Proteasome Impairment Research Articles

Early-onset impairment of the ubiquitin-proteasome system in dopaminergic neurons caused by \u03b1-synuclein

Parkinson’s disease is a progressive neurodegenerative disorder characterised by the accumulation of misfolded α-synuclein in selected brain regions, including the substantia nigra pars compacta (SNpc), where marked loss of dopaminergic neurons is also observed. Yet, the relationship between misfolded α-synuclein and neurotoxicity currently remains unclear. As the principal route for degradation of misfolded proteins in mammalian cells, the ubiquitin-proteasome system (UPS) is critical for maintenance of cellular proteostasis. Misfolded α-synuclein impairs UPS function and contributes to neuronal death in vitro. Here, we examine its effects in vivo using adeno-associated viruses to co-express A53T α-synuclein and the ubiquitinated reporter protein UbG76V-GFP in rat SNpc. We found that α-synuclein over-expression leads to early-onset catalytic impairment of the 26S proteasome with associated UPS dysfunction, preceding the onset of behavioural deficits and dopaminergic neurodegeneration. UPS failure in dopaminergic neurons was also associated with selective accumulation of α-synuclein phosphorylated at the serine 129 residue, which has previously been linked to increased neurotoxicity. Our study highlights a role for α-synuclein in disturbing proteostasis which may contribute to neurodegeneration in vivo.

Molecular Insight Into the IRE1α-Mediated Type I Interferon Response Induced by Proteasome Impairment in Myeloid Cells of the Brain.

Proteostasis is critical for cells to maintain the balance between protein synthesis, quality control, and degradation. This is particularly important for myeloid cells of the central nervous system as their immunological function relies on proper intracellular protein turnover by the ubiquitin-proteasome system. Accordingly, disruption of proteasome activity due to, e.g., loss-of-function mutations within genes encoding proteasome subunits, results in systemic autoinflammation. On the molecular level, pharmacological inhibition of proteasome results in endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR) as well as an induction of type I interferons (IFN). Nevertheless, our understanding as to whether and to which extent UPR signaling regulates type I IFN response is limited. To address this issue, we have tested the effects of proteasome dysfunction upon treatment with proteasome inhibitors in primary murine microglia and microglia-like cell line BV-2. Our data show that proteasome impairment by bortezomib is a stimulus that activates all three intracellular ER-stress transducers activation transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and inositol-requiring protein 1 alpha (IRE1α), causing a full activation of the UPR. We further demonstrate that impaired proteasome activity in microglia cells triggers an induction of IFNβ1 in an IRE1-dependent manner. An inhibition of the IRE1 endoribonuclease activity significantly attenuates TANK-binding kinase 1-mediated activation of type I IFN. Moreover, interfering with TANK-binding kinase 1 activity also compromised the expression of C/EBP homologous protein 10, thereby emphasizing a multilayered interplay between UPR and type IFN response pathway. Interestingly, the induced protein kinase R-like endoplasmic reticulum kinase-activation transcription factor 4-C/EBP homologous protein 10 and IRE1-X-box-binding protein 1 axes caused a significant upregulation of proinflammatory cytokine interleukin 6 expression that exacerbates STAT1/STAT3 signaling in cells with dysfunctional proteasomes. Altogether, these findings indicate that proteasome impairment disrupts ER homeostasis and triggers a complex interchange between ER-stress sensors and type I IFN signaling, thus inducing in myeloid cells a state of chronic inflammation.

