is a selective and irreversible proteasome inhibitor that is structurally and mechanistically distinct from bortezomib. Single agent treatment with CFZ has demonstrated strong activity in relapsed and refractory myeloma and a favorable safety profile in Phase 2 trials (ASH2008:864 & 865). Importantly treatment-emergent PN was seen at low levels and did not result in dose modifications or discontinuations. The disparate safety data for these proteasome inhibitors suggest that non-proteasomal mechanisms may underlie BIPN. Using activity-based probes in peripheral blood mononuclear cell (PBMC) lysates, we previously demonstrated inhibition of non-proteasomal proteases by BTZ and other proteasome inhibitors with a boronate pharmacophore (EHA2009:0939). However, the involvement of the proteasome in the peripheral nerve degeneration and BIPN in BTZ-treated myeloma patients remains to be established. AIMS: To establish an in vitro model of peripheral nerve degeneration and to determine the effects of proteasome inhibition by BTZ and CFZ on neurite outgrowth and cell survival. METHODS: SH-SY5Y neuroblastoma cells were differentiated by long term culture in retinoic acid and brain derived nerve growth factor to induce neurite outgrowth. The effects of proteasome inhibitors were measured by high content image analysis of fluorescent images for cell survival (Hoechst nuclear counterstain) and neurite degeneration (FITC-mouse anti-beta-III-tubulin). Phase contrast images were also collected to observe morphological effects and gross cell death. Cell viability and proteasome inhibition was measured in undifferentiated and differentiated cells. The MEROPS (peptidase) database was mined for candidate serine proteases with a P1 selectivity of Leu/Phe/Tyr to identify candidate off-targets CFZ and BTZ and candidate proteases were validated by standard biochemical and cell biology techniques. RESULTS: In differentiated SH-SY5Y cells, the average neurite length decreased by 33% following 24 hr exposure to 10nM BTZ but was unaffected by the same concentration of CFZ. Proteasome inhibition as determined by a fluorescent substrate for the chymotrypsin-like activity was equivalent (∼70%) after a 24 hr exposure for both compounds in differentiated cells, suggesting that neurodegeneration involves non-proteasomal pathways. With 72 hrs continuous exposure, BTZ was 10-fold more potent than CFZ at inducing neurodegeneration. Furthermore, in both undifferentiated and differentiated SH-SY5Y cells, BTZ was 5-fold more cytotoxic than CFZ. Database mining for serine proteases with a selectivity for Leu/Phe/Tyr at P1 was used to identify other potential BTZ targets that might underlie neurotoxicity. One candidate is HtrA2 (also called Omi), an inducible mitochondrial serine protease whose activity protects neurons from stress induced apoptosis (Hum Mol Genet (2005) 14(5):2099-2111). HtrA2 levels increased 2-fold in SH-SY5Y cells treated with either BTZ or CFZ for 6 hrs at 40 nM. Using a gel based assay and purified enzyme preparations, BTZ inhibited HtrA2 activity with an IC50 ∼ 4 nM, equivalent to its activity against the proteasome. In contrast, Carfilzomib did not inhibit HtrA2 at the highest concentration tested (10 mM). CONCLUSIONS: These data demonstrate that BTZ induces neuronal cell death and neurite degeneration in vitro by proteasome-independent mechanisms. We propose that combined inhibition of the proteasome and HtrA2 by BTZ may underlie peripheral nerve toxicities in vitro and may be involved in BIPN in myeloma patients. In this model, CFZ, which mediates equivalent proteasome inhibition to BTZ in neurons, does not induce neurodegeneration due to inactivity against HtrA2. Future profiling of non-proteasomal targets of BTZ, including HtrA2 activity, in patient samples is merited. These results suggest that the favorable safety profile of CFZ in myeloma patients may be a result of its high selectivity for proteasomal proteases. Disclosures: Arastu-Kapur: Proteolix, Inc: Employment. Ball:Millipore Corp: Employment. Anderl:Millipore Corp: Employment. Bennett:Proteolix: Employment. Kirk:Proteolix, Inc: Employment.

