NFE2L1-mediated proteasome function protects from ferroptosis

Ferroptosis continues to emerge as a novel modality of cell death with important therapeutic implications for a variety of diseases, most notably cancer and degenerative diseases. While susceptibility, initiation, and execution of ferroptosis have been linked to reprogramming of cellular lipid metabolism, imbalances in iron-redox homeostasis, and aberrant mitochondrial respiration, the detailed mechanisms of ferroptosis are still insufficiently well understood. Here we show that diminished proteasome function is a new mechanistic feature of ferroptosis. The transcription factor nuclear factor erythroid-2, like-1 (NFE2L1) protects from ferroptosis by sustaining proteasomal activity. In cellular systems, loss of NFE2L1 reduced cellular viability after the induction of both chemically and genetically induced ferroptosis, which was linked to the regulation of proteasomal activity under these conditions. Importantly, this was reproduced in a Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) patient-derived cell line carrying mutated glutathione peroxidase-4 (GPX4), a critical regulator of ferroptosis. Also, reduced proteasomal activity was associated with ferroptosis in Gpx4-deficient mice. In a mouse model for genetic Nfe2l1 deficiency, we observed brown adipose tissue (BAT) involution, hyperubiquitination of ferroptosis regulators, including the GPX4 pathway, and other hallmarks of ferroptosis. Our data highlight the relevance of the NFE2L1-proteasome pathway in ferroptosis. Manipulation of NFE2L1 activity might enhance ferroptosis-inducing cancer therapies as well as protect from aberrant ferroptosis in neurodegeneration, general metabolism, and beyond.

Osteocrin, a novel myokine, prevents diabetic cardiomyopathy via restoring proteasomal activity

Proteasomal activity is compromised in diabetic hearts that contributes to proteotoxic stresses and cardiac dysfunction. Osteocrin (OSTN) acts as a novel exercise-responsive myokine and is implicated in various cardiac diseases. Herein, we aim to investigate the role and underlying molecular basis of OSTN in diabetic cardiomyopathy (DCM). Mice received a single intravenous injection of the cardiotrophic adeno-associated virus serotype 9 to overexpress OSTN in the heart and then were exposed to intraperitoneal injections of streptozotocin (STZ, 50 mg/kg) for consecutive 5 days to generate diabetic models. Neonatal rat cardiomyocytes were isolated and stimulated with high glucose to verify the role of OSTN in vitro. OSTN expression was reduced by protein kinase B/forkhead box O1 dephosphorylation in diabetic hearts, while its overexpression significantly attenuated cardiac injury and dysfunction in mice with STZ treatment. Besides, OSTN incubation prevented, whereas OSTN silence aggravated cardiomyocyte apoptosis and injury upon hyperglycemic stimulation in vitro. Mechanistically, OSTN treatment restored protein kinase G (PKG)-dependent proteasomal function, and PKG or proteasome inhibition abrogated the protective effects of OSTN in vivo and in vitro. Furthermore, OSTN replenishment was sufficient to prevent the progression of pre-established DCM and had synergistic cardioprotection with sildenafil. OSTN protects against DCM via restoring PKG-dependent proteasomal activity and it is a promising therapeutic target to treat DCM.

Role of Proteasomes in Inflammation

The ubiquitin–proteasome system (UPS) is involved in multiple cellular functions including the regulation of protein homeostasis, major histocompatibility (MHC) class I antigen processing, cell cycle proliferation and signaling. In humans, proteasome loss-of-function mutations result in autoinflammation dominated by a prominent type I interferon (IFN) gene signature. These genomic alterations typically cause the development of proteasome-associated autoinflammatory syndromes (PRAAS) by impairing proteasome activity and perturbing protein homeostasis. However, an abnormal increased proteasomal activity can also be found in other human inflammatory diseases. In this review, we cast a light on the different clinical aspects of proteasomal activity in human disease and summarize the currently studied therapeutic approaches.