Contribution of the Unfolded Protein Response (UPR) to the Pathogenesis of Proteasome-Associated Autoinflammatory Syndromes (PRAAS)

Type I interferonopathies cover a phenotypically heterogeneous group of rare genetic diseases including the recently described proteasome-associated autoinflammatory syndromes (PRAAS). By definition, PRAAS are caused by inherited and/or de novo loss-of-function mutations in genes encoding proteasome subunits such as PSMB8, PSMB9, PSMB7, PSMA3, or proteasome assembly factors including POMP and PSMG2, respectively. Disruption of any of these subunits results in perturbed intracellular protein homeostasis including accumulation of ubiquitinated proteins which is accompanied by a type I interferon (IFN) signature. The observation that, similarly to pathogens, proteasome dysfunctions are potent type I IFN inducers is quite unexpected and, up to now, the underlying molecular mechanisms of this process remain largely unknown. One promising candidate for triggering type I IFN under sterile conditions is the unfolded protein response (UPR) which is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) (also referred to as ER stress). The recent observation that the UPR is engaged in subjects carrying POMP mutations strongly suggests its possible implication in the cause-and-effect relationship between proteasome impairment and interferonopathy onset. The purpose of this present review is therefore to discuss the possible role of the UPR in the pathogenesis of PRAAS. We will particularly focus on pathways initiated by the four ER-membrane proteins ATF6, PERK, IRE1-α, and TCF11/Nrf1 which undergo activation under proteasome inhibition. An overview of the current understanding of the mechanisms and potential cross-talk between the UPR and inflammatory signaling casacades is provided to convey a more integrated picture of the pathophysiology of PRAAS and shed light on potential biomarkers and therapeutic targets.

Negative Regulator of Ubiquitin-Like Protein 1 modulates the autophagy–lysosomal pathway via p62 to facilitate the extracellular release of tau following proteasome impairment

Negative regulator of ubiquitin-like protein 1 (NUB1) and its longer isoform NUB1L are ubiquitin-like (UBL)/ubiquitin-associated (UBA) proteins that facilitate the targeting of proteasomal substrates, including tau, synphilin-1 and huntingtin. Previous data revealed that NUB1 also mediated a reduction in tau phosphorylation and aggregation following proteasome inhibition, suggesting a switch in NUB1 function from targeted proteasomal degradation to a role in autophagy. Here, we delineate the mechanisms of this switch and show that NUB1 interacted specifically with p62 and induced an increase in p62 levels in a manner facilitated by inhibition of the proteasome. NUB1 moreover increased autophagosomes and the recruitment of lysosomes to aggresomes following proteasome inhibition. Autophagy flux assays revealed that NUB1 affected the autophagy-lysosomal pathway primarily via the UBA domain. NUB1 localized to cytosolic inclusions with pathological forms of tau, as well as LAMP1 and p62 in the hippocampal neurons of tauopathy mice. Finally, NUB1 facilitated the extracellular release of tau following proteasome inhibition. This study thus shows that NUB1 plays a role in regulating the autophagy-lysosomal pathway when the ubiquitin proteasome system is compromised, thus contributing to the mechanisms targeting the removal of aggregation-prone proteins upon proteasomal impairment.

Proteasome Inhibition Activates Autophagy-Lysosome Pathway Associated With TFEB Dephosphorylation and Nuclear Translocation.

Ubiquitin-proteasome pathway (UPS) and autophagy-lysosome pathway (ALP) are the two major protein degradation pathways, which are critical for proteostasis. Growing evidence indicates that proteasome inhibition-induced ALP activation is an adaptive response. Transcription Factor EB (TFEB) is a master regulator of ALP. However, the characteristics of TFEB and its role in proteasome inhibition-induced ALP activation are not fully investigated. Here we reported that the half-life of TFEB is around 13.5 h in neuronal-like cells, and TFEB is degraded through proteasome pathway in both neuronal-like and non-neuronal cells. Moreover, proteasome impairment not only promotes TFEB accumulation but also facilitates its dephosphorylation and nuclear translocation. In addition, proteasome inhibition-induced TFEB accumulation, dephosphorylation and nuclear translocation significantly increases the expression of a number of TFEB downstream genes involved in ALP activation, including microtubule-associated protein 1B light chain-3 (LC3), particularly LC3-II, cathepsin D and lysosomal-associated membrane protein 1 (LAMP1). Furthermore, we demonstrated that proteasome inhibition increases autophagosome biogenesis but not impairs autophagic flux. Our study advances the understanding of features of TFEB and indicates that TFEB might be a key mediator of proteasome impairment-induced ALP activation.