Phase 1 Clinical Trial of the Novel Structure Proteasome Inhibitor NPI-0052.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2693-2693 ◽  
Author(s):  
Andrew Spencer ◽  
Michael Millward ◽  
Paul Mainwaring ◽  
Simon Harrison ◽  
Laurence Catley ◽  
...  
Keyword(s):  
Solid Tumor ◽  
Whole Blood ◽  
Proteasome Inhibitor ◽  
Mononuclear Cells ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Pharmacokinetic Data ◽  
Employment Equity ◽  
Cognitive Changes ◽  
Equity Ownership

Abstract Abstract 2693 Poster Board II-669 Background: NPI-0052 is a proteasome inhibitor with a novel bicyclic structure (other proteasome inhibitors in clinical use are peptide based). Preclinical studies indicate rapid, broad and prolonged inhibition of all 3 catalytic sites of the proteasome, and subsequently unique proteasome inhibition, signal transduction, toxicology and efficacy profiles. Taken together these suggest the potential for improvements in therapeutic ratio and activity in hematologic and solid tumor malignancies. Materials and Methods: Patients with solid tumor, lymphoma, leukemia or myeloma diagnoses without standard treatment options have been treated with IV NPI-0052 on one of two arms (weekly or twice weekly) in this 3+3 design dose escalation study. This is followed by 10 patient Recommended Phase 2 dose Cohorts of patients with lymphomas, CLL and myeloma respectively. Proteasome inhibition (pharmacodynamics) and pharmacokinetics are also assayed in whole blood, and proteasome inhibition in peripheral blood mononuclear cells (PBMC). Results: 44 patients have been treated with NPI-0052 at doses ranging from 0.075 mg/m2 to 0.9 mg/m2. Common adverse events include fatigue, parosmia/dysgeusia, transient peri-infusion site pain, lymphopenia, headaches, dizziness / unsteady gait, closed-eye visuals, cognitive changes. Incidence and grade of these events correlate with dose, being quite tolerable at the MTD of 0.7 mg/m2 on the weekly dosing arm. An MTD has not yet been determined for the twice weekly dosing arm. Pharmacokinetic data has demonstrated a rapid elimination half-life (<20 minutes) and relatively large volume of distribution. Assessment of proteasome inhibition has demonstrated increasing inhibition of chymotrypsin-like activity of up to 88% Day 1 and 100% Day 15. Inhibition of caspase-like and trypsin-like activity of up to 52% and 71% respectively has also been seen. Inhibition remains between doses in whole blood (principally RBC), but recovers between doses in PBMC. Clinical benefit, including stable disease, regression or response, was reported in patients with mantle cell lymphoma, myeloma, Hodgkin's lymphoma, cutaneous marginal zone lymphoma, follicular lymphoma, sarcoma, prostate carcinoma and melanoma. Conclusions: NPI-0052 produces dose-dependent pharmacologic effects through the predicted efficacious range, while producing a toxicity profile that is dissimilar to what is reported with other proteasome inhibitors (notably deficient in peripheral neuropathy, neutropenia and thrombocytopenia) in spite of producing equal or greater proteasome inhibition. These data indicate a broad range of potential uses, and led to additional studies in hematologic malignancies and solid tumors alone and in combination. Disclosures: Longenecker: Nereus Pharmaceuticals: Employment. Palladino:Nereus Pharmaceuticals: Employment, Equity Ownership. Lloyd:Nereus Pharmaceuticals: Employment, Equity Ownership. Neuteboom:Nereus Pharmaceuticals: Employment, Equity Ownership. Spear:Nereus Pharmaceuticals: Employment, Equity Ownership.