Sensitivity of Osteosarcoma Cells to Concentration-Dependent Bioactivities of Lipid Peroxidation Product 4-Hydroxynonenal Depend on Their Level of Differentiation

4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological effects. Normal and malignant cells of the same origin express different sensitivity to HNE. We used human osteosarcoma cells (HOS) in different stages of differentiation in vitro, showing differences in mitosis, DNA synthesis, and alkaline phosphatase (ALP) staining. Differentiated HOS cells showed decreased proliferation (3H-thymidine incorporation), decreased viability (thiazolyl blue tetrazolium bromide-MTT), and increased apoptosis and necrosis (nuclear morphology by staining with 4′,6-diamidino-2-phenylindole-DAPI). Differentiated HOS also had less expressed c-MYC, but the same amount of c-FOS (immunocytochemistry). When exposed to HNE, differentiated HOS produced more reactive oxygen species (ROS) in comparison with undifferentiated HOS. To clarify this, we measured HNE metabolism by an HPLC method, total glutathione (GSH), oxidized GSH (ox GSH), glutathione transferase activity (GST), proteasomal activity by enzymatic methods, HNE-protein adducts by genuine ELISA and fatty acid composition by GC-MS in these cell cultures. Differentiated HOS cells had less GSH, lower HNE metabolism, increased formation of HNE-protein adducts, and lower proteasomal activity, in comparison to undifferentiated counterpart cells, while GST and oxGSH were the same. Fatty acids analyzed by GC-MS showed that there is an increase in C20:3 in differentiated HOS while the amount of C20:4 remained the same. The results showed that the cellular machinery responsible for protection against toxicity of HNE was less efficient in differentiated HOS cells. Moreover, differentiated HOS cells contained more C20:3 fatty acid, which might make them more sensitive to free radical-initiated oxidative chain reactions and more vulnerable to the effects of reactive aldehydes such as HNE. We propose that HNE might act as natural promotor of decay of malignant (osteosarcoma) cells in case of their differentiation associated with alteration of the lipid metabolism.

The Nrf2/PGC1α Pathway Regulates Antioxidant and Proteasomal Activity to Alter Cisplatin Sensitivity in Ovarian Cancer

Drug resistance remains a barrier in the clinical treatment of ovarian cancer. Proteasomal and antioxidant activities play important roles in tumor drug resistance, and increasing evidence suggests the existence of an interaction between antioxidant and proteasomal activities. However, the mechanism of the synergistic effects of proteasomal activity and antioxidation on tumor drug resistance is not completely clear. In this study, we compared two ovarian cancer cells, A2780 and SKOV3 cells. Among them, SKOV3 cell is a human clear cell carcinoma cell line that is resistant to platinum. We found that compared with the findings in A2780 cells, SKOV3 cells were less sensitive to both proteasomal inhibitor and cisplatin. Proteasomal inhibition enhanced the sensitivity of A2780 cells, but not SKOV3 cells, to cisplatin. Notably, the Nrf2-mediated antioxidant pathway was identified as a resistance mechanism in proteasome inhibitor-resistant cells, but this was not the only factor identified in our research. In SKOV3 cells, PGC1α regulated the antioxidant activity of Nrf2 by increasing the phosphorylation of GSK3β, and in turn, Nrf2 regulated the transcriptional activity of PGC1α. Thus, Nrf2 and PGC1α synergistically participate in the regulation of proteasomal activity. Furthermore, the Nrf2/PGC1α pathway participated in the regulation of mitochondrial function and homeostasis, further regulating proteasomal activity in SKOV3 cells. Therefore, exploring the roles of PGC1α and Nrf2 in the regulation of proteasomal activity by antioxidant and mitochondrial functions may provide new avenues for reversing drug resistance in ovarian cancer.

Investigating the impact of aging and hyperlipidaemia on metformin cardioprotection against carfilzomib-induced cardiotoxicity in vivo