Abstract 100: Dual Activation of PKA and PKG by PDE1 Inhibition Facilitates Proteasomal Degradation of Misfolded Proteins and Protects Against Proteinopathy-Based HFpEF

No current treatment is intended to target cardiac proteotoxicity or can reduce mortality of heart failure with preserved ejection fraction (HFpEF), a prevalent form of heart failure (HF). Selective degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is vital to the cell. Proteasome impairment is recently implicated in HF genesis. Activation of the cGMP-protein kinase G (PKG) or the cAMP-protein kinase A (PKA) pathways facilitates proteasome functioning. Phosphodiesterase 1 (PDE1) hydrolyzes both cyclic nucleotides and accounts for the majority of PDE activities in human myocardium. Here we report the preclinical therapeutic efficacy and a new mechanism of action of PDE1 inhibition (IC86430) for cardiac proteinopathy caused by Arg120Gly missense mutant αB-crystallin (CryAB R120G ). In mice expressing GFPdgn, an inverse reporter of UPS proteolytic activity, IC86430 treatment increased myocardial 26S proteasome activities and substantially decreased GFPdgn protein levels. Myocardial PDE1A expression was highly upregulated in CryAB R120G mice. HFpEF was detected in CryAB R120G mice at 4 months; IC86430 treatment initiated at this stage markedly attenuated HFpEF, substantially delayed mouse premature death, increased myocardial levels of Ser14-phosphorylated Rpn6 (the primary proteasome target of PKA), and reduced the steady state level of the misfolded CryAB species in these mice. In cultured cardiomyocytes, IC86430 treatment increased proteasome activities and accelerated proteasomal degradation of GFPu and CryAB R120G in a PKA- and PKG- dependent manner. We conclude that PDE1 inhibition induces PKA- and PKG-mediated promotion of proteasomal degradation of misfolded proteins in cardiomyocytes and effectively treats HFpEF caused by CryAB R120G ; hence, PDE1 inhibition represents a potentially new therapeutic strategy for HFpEF and heart disease with increased proteotoxic stress.

Cullin-4B E3 ubiquitin ligase mediates Apaf-1 ubiquitination to regulate caspase-9 activity.

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington’s disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.

FAM48A mediates compensatory autophagy induced by proteasome impairment.

Maintaining protein homeostasis is central to cell survival. The ubiquitin-proteasome system and autophagy play pivotal roles in protein quality control through protein degradation. Activities of these degradative pathways are carefully orchestrated, and autophagy is up-regulated during proteasome dysfunction for cellular homeostasis. However, the mechanism by which proteasome impairment induces compensatory autophagy has remained largely elusive. Here, we show that FAM48A mediates autophagy induction during proteasome inhibition. FAM48A is degraded by the proteasome and accumulates in cells by proteasome inhibition. Knockdown of FAM48A led to defective induction of autophagy during proteasome inhibition and accompanied by defective localization of Atg9 on recycling endosomes. Our results indicate that FAM48A is a kind of sensor that is required for compensatory autophagy induction upon proteasome impairment.

Proteasome and Autophagy-Mediated Impairment of Late Long-Term Potentiation (l-LTP) after Traumatic Brain Injury in the Somatosensory Cortex of Mice.

Traumatic brain injury (TBI) can lead to impaired cognition and memory consolidation. The acute phase (24–48 h) after TBI is often characterized by neural dysfunction in the vicinity of the lesion, but also in remote areas like the contralateral hemisphere. Protein homeostasis is crucial for synaptic long-term plasticity including the protein degradation systems, proteasome and autophagy. Still, little is known about the acute effects of TBI on synaptic long-term plasticity and protein degradation. Thus, we investigated TBI in a controlled cortical impact (CCI) model in the motor and somatosensory cortex of mice ex vivo-in vitro. Late long-term potentiation (l-LTP) was induced by theta-burst stimulation in acute brain slices after survival times of 1–2 days. Protein levels for the plasticity related protein calcium/calmodulin-dependent protein kinase II (CaMKII) was quantified by Western blots, and the protein degradation activity by enzymatical assays. We observed missing maintenance of l-LTP in the ipsilateral hemisphere, however not in the contralateral hemisphere after TBI. Protein levels of CaMKII were not changed but, interestingly, the protein degradation revealed bidirectional changes with a reduced proteasome activity and an increased autophagic flux in the ipsilateral hemisphere. Finally, LTP recordings in the presence of pharmacologically modified protein degradation systems also led to an impaired synaptic plasticity: bath-applied MG132, a proteasome inhibitor, or rapamycin, an activator of autophagy, both administered during theta burst stimulation, blocked the induction of LTP. These data indicate that alterations in protein degradation pathways likely contribute to cognitive deficits in the acute phase after TBI, which could be interesting for future approaches towards neuroprotective treatments early after traumatic brain injury.