Selective Inhibition of the proteasome's β2 Catalytic Subunit Alone Does Not Induce Cytotoxicity, but Resensitizes Bortezomib-Refractory Myeloma Cells for Bortezomib Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2915-2915
Author(s):  
Marianne Kraus ◽  
Bobby Florea ◽  
Jürgen Bader ◽  
Nan Li ◽  
Paul Geurink ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Active Site ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Selective Inhibition ◽  
Catalytic Site ◽  
Myeloma Cells ◽  
Bortezomib Treatment

Abstract Abstract 2915 Bortezomib is a reversible first-generation proteasome inhibitor that inhibits the β5 and to a lesser extent the β1 catalytic site of the proteasome. However, bortezomib does not inhibit the β2 catalytic proteasomal site at clinically relevant concentrations, and bortezomib-resistance is accompanied by upregulation of the β2 subunit, suggesting that increased β2 activity may compensate for the loss of β1/ β5 activity during bortezomib-treatment. The second generation proteasome inhibitor carfilzomib, due to the chemistry of its epoxyketone warhead, has a higher substrate specificity and functions as an irreversible proteasome inhibitor, but is still a β1/ β5 inhibitor that does not affect the β2 active site. We investigated the effect of β2-specific proteasome inhibition on myeloma and acute myeloid leukemia (AML) cells and tested the hypothesis that β2-selective proteasome inhibition may overcome bortezomib-resistance. To this end we have developed a set of epoxyketone- and vinylsulfone-based, cell permeable proteasome inhibitors of which we selected the compounds PR523A and PR671A for further testing in cell-based assays. PR671A is a peptide-vinylsulfone that selectively inhibits the proteasome's β2/ β2i subunit in an irreversible fashion in human cell lines and primary cells at low micromolar concentrations without inhibition of other protease species. PR523A is a β5-selective peptide-epoxyketone with otherwise similar properties. Treatment of myeloma and AML cell lines (AMO-1, U-266, HL-60, THP-1) with PR523A induced ER-stress mediated apoptosis, very similar to bortezomib. The combination of bortezomib with PR523A led to additive, but not synergistic induction of apoptosis, as expected. Selective β2 inhibition by PR671A resulted in the induction of ER stress and the accumulation of poly-ubiquitinated protein, however, this was not effectively translated into apoptotic cell death. This indicates that selective inhibition of the β2 proteasome subunit alone has only a poor cytotoxic effect on myeloma and AML cell lines, suggesting that the function of β2 is largely redundant and can be compensated when the remaining proteasome catalytic subunits (β1 and β5) remain active. However, when the β2 inhibitor PR671A was combined with agents that target the proteasome's β5 active site (PR523A) or the β5 and the β1 site (bortezomib), the combination of either inhibitor with the β2 inhibitor PR671A was highly synergistic for both activation of ER stress and the induction of apoptotic death. Importantly, the bortezomib-resistance in bortezomib-adapted myeloma and AML cell lines could be overcome by combining PR671A with either bortezomib or PR523A, while β2 inhibition by PR671A alone had no effect on the viability of bortezomib-adapted cells. We conclude that PR671A is a β2 selective proteasome inhibitor. Selective Inhibition of the proteasome's β2 subunit has little effect on viability or ER stress both in normal and bortezomib-resistant myeloma and leukemia cells, suggesting that the function of the β2 catalytic site is largely redundant. However, when β1/ β5 proteasome activity is inhibited by drugs like bortezomib or carfilzomib, proper function of the β2 proteasome active site is crucial for cell survival, also in bortezomib-resistant myeloma cells. The use of specific β2 inhibitors like PR671A in combination with β1/ β5 inhibitors like bortezomib or carfilzomib is therefore a promising strategy to overcome resistance against β1/ β5-selective proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

