Proteasome Impairment Research Articles

Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice.

A reduction in proteasome activity, loss of synapses and increased neuroinflammation in the brain are hallmarks of aging and many neurodegenerative disorders, including Alzheimer disease (AD); however, whether proteasome dysfunction is causative to neuroinflammation remains less understood. In this study, we investigated the impact of 26S proteasome deficiency on neuroinflammation in the Psmc1 knockout (KO) mice deficient in a 19S proteasome subunit limited to the forebrain region. Our results revealed that impaired 26S proteasome led to reduced learning and memory capability and overt neuroinflammation in the synapses of the Psmc1 KO brain at eight weeks of age. Moreover, pronounced neuroinflammation was also found in the whole brain cortex, which was confirmed by increased levels of several key immune response-related proteins, including Stat1, Trem2 and NF-κB, and by activation of astrocytes and microglia in the KO brain. To validate NF-κB mediating neuroinflammation, we administered a selective NF-κB inhibitor to the KO animals at 5 weeks of age for three weeks, and then, animal behaviors and neuroinflammation were assessed when they reached eight weeks of age. Following the treatment, the KO mice exhibited improved behaviors and reduced neuroinflammation compared to the control animals. These data indicate that impaired 26S proteasome causes AD-like cognitive deficiency and induces neuroinflammation mediated largely by NF-κB. These results may aid development of effective therapeutics and better understanding of the pathogenesis of AD and many other neurodegenerative disorders where impaired proteasome is consistently coupled with neuroinflammation.

SOX17 expression in ovarian clear cell carcinoma

Recent studies have revealed that the Sry-related HMG box gene 17 (SOX17) plays an important role in ovarian carcinogenesis. Unlike other types of ovarian cancer, ovarian clear cell carcinoma (OCCC) has a distinct pathobiological phenotype, often harboring an AT-rich interaction domain 1 A (ARID1A) mutation. In the present study, to determine the SOX17 in OCCC cells, we immunohistochemically examined SOX17 expression in 47 whole-tissue specimens of OCCC. Although not statistically significant, SOX17-high immunoreactivity tended to be related to unfavorable patient outcomes. We also aimed to determine the relationship of SOX17 with ARID1A. Double immunofluorescence staining demonstrated that SOX17 immunoreactivity was not associated with ARID1A immunoreactivity. Immunoblotting revealed that SOX17 was abundantly expressed in cultured OVISE and RMG-V OCCC cells, but not in OVTOKO OCCC cells. Polyubiquitinated bands of SOX17 were observed in MG132 treated OVTOKO, but not in OVISE or RMG-V OCCC cells. Notably, si-RNA-mediated knockdown of a deubiquitinase enzyme, ubiquitin C-terminal hydrolase L1, increased polyubiquitination followed by proteasome degradation of SOX17 in OVISE. These findings indicate that SOX17 is not uniformly and heterogeneously expressed in OCCCs, independent of ARID1A deficiency. Impaired ubiquitin-mediated proteasome degradation may stabilize SOX17 in some OCCC cells.

β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits.

Alzheimer's Disease (AD) is the leading cause of dementia globally, affecting around 50 million people and marked by cognitive decline and the accumulation of β-amyloid plaques and hyperphosphorylated tau. The limited treatment options and numerous failed clinical trials targeting β-amyloid (Aβ) highlight the need for novel approaches. Lowered proteasome activity is a consistent feature in AD, particularly in the hippocampus. Impaired proteasome function in AD is hypothesized to stem from direct inhibition by β-amyloid or hyperphosphorylated tau, disrupting critical neuronal processes such as memory formation and synaptic plasticity. This study tests the hypothesis that AD related deficits are driven in part by impaired proteasome function as a consequence of inhibition by Aβ. We evaluated how proteasome function is modulated by Aβ and the capacity of two proteasome-activating compounds, TAT1-8,9-TOD and TAT1-DEN to rescue Aβ-induced impairment in vitro, as well as survival deficits in cell culture and Aβ-induced cognitive deficits in Drosophila and mouse models. Our study demonstrates that oligomeric β-amyloid binds to the 20S proteasome and impairs its activity and conformational stability. The oligomers also destabilize the 26S proteasome to release the free 20S proteasome. Treatment with proteasome activators TAT1-8,9TOD and TAT1-DEN rescue the 20S proteasome function and reduces cell death caused by Aβ42 toxicity in SK-N-SH cells. In Drosophila models overexpressing Aβ42, oral administration of proteasome agonists delayed mortality and restored cognitive function. Chronic treatment with TAT1-DEN protected against deficits in working memory caused by Aβ42 in mice and in hAPP(J20) mice with established deficits, acute TAT1-DEN treatment significantly improved spatial learning, with treated mice performing comparably to controls. Aβ has dual impacts on 20S and 26S proteasome function and stability. Proteasome activation using TAT1-8,9TOD and TAT1-DEN shows promise in mitigating AD-like deficits by protecting against amyloid toxicity and enhancing proteasome function. These findings suggest that targeting proteasome activity could be a viable therapeutic approach for AD, warranting further investigation into the broader impacts of proteasome modulation on AD pathology.

DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment

Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment.

Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

PSMD11 loss-of-function variants correlate with a neurobehavioral phenotype, obesity, and increased interferon response

Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, we have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. Our findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Our investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health.

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

Abstract 4316: Investigation the role of DDI2 in autophagy

Abstract Background: The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are critical intracellular protein degradation pathways. In response to proteasome dysfunction, cells initiate compensatory autophagy to manage proteotoxic stress, which impacts cancer therapy efficacy. Despite current insights, the exact molecular mechanisms underlying the induction of autophagy due to impaired proteasome function remain unclear. The transcription factor nuclear factor erythroid-2-related factor 1 (NRF1) is the master regulator of proteasome subunit genes, which is activated by the protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome inhibition. DDI2 also acts as an ubiquitin shuttling factor, and its depletion causes the accumulation of highly ubiquitinated proteins, resulting in increased sensitivity to proteasome inhibition in cancer cells. However, cancer cells might trigger compensatory autophagy in response to DDI2 depletion to degrade ubiquitinated protein aggregates. Here, we demonstrated that DDI2 depletion plays a role in autophagy induction. Methods: A panel of cell lines of different origins (EW16 and ES1 human Ewing sarcoma, MIA PaCa-2 human pancreatic cancer, and NIH-3T3 murine fibroblasts) either control or DDI2-deficient were examined for autophagy activity following treatment with the autophagy inhibitor chloroquine (CQ). Through total proteome analysis, we identified an increase in cellular communication network factor 1 (CCN1) protein levels in DDI2-depleted cells, which plays a role in autophagy induction. To determine whether CCN1 is required and sufficient to induce autophagy, we applied small interfering RNA (siRNA), and viral constructs to knockdown and overexpress CCN1. Results: The autophagic flux measurements in four different DDI2-deficient cell lines revealed elevated LC3B-II protein levels, a known autophagy marker, following CQ treatment compared to their control. Western blotting analysis confirmed increased CCN1 protein levels in the same cell lines. The autophagy induction observed in DDI2-deficient cells was diminished by CCN1-siRNA, demonstrating that CCN1 may be required for autophagy induction. Furthermore, overexpressing CCN1 in wild-type cells resulted in increased LC3B-II protein levels, indicating that CCN1 alone is sufficient to induce autophagy. Conclusion: Targeting autophagy is a promising therapeutic strategy to improve the effects of anti-cancer therapies. We demonstrated that upon depletion of ubiquitin shuttle factor DDI2, secretory protein CCN1 accumulates leading to the subsequent induction of autophagy. These findings improve our understanding of the molecular basis governing DDI2’s role in crosstalk between protein degradation pathways. Targeting DDI2 in combination with autophagy inhibition could further potentiate these drugs as anti-cancer therapies. Citation Format: Nayyerehalsadat Hosseini, Holly A. Byers, Senthil K. Radhakrishnan. Investigation the role of DDI2 in autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4316.

Decreased proteasome function increases oxidative stress in the early stage of pressure ulcer development

The aging process in the elderly results in heightened skin fragility associated with various disorders, including pressure ulcers (PUs). Despite the high incidence of PUs in the elderly population, there is a limited body of research specifically examining the impact of aging on the development of pressure ulcers. Therefore, investigating age-related physiological abnormalities is essential to elucidate the pathogenesis of PUs. Ischemia-reperfusion (I/R) injury and the subsequent oxidative stress caused by reactive oxygen species (ROS) play essential roles in the early stage of PUs. In this study, we used a mouse model of proteasomal dysfunction with an age-related phenotype to examine the role of proteasome activity in cutaneous I/R injury in vivo. Decreased proteasome function did not affect the expression of inflammatory cytokines and adhesion molecules in the I/R area in transgenic mice; however, proteasome inhibition increased oxidative stress that was not attenuated by activation of the oxidative stress response mediated by NF-E2-related factor 2 (Nrf2). In dermal fibroblasts (FCs) subjected to hypoxia-reoxygenation (H/R), proteasome inhibition induced oxidative stress and ROS production, and Nrf2 activation did not adequately upregulate antioxidant enzyme expression, possibly leading to antioxidant/oxidant imbalance. The free radical scavenger edaravone had protective effects against I/R injury in vivo and decreased oxidative stress in FCs treated with a proteasome inhibitor and subjected to H/R in vitro. The results suggest that the age-related decline in proteasome activity promotes cutaneous I/R injury-induced oxidative stress, and free radical scavengers may exert protective effects by preventing oxidative stress in the early stage of PUs.