A Bird's-Eye View of the Multiple Biochemical Mechanisms that Propel Pathology of Alzheimer's Disease: Recent Advances and Mechanistic Perspectives on How to Halt the Disease Progression Targeting Multiple Pathways.

Neurons consume the highest amount of oxygen, depend on oxidative metabolism for energy, and survive for the lifetime of an individual. Therefore, neurons are vulnerable to death caused by oxidative-stress, accumulation of damaged and dysfunctional proteins and organelles. There is an exponential increase in the number of patients diagnosed with neurodegenerative diseases such as Alzheimer’s (AD) as the number of elderly increases exponentially. Development of AD pathology is a complex phenomenon characterized by neuronal death, accumulation of extracellular amyloid-β plaques and neurofibrillary tangles, and most importantly loss of memory and cognition. These pathologies are most likely caused by mechanisms including oxidative stress, mitochondrial dysfunction/stress, accumulation of misfolded proteins, and defective organelles due to impaired proteasome and autophagy mechanisms. Currently, there are no effective treatments to halt the progression of this disease. In order to treat this complex disease with multiple biochemical pathways involved, a complex treatment regimen targeting different mechanisms should be investigated. Furthermore, as AD is a progressive disease-causing morbidity over many years, any chemo-modulator for treatment must be used over long period of time. Therefore, treatments must be safe and non-interfering with other processes. Ideally, a treatment like medicinal food or a supplement that can be taken regularly without any side effect capable of reducing oxidative stress, stabilizing mitochondria, activating autophagy or proteasome, and increasing energy levels of neurons would be the best solution. This review summarizes progress in research on different mechanisms of AD development and some of the potential therapeutic development strategies targeting the aforementioned pathologies.

Effects of proteasome inhibitors on cytokines, metalloproteinases and their inhibitors and collagen type-I expression in periprosthetic tissues and fibroblasts from loose arthroplasty endoprostheses

ABSTRACTObjective: Aseptic loosening is a major problem in total joint replacement. Implant wear debris provokes a foreign body host response and activates cells to produce a variety of mediators and ROS, leading to periprosthetic osteolysis. Elevated ROS levels can harm proteasome function. Proteasome inhibitors have been reported to alter the secretory profile of cells involved in inflammation and also to induce ROS production. In this work, we aimed to document the effects of proteasome inhibitors MG-132 and Epoxomicin, on the production of factors involved in aseptic loosening, in periprosthetic tissues and fibroblasts, and investigate the role of proteasome impairment in periprosthetic osteolysis.Materials and methods: IL-6 levels in tissue cultures were determined by sandwich ELISA. MMP-1, -3, -13, -14 and TIMP-1 levels in tissue or cell cultures were determined by indirect ELISA. Results for MMP-1 and TIMP-1 in tissue cultures were confirmed by Western blotting. MMP-2 and MMP-9 levels were determined by gelatin zymography. Gene expression of IL-6, MMP-1,-3,-14, TIMP-1 and collagen type-I was determined by RT-PCR.Results: Results show that proteasome inhibition induces the expression of ΜΜΡ-1, -2, -3, -9 and suppresses that of IL-6, MMP-14, -13, TIMP-1 and collagen type I, enhancing the collagenolytic and gelatinolytic activity already present in periprosthetic tissues, as documented in various studies.Conclusions: These findings suggest that proteasome impairment could be a contributing factor to aseptic loosening. Protection and enhancement of proteasome efficacy could thus be considered as an alternative strategy toward disease treatment.