The Novel Proteasome Inhibitors Carfilzomib and Oprozomib Induce Milder Degenerative Effects Compared To Bortezomib When Administered Via Oral Feeding In An In Vivo Drosophila Experimental Model: A Biological Platform To Evaluate Safety/Efficacy Of Proteasome Inhibitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1930-1930
Author(s):  
Evangelos Terpos ◽  
Eleni N. Tsakiri ◽  
Efstathios Kastritis ◽  
Tina Bagratuni ◽  
Vassilis G. Gorgoulis ◽  
...  
Keyword(s):  
Oral Administration ◽  
Proteasome Inhibitor ◽  
Germ Line ◽  
Conflicts Of Interest ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Somatic Tissue ◽  
Oral Feeding ◽  
Somatic Tissues

Abstract The proteasome is involved in the degradation of both normal, short-lived ubiquitinated proteins and mutated or damaged proteins. Carfilzomib is a tetrapeptide epoxyketone–based proteasome inhibitor and oprozomib is an orally bioavailable tripeptide epoxyketone-based proteasome inhibitor. The primary target for both agents is the chymotrypsin-like β5 subunit of the constitutive proteasome and immunoproteasome. Oprozomib is 5-fold less potent than carfilzomib, but displays similar cytotoxic potential with longer exposure times due to its time-dependent proteasome inhibition. In contrast, bortezomib is a slowly reversible proteasome inhibitor with potency of proteasome inhibition similar to carfilzomib. We propose the fruit fly Drosophila melanogaster as an in vivo platform for screening and characterizing proteasome inhibitors at the whole organism level. Drosophilais well-suited to this line of investigation, due to its powerful genetics, its similarities in key metabolic and aging pathways with humans, the fact that it expresses proteasomes that structurally resemble those from mammals, and also because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cells. Moreover, flies live for few months and thus, drug screening on large cohorts can be completed in a reasonable time. We validate our model by investigating the effects of orally administered carfilzomib and oprozomib vs. bortezomib. In isolated Drosophila proteasome in vitroassays, carfilzomib showed a pattern of inhibitory activity similar to bortezomib, whereas oprozomib was less effective. After continuous oral administration of the inhibitors (∼50 μM of carfilzomib and ∼300-400 μM of oprozomib) to young flies (by adding the inhibitor in the flies’ culture medium) a proteasome inhibitory effect in somatic tissues roughly similar to 1 μM bortezomib was induced. Similar findings were noted when we analyzed distinct somatic tissue parts (i.e., head, thorax and abdomen), indicating that orally administered proteasome inhibitors are equally distributed to different body parts. As in the case of bortezomib, the effects of the inhibitors were less pronounced in the reproductive tissues. At the molecular level, carfilzomib (as compared to bortezomib) induced a milder disruption of fly somatic tissue proteostasis, lower rates of somatic tissue oxidative stress and less intense activation of genomic antioxidant response elements that correlated with reduced intensities of proteasome genes and protein subunit upregulation. Proteasome subunit induction was found to depend on the activity of the transcription factor Nrf2, a master regulator of cellular anti-oxidant responses. Furthermore, carfilzomib promoted the induction of lysosomal enzymes (e.g. cathepsins) and autophagy-related genes but less intensively compared to bortezomib. At concentrations that induced rates of proteasome inhibition that were similar to bortezomib, there were no significant toxic effects of either carfilzomib or oprozomib to oogenesis or to embryogenesis. Compared to bortezomib, both inhibitors exerted a significantly milder impact on the neuromusculatory system (locomotor performance) of the flies. Finally, we found that sustained oral administration of either carfilzomib or oprozomib exerted significantly milder effects (as compared to bortezomib) on flies’ mortality rate, healthspan and overall longevity. Our in vivo data support that carfilzomib is significantly less toxic compared to bortezomib, including neuromusculatory toxicity. Oprozomib was also less toxic but it is worth noting that it showed reduced activity against fly proteasomes. In support, our preliminary analyses indicated that in comparison to bortezomib and carfilzomib, oprozomib was less potent when tested in human osteosarcoma cancer cell lines. The validity of our in vivo pharmacological model is exemplified by the observed similarities with the reported clinical adverse effects, while the ratio of the different doses used to achieve similar rates of proteasome inhibition in Drosophila somatic tissues (i.e. ∼1 μM bortezomib, ∼50 μM carfilzomib) is reminiscent of the doses used in the clinic (i.e. ∼1.3 mg/m2 bortezomib and ∼25-56 mg/m2 carfilzomib). We conclude that fruit flies represent a valid biological platform for evaluating the efficacy and toxicity of proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals O-GlcNAcylation Signal Mediates Proteasome Inhibitor Resistance in Cancer Cells by Stabilizing NRF1