High methionine diet mediated oxidative stress and proteasome impairment causes toxicity in liver

Methionine (Met) rich diet inducing oxidative stress is reported to alter many organs. Proteasome as a regulator of oxidative stress can be targeted. This study was performed to investigate if excessive methionine supplementation causes hepatotoxicity related to proteasome dysfunction under endogenous oxidative stress in rats. Male Wistar albino rats (n = 16) were divided into controls and treated groups. The treated rats (n = 08) received orally l-methionine (1 g/kg/day) for 21 days. Total homocysteine (tHcy), total oxidant status (TOS), total antioxidant status (TAS), hepatic enzymes levels: aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), with total bilirubin (TBil), albumin (Alb), and C-reactive protein (CRP) were determined in plasma by biochemical assays. Liver supernatants were used for malondialdehyde (MDA), protein carbonyls (PC), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), 20S proteasome activities and their subunits expression, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) evaluation by appropriate methods and light microscopy for liver histological examination. Methionine treatment increased homocysteine, TOS, oxidative stress index (OSI), MDA and PC but decreased TAS, GSH, CAT, SOD, GPx with the 20S proteasome activities and their β subunits expression. Liver proteins: AST, ALT, LDH, ALP, TBil and CRP were increased but Alb was decreased. Liver histology was also altered. An increase in liver TNF-α and IL-6 levels were observed. These findings indicated that methionine supplementation associated oxidative stress and proteasome dysfunction, caused hepatotoxicity and inflammation in rat. Further investigations should be to better understand the relation between methionine, oxidative stress, proteasome, and liver injuries.

Decreased Proteasomal Function Exacerbates Endoplasmic Reticulum Stress-Induced Chronic Inflammation in Obese Adipose Tissue

Low-grade chronic inflammation contributes to both aging and the pathogenesis of age-related diseases. White adipose tissue (WAT) in obese individuals exhibits chronic inflammation, which is associated with obesity-related disorders. Aging exacerbates obesity-related inflammation in WAT; however, the molecular mechanisms underlying chronic inflammation and its exacerbation by aging remain unclear. Age-related decline in activity of the proteasome, a multi-subunit proteolytic complex, has been implicated in age-related diseases. This study employed a mouse model with decreased proteasomal function that exhibits age-related phenotypes to investigate the impact of adipocyte senescence on WAT inflammation. Transgenic mice expressing proteasomal subunit β5t with weak chymotrypsin-like activity suffer from reduced lifespan and develop age-related phenotypes. Mice fed with a high-fat diet (HFD) and suffering proteasomal dysfunction exhibited increased WAT inflammation, increased infiltration of pro-inflammatory M1-like macrophages, and increased pro-inflammatory adipocytokines like monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and tumor necrosis factor αwhich are all associated with activation of endoplasmic reticulum (ER) stress-related pathways. Impaired proteasomal activity also activated ER stress-related molecules and induced expression of pro-inflammatory adipocytokines in adipocyte-like cells differentiated from 3T3-L1 cells. Collective evidence suggests that impaired proteasomal activity increases ER stress and that subsequent inflammatory pathways play pivotal roles in WAT inflammation. Since proteasomal function declines with age, age-related proteasome impairment may be involved in obesity-related inflammation among the elderly.

Fibroblast growth factor-21 alleviates proteasome injury via activation of autophagy flux in Parkinson's disease.