Background Carfilzomib (Cfz) is an irreversible proteasome inhibitor, indicated for the treatment of Multiple Myeloma (MM); however, is associated with cardiotoxicity in humans. Metformin (Met) has been suggested as a prophylactic therapy against Cfz-induced cardiotoxicity in mice. Purpose Considering that MM is an elderly disease, manifested in presence of cardiovascular comorbidities, we investigated the cardioprotective effect of Met against Cfz-cardiomyopathy on the age- or hyperlipidaemia-burdened myocardium in vivo. Methods Aging model: Male C57BL/6 mice, 15-month old, were randomized into: 1. Control (N/S 0.9%), 2. Met (140 mg/kg, po), 3. Cfz (8mg/kg, ip) and 4. Cfz+Met (8 and 140 mg/kg respectively) (n=5–7/group). Hyperlipidaemic model: ApoE−/− male mice, 20-weeks old were randomized into: 1. Control (N/S 0.9%), 2. Met (140 mg/kg, po), 3. Cfz (8mg/kg, ip) and 4. Cfz+Met (8 and 140 mg/kg respectively) (n=6/group). Cfz was administered every 48 hours and Met every 24 hours for 6 days. At baseline and at the end of treatments mice underwent echocardiography. Proteasomal activity and molecular signaling mechanisms were assessed in myocardial tissue and in peripheral blood mononuclear cells (PBMCs). Results Aging model: Proteasome activity was significantly reduced in myocardial tissue of aged vs young mice (P<0.05). Cfz-treated mice showed an additional decrease in proteasomal activity compared to Control (P<0.05) and Met (P<0.01) groups, in heart and PBMCs. A significant fractional shortening (FS%) reduction was observed in Cfz group vs controls at day 6 (29.90±1.7 vs 37.60±0.2, respectively, P<0.05) while Met in both Met and Cfz+Met groups maintained FS% vs controls (34.88±1.6, 33.20±0.6 vs 37.60±0.2, respectively, P=NS). By investigating molecular signaling pathways involved in cardioprotection, we observed that Cfz led to a decrease of Akt and AMPKα phosphorylation (P<0.05 vs Control), whereas Cfz+Met co-administration restored Akt and AMPKα phosphorylation and upregulated t-eNOS and t-Akt (P<0.05 vs Control). Hyperlipidaemic model: In ApoE−/− mice, no significant reduction of myocardial proteasomal activity was observed, whilst PBMCs proteasomal activity was decreased compared to controls (P<0.05). Cfz treated ApoE−/− mice exhibited a reduction of myocardial (P<0.01 in Cfz and P<0.001 in Cfz+Met) and PBMCs' proteasome activity (P<0.001) vs controls. Interestingly, Cfz, Met as well as the combination led to a decrease in FS% versus controls (44.00±1.2, 42.33±0.92, 42.33±1.0 vs 47.50±0.72 respectively, P<0.05). Conclusions Our results demonstrate that Met retains its prophylactic potency against Cfz-induced cardiotoxicity in aged mice and activates cardioprotective signaling molecules. In the ApoE−/− mice, Met failed to exert cardioprotection against Cfz induced cardiomyopathy. Therefore, the monitoring of the cardiovascular burden seems to be of outmost importance in managing Cfz cardiac adverse effects. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Amgen

Aggresomal sequestration and STUB1-mediated ubiquitylation during mammalian proteaphagy of inhibited proteasomes

The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.

Age-Related Maintenance of the Autophagy-Lysosomal System Is Dependent on Skeletal Muscle Type

The skeletal muscle plays an important role in maintaining whole-body mechanics, metabolic homeostasis, and interorgan crosstalk. However, during aging, functional and structural changes such as fiber integrity loss and atrophy can occur across different species. A commonly observed hallmark of aged skeletal muscle is the accumulation of oxidatively modified proteins and protein aggregates which point to an imbalance in proteostasis systems such as degradation machineries. Recently, we showed that the ubiquitin-proteasomal system was impaired. Specifically, the proteasomal activity, which was declining in aged M. soleus (SOL) and M. extensor digitorum longus (EDL). Therefore, in order to understand whether another proteolytic system would compensate the decline in proteasomal activity, we aimed to investigate age-related changes in the autophagy-lysosomal system (ALS) in SOL, mostly consisting of slow-twitch fibers, and EDL, mainly composed of fast-twitch fibers, from young (4 months) and old (25 months) C57BL/6JRj mice. Here, we focused on changes in the content of modified proteins and the ALS. Our results show that aged SOL and EDL display high levels of protein modifications, particularly in old SOL. While autophagy machinery appears to be functional, lysosomal activity declines gradually in aged SOL. In contrast, in old EDL, the ALS seems to be affected, demonstrated by an increased level of key autophagy-related proteins, which are known to accumulate when their delivery or degradation is impaired. In fact, lysosomal activity was significantly decreased in old EDL. Results presented herein suggest that the ALS can compensate the high levels of modified proteins in the more oxidative muscle, SOL, while EDL seems to be more prone to ALS age-related alterations.