An SOD1 deficiency aggravates proteasome inhibitor bortezomib-induced testicular damage in mice

Proteasomes play a key role in maintaining cellular homeostasis by the proteolytic removal of proteins, including ubiquitinated proteins and/or oxidatively-damaged proteins. The proteasome inhibitor bortezomib (BTZ) has been reported to exert testicular toxicity in mice. In the current study, we treated SOD1-knockout (KO) mice with BTZ and investigated the issue of whether oxidative stress is involved in the development of testicular toxicity. The BTZ treatment significantly increased superoxide production and cell death in the testes of SOD1-KO mice compared to wild-type (WT) mice. We also found that high levels of both ubiquitinated proteins and p62 accumulated and underwent aggregation in the seminiferous tubules of BTZ-injected SOD1-KO mice. Furthermore, the proteolytic activities of proteasomes were significantly decreased in the testes of BTZ-injected SOD1-KO mice compared to their WT counterparts. These results suggest that a combination of oxidative stress caused by an SOD1 deficiency and proteasome inhibition by BTZ accelerates the impairment of proteasomes, which results in severe testicular damage in SOD1-KO mice.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) loss causes neurodegeneration by altering protein turnover in the first postnatal weeks

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

Altered unfolded protein response and proteasome impairment in pseudoexfoliation pathogenesis

Pseudoexfoliation (PEX), an ocular disorder involving deposition of proteinaceous fibrils on the surface of anterior eye tissues, is a major contributing factor to worldwide glaucoma. Excessive production and accumulation of fibrillar materials in PEX could be an indication of proteostasis imbalance. This study aims at investigating the differential expression of various genes involved in unfolded protein response and ubiquitin proteasome pathway in pseudoexfoliation (PEX) patients compared to non-PEX controls using lens capsule tissue as the study material. The custom RT2 Profiler PCR array was used to identify a set of stress-related candidate genes that were differentially expressed in PEX. The expression of the highly deregulated genes was validated by qRT-PCR and subsequently their protein expression was checked through immunoblotting and immunostaining. Proteasome-Glo based assay and TUNEL assay were employed to detect specific proteasomal activity and apoptotic activity, respectively in the study subjects. Increased ER stress markers, Synoviolin1, Eukaryotic initiation factor 2-alpha kinase 3, DnaJ (Hsp40) homolog, subfamily B, member 11, Caspase 12, Heat shock 70 kDa protein 5, Heat shock 60 kDa protein 1 and Calnexin were observed in the lens capsule of PEX individuals compared to age-matched controls. On the other hand, increased ubiquitin B mRNA expression followed by significant downregulation of proteasome subunits; 26 S proteasome non-ATPase regulatory subunit 1, and proteasome subunit alpha-type 5 was found in pseudoexfoliation syndrome (PEXS) individuals. Decrease in chymotrypsin-like proteasome activity and increased apoptosis were also observed in PEX subjects.The present findings provide evidence for alterations in endoplasmic reticulum–related stress response and ubiquitin proteasome function in lens capsule of PEX individuals. Altogether, our study has identified deregulated expression of candidate genes in ER-UPR pathway and implicates proteasome impairment as a causative factor in PEX pathogenesis.

Transcriptional regulation of the 26S proteasome by Nrf1.

The 26S proteasome is a large protease complex that selectively degrades ubiquitinated proteins. It comprises 33 distinct subunits, each of which differ in function and structure, and which cannot be substituted by the other subunits. Owing to its complicated structure, the biogenesis of the 26S proteasome is elaborately regulated at the transcription, translation, and molecular assembly levels. Recent studies revealed that Nrf1 (NFE2L1) is a transcription factor that upregulates the expression of all the proteasome subunit genes in a concerted manner, especially during proteasome impairment in mammalian cells. In this review, we summarize current knowledge regarding the transcriptional regulation of the proteasome and recent findings concerning the regulation of Nrf1 transcription activity.