2018 ◽  
Vol 38 (17) ◽  
Author(s):  
Hiroki Sekine ◽  
Keito Okazaki ◽  
Koichiro Kato ◽  
M. Morshedul Alam ◽  
Hiroki Shima ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Proteasome Inhibitor ◽  
Cancer Metabolism ◽  
Meta Analysis ◽  
Strong Dependence ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Proteasome Activity ◽  
Proteasome Subunit ◽  
Culture Cells

ABSTRACTCancer cells often heavily depend on the ubiquitin-proteasome system (UPS) for their growth and survival. Irrespective of their strong dependence on the proteasome activity, cancer cells, except for multiple myeloma, are mostly resistant to proteasome inhibitors. A major cause of this resistance is the proteasome bounce-back response mediated by NRF1, a transcription factor that coordinately activates proteasome subunit genes. To identify new targets for efficient suppression of UPS, we explored, using immunoprecipitation and mass spectrometry, the possible existence of nuclear proteins that cooperate with NRF1 and identifiedO-linkedN-acetylglucosamine transferase (OGT) and host cell factor C1 (HCF-1) as two proteins capable of forming a complex with NRF1.O-GlcNAcylation catalyzed by OGT was essential for NRF1 stabilization and consequent upregulation of proteasome subunit genes. Meta-analysis of breast and colorectal cancers revealed positive correlations in the relative protein abundance of OGT and proteasome subunits. OGT inhibition was effective at sensitizing cancer cells to a proteasome inhibitor both in culture cells and a xenograft mouse model. Since activeO-GlcNAcylation is a feature of cancer metabolism, our study has clarified a novel linkage between cancer metabolism and UPS function and added a new regulatory axis to the regulation of the proteasome activity.

scholarly journals Proteasome inhibition improves diaphragm function in congestive heart failure rats

2008 ◽  
Vol 294 (6) ◽  
pp. L1260-L1268 ◽  
Author(s):  
Hieronymus W. H. van Hees ◽  
Yi-Ping Li ◽  
Coen A. C. Ottenheijm ◽  
Bingwen Jin ◽  
Cindy J. C. Pigmans ◽  
...  
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Proteasome Inhibitor ◽  
Caspase 3 ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Cross Bridge ◽  
Bortezomib Treatment ◽  
Diaphragm Function ◽  
Generating Capacity

In congestive heart failure (CHF), diaphragm weakness is known to occur and is associated with myosin loss and activation of the ubiquitin-proteasome pathway. The effect of modulating proteasome activity on myosin loss and diaphragm function is unknown. The present study investigated the effect of in vivo proteasome inhibition on myosin loss and diaphragm function in CHF rats. Coronary artery ligation was used as an animal model for CHF. Sham-operated rats served as controls. Animals were treated with the proteasome inhibitor bortezomib (intravenously) or received saline (0.9%) injections. Force generating capacity, cross-bridge cycling kinetics, and myosin content were measured in diaphragm single fibers. Proteasome activity, caspase-3 activity, and MuRF-1 and MAFbx mRNA levels were determined in diaphragm homogenates. Proteasome activities in the diaphragm were significantly reduced by bortezomib. Bortezomib treatment significantly improved diaphragm single fiber force generating capacity (∼30–40%) and cross-bridge cycling kinetics (∼20%) in CHF. Myosin content was ∼30% higher in diaphragm fibers from bortezomib-treated CHF rats than saline. Caspase-3 activity was decreased in diaphragm homogenates from bortezomib-treated rats. CHF increased MuRF-1 and MAFbx mRNA expression in the diaphragm, and bortezomib treatment diminished this rise. The present study demonstrates that treatment with a clinically used proteasome inhibitor improves diaphragm function by restoring myosin content in CHF.