Parkinson's disease (PD) is one of the most common and complex Neurodegeneration, with an inherited metabolic disorder. Fibroblast growth factor 21 (FGF21), an endocrine hormone that belongs to the fibroblast growth factor superfamily, plays an extensive role in metabolic regulation. However, our understandings of the specific function and mechanisms of FGF21 on PD are still quite limited. Here, we aimed to elucidate the actions and the underlying mechanisms of FGF21 on dopaminergic neurodegeneration using cellular models of parkinsonism. To investigate the effects of FGF21 on dopaminergic neurodegeneration in vitro, proteasome impairment models of PD were utilized. Human dopaminergic neuroblastoma SH-SY5Y cells were treated with the proteasome inhibitor lactacystin (5 μmol/L) for 12h, then with 50ng/ml FGF-21 with or without 5 mmol/L of 3-methyladenine.The cells were dissected to assess alterations in autophagy using immunofluorescence, immunoblotting and electron microscopy assays. Our data indicate that FGF21 prevents dopaminergic neuron loss and shows beneficial effects against proteasome impairment induced PD syndrome, indicating it might be a potent candidate for developing novel drugs to deal with PD.

The histone acetyl transferases CBP and p300 regulate stress response pathways in synovial fibroblasts at transcriptional and functional levels

The activation of stress response pathways in synovial fibroblasts (SF) is a hallmark of rheumatoid arthritis (RA). CBP and p300 are two highly homologous histone acetyl transferases and writers of activating histone 3 lysine 27 acetylation (H3K27ac) marks. Furthermore, they serve as co-factors for transcription factors and acetylate many non-histone proteins. Here we showed that p300 but not CBP protein expression was down regulated by TNF and 4-hydroxynonenal, two factors that mimic inflammation and oxidative stress in the synovial microenvironment. We used existing RNA-sequencing data sets as a basis for a further in-depth investigation of individual functions of CBP and p300 in regulating different stress response pathways in SF. Pathway enrichment analysis pointed to a profound role of CBP and/ or p300 in regulating stress response-related gene expression, with an enrichment of pathways associated with oxidative stress, hypoxia, autophagy and proteasome function. We silenced CBP or p300, and performed confirmatory experiments on transcriptome, protein and functional levels. We have identified some overlap of CBP and p300 target genes in the oxidative stress response pathway, however, with several genes being regulated in opposite directions. The majority of stress response genes was regulated by p300, with a specific function of p300 in regulating hypoxia response genes and genes encoding proteasome subunits. Silencing of p300 suppressed proteasome enzymatic activities. CBP and p300 regulated autophagy on transcriptome and functional levels. Whereas CBP was indispensable for autophagy synthesis, silencing of p300 affected late-stage autophagy. In line with impaired autophagy and proteasome function, poly-ubiquitinated proteins accumulated after silencing of p300.

C9orf72 proline-arginine dipeptide repeats disrupt the proteasome and perturb proteolytic activities.

The hexanucleotide G4C2 repeat expansion in C9orf72 is the most frequent genetic cause of familial amyotrophic lateral sclerosis (ALS). Aberrant translation of this hexanucleotide sequence leads to production of 5 dipeptide repeats (DPRs). One of these DPRs is proline-arginine (polyPR), which is found in C9orf72-expanded ALS (C9ALS) patient brain tissue and is neurotoxic across multiple model systems. PolyPR was previously reported to bind and impair proteasomes in vitro. Nevertheless, the clinical relevance of the polyPR-proteasome interaction and its functional consequences in vivo are yet to be established. Here, we aim to confirm and functionally characterize polyPR-induced impairment of proteolysis in C9ALS patient tissue and an in vivo model system. Confocal microscopy and immunofluorescence studies on both human and Drosophila melanogaster brain tissues revealed sequestration of proteasomes by polyPR into inclusion-like bodies. Co-immunoprecipitation in D. melanogaster showed that polyPR strongly binds to the proteasome. In vivo, functional evidence for proteasome impairment is further shown by the accumulation of ubiquitinated proteins along with lysosomal accumulation and hyper-acidification, which can be rescued by a small-molecule proteasomal enhancer. Together, we provide the first clinical report of polyPR-proteasome interactions and offer in vivo evidence proposing polyPR-induced proteolytic dysfunction as a pathogenic mechanism in C9ALS.

The transcription factor NRF1 (NFE2L1) activates aggrephagy by inducing p62 and GABARAPL1 after proteasome inhibition to maintain proteostasis

The ubiquitin‒proteasome system (UPS) and autophagy are the two primary cellular pathways of misfolded or damaged protein degradation that maintain cellular proteostasis. When the proteasome is dysfunctional, cells compensate for impaired protein clearance by activating aggrephagy, a type of selective autophagy, to eliminate ubiquitinated protein aggregates; however, the molecular mechanisms by which impaired proteasome function activates aggrephagy remain poorly understood. Here, we demonstrate