Functional proteasome complex is required for turnover of islet amyloid polypeptide in pancreatic β-cells

Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed β-cells evoked by chronic hyperglycemia. Moderate inhibition (10-35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed β-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma β-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in β-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.

Impaired Proteasomal Function in Human Osteoarthritic Chondrocytes Can Contribute to Decreased Levels of SOX9 and Aggrecan.

Osteoarthritis (OA) chondrocytes exhibit impairment of autophagy, one arm of the proteostasis network that coordinates proteome and organelle quality control and degradation. Deficient proteostasis impacts differentiation and viability, and inflammatory processes in aging and disease. The present study was undertaken to assess ubiquitin proteasome system proteasomal function in OA chondrocytes. We evaluated human knee cartilage by immunohistochemistry, and assessed proteasomal function, levels of proteasomal core subunits and chaperones, and autophagy in cultured chondrocytes. Assays included Western blotting, quantitative reverse transcription-polymerase chain reaction, proteasomal protease activity assessment, and cell immunofluorescence analysis. Human knee OA cartilage exhibited polyubiquitin accumulation, with increased ubiquitin K48-linked polyubiquitinated proteins insitu, suggesting proteasomal impairment. Cultured OA chondrocytes demonstrated accumulation of K48 polyubiquitinated proteins, significantly reduced 20S proteasome core protease activity, and decreased levels of phosphorylated FOXO4 and proteasome 26S subunit, non-ATPase 11 (PSMD11), a FOXO4-inducible promoter of proteasomal activation. Levels of proteasome subunit β type 3 (PSMB3), PSMB5, PSMB6, and proteasome assembly chaperone 1 were not decreased in OA chondrocytes. In normal chondrocytes, PSMD11 small interfering RNA knockdown stimulated certain autophagy machinery elements, increased extracellular nitric oxide (NO) levels, and reduced chondrocytic master transcription factor SOX9 protein and messenger RNA (mRNA) and aggrecan (AGC1) mRNA. PSMD11 gain-of- function by transfection increased proteasomal function, increased levels of SOX9-induced AGC1 mRNA, stimulated elements of the autophagic machinery, and inhibited extracellular levels of interleukin-1-induced NO and matrix metalloproteinase 13 in OA chondrocytes. Deficient PSMD11, associated with reduced phosphorylated FOXO4, promotes impaired proteasomal function in OA chondrocytes, dysregulation of chondrocytic homeostasis, and decreased levels of SOX9 mRNA, SOX9 protein, and AGC1 mRNA. Chondrocyte proteasomal impairment may be a therapeutic target for OA.

Inhibition of HIF1α-Dependent Upregulation of Phospho-l-Plastin Resensitizes Multiple Myeloma Cells to Frontline Therapy.

The introduction of novel frontline agents in multiple myeloma (MM), like immunomodulatory drugs and proteasome inhibitors, has improved the overall survival of patients. Yet, MM is still not curable, and drug resistance (DR) remains the main challenge. To improve the understanding of DR in MM, we established a resistant cell line (MOLP8/R). The exploration of DR mechanisms yielded an overexpression of HIF1α, due to impaired proteasome activity of MOLP8/R. We show that MOLP8/R, like other tumor cells, overexpressing HIF1α, have an increased resistance to the immune system. By exploring the main target genes regulated by HIF1α, we could not show an overexpression of these targets in MOLP8/R. We, however, show that MOLP8/R cells display a very high overexpression of LCP1 gene (l-Plastin) controlled by HIF1α, and that this overexpression also exists in MM patient samples. The l-Plastin activity is controlled by its phosphorylation in Ser5. We further show that the inhibition of l-Plastin phosphorylation restores the sensitivity of MOLP8/R to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). Our results reveal a new target gene of DR, controlled by HIF1α.