scholarly journals Development of noninvasive biomarkers of response to proteasome inhibitor therapy (ixazomib) by imaging disrupted protein homeostasis in mouse models of solid tumors

2017 ◽  
Author(s):  
Yanan Zhu ◽  
Rajiv Ramasawmy ◽  
Sean Peter Johnson ◽  
Valerie Taylor ◽  
Alasdair Gibb ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Solid Tumors ◽  
Proteasome Inhibitor ◽  
Chemical Exchange ◽  
Proteasome Inhibitors ◽  
Surrogate Marker ◽  
Standard Of Care ◽  
Proteasome Inhibition ◽  
Protein Homeostasis

AbstractWith clinically-approved proteasome inhibitors now a standard of care for multiple myeloma, and increasing interest in their use in solid tumors, methods for monitoring therapeutic response in vivo are critically required. Here, we show that tumor protein homeostasis can be noninvasively monitored, using chemical exchange (CEST) magnetic resonance imaging (MRI) as a surrogate marker for proteasome inhibition, alongside diffusion MRI and relaxometry. We show that the in vivo CEST signal associated with amides and amines increases in proportion to proteasome inhibitor dose (ixazomib) and the magnitude of therapeutic effect in colorectal cancer xenografts. Moreover, we show that SW1222 and LS174T human colorectal cancer cell lines demonstrate differing sensitivities to ixazomib, which was reflected in our MRI measurements. We also found evidence of a mild stimulation in tumor growth at low ixazomib doses. Our results therefore identify CEST MRI as a promising method for safely and noninvasively monitoring changes in tumor protein homeostasis.

Proteasome inhibition improves diaphragm function in an animal model for COPD

2011 ◽  
Vol 301 (1) ◽  
pp. L110-L116 ◽  
Author(s):  
Hieronymus van Hees ◽  
Coen Ottenheijm ◽  
Leo Ennen ◽  
Marianne Linkels ◽  
Richard Dekhuijzen ◽  
...  
Keyword(s):  
Animal Model ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Contractile Protein ◽  
Specific Force ◽  
Bortezomib Treatment ◽  
Diaphragm Function ◽  
Generating Capacity ◽  
Diaphragm Weakness

Diaphragm muscle weakness in patients with chronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality. Recent studies indicate that increased contractile protein degradation by the proteasome contributes to diaphragm weakness in patients with COPD. The aim of the present study was to investigate the effect of proteasome inhibition on diaphragm function and contractile protein concentration in an animal model for COPD. Elastase-induced emphysema in hamsters was used as an animal model for COPD; normal hamsters served as controls. Animals were either treated with the proteasome inhibitor Bortezomib (iv) or its vehicle saline. Nine months after induction of emphysema, specific force-generating capacity of diaphragm bundles was measured. Proteolytic activity of the proteasome was assayed spectrofluorometrically. Protein concentrations of proteasome, myosin, and actin were measured by means of Western blotting. Proteasome activity and concentration were significantly higher in the diaphragm of emphysematous hamsters than in normal hamsters. Bortezomib treatment reduced proteasome activity in the diaphragm of emphysematous and normal hamsters. Specific force-generating capacity and myosin concentration of the diaphragm were reduced by ∼25% in emphysematous hamsters compared with normal hamsters. Bortezomib treatment of emphysematous hamsters significantly increased diaphragm-specific force-generating capacity and completely restored myosin concentration. Actin concentration was not affected by emphysema, nor by bortezomib treatment. We conclude that treatment with a proteasome inhibitor improves contractile function of the diaphragm in emphysematous hamsters through restoration of myosin concentration. These findings implicate that the proteasome is a potential target of pharmacological intervention on diaphragm weakness in COPD.

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