Protein kinase C activator bryostatin‐1 modulates proteasome function

Proteasome activity in ubiquitin-proteasome pathway plays a pivotal role in degradation and clearance of aggregated, oxidized, damaged, and misfolded unwanted proteins to control protein homeostasis or proteostasis. Proteasome activity decreases with cellular senescence, aging, and age-related diseases. Therefore, enhancement of impaired proteasome function by molecular biological and/or pharmacological intervention is an active area of research. Bryostatin-1, a naturally occurring macrocyclic lactone, activates PKC isozymes (specifically, -α and -ϵ) at sub-nanomolar concentrations, but downregulates at higher concentrations. Here, we present bryostatin-1 increased chymotrypsin-like proteasome activity of 20S assembly at sub-nanomolar to nanomolar concentrations (0.3-30 nM). However, proteasome activity decreased at a micromolar concentration of bryostatin-1 (AG08044 cultured skin: P < 0.005; differentiated SH-SY5Y cells: P < 0.02). Modulation of proteasome function by bryostatin-1 was studied in six dermal fibroblast primary cell lines developed both from freshly taken biopsies from healthy donors (n = 2) and obtained from well-characterized cell repositories (n = 4; without any diseases). Bryostatin-1 enhanced proteasome activity in cultured skin fibroblasts obtained from banked and freshly isolated skin fibroblasts from skin biopsies at the sub-nanomolar concentration (P < 0.015). Modulation of proteasome function by bryostatin-1 was confirmed in neuron-like differentiated SH-SY5Y cells. Direct additions of bryostatin-1 into cell lysates prepared from neuron-like differentiated SH-SY5Y, Jurkat cells, and cultured skin fibroblasts were unable to increase proteasome activity indicating that bryostatin-1 can only modulate proteasome activity when added to live cell culture systems. Standard PKC inhibitors blocked bryostatin-1 induced proteasome activity modulation suggesting that enhancement of proteasome activity was mediated by PKC modulation.

Gender Differences on the Effects of Ibuprofen on Proteasome Function in Mice Heart

Introduction and objectivesIbuprofen, a cyclo‐oxygenase inhibitor, is a commonly used non‐steroidal anti‐inflammatory (NSAID) agent used in the treatment of pain, fever and inflammation. The long‐term use of ibuprofen is associated with increased risks for cardiovascular events and stroke. The effect of ibuprofen on cardiac function as well as if gender differences exist with respect to ibuprofen's effect on cardiac function are not well understood. Accordingly, the aim of this study was to explore the effects of ibuprofen on protein homeostasis in mice cardiac tissue and to determine if ibuprofen affects male and female cardiac proteasome activity in a similar manner.Method/ResultsAged matched male and female C57BL/6J mice were randomly assigned to two groups: (A) control (B) group treated with ibuprofen. Mice were treated with ibuprofen (100 mg/kg) dissolved in drinking water for 6 weeks.Since impaired proteasome function is associated with cardiovascular disorders, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like proteasome activities were measured using bortezomib as a specific inhibitor of proteasome in cardiac tissue of both male and female mice. In female mice chronic treatment with ibuprofen showed decreased proteasome function as decreased β1, β2, and β5 activity was observed in ibuprofen treated female animals. However, proteasome activity was not affected in male animals treated with ibuprofen. Excess accumulation of misfolded and oxidized proteins alters proteasome function, therefore levels of oxidized proteins in heart lysates was measured by oxyblot method using dinitrophenylhydrazine (DNPH). The levels of oxidized proteins increased in only female animals treated with ibuprofen. The levels of polyubiquitinated proteins was similar in male and female ibuprofen treated hearts compared to control hearts. An increase in free ubiquitin levels was only observed in the hearts of female animals treated with ibuprofen when compared to control animals and ibuprofen treated male animals.ConclusionOur findings suggest, for the first time, that gender differences on proteasome function occur in the heart of ibuprofen treated animals. These data show that chronic treatment of ibuprofen decreased proteasome activities in females as well as increased the levels of oxidized proteins and free ubiquitin levels in only ibuprofen treated female mice hearts.Support or Funding InformationAmerican Heart Association Award #16GRNT31350040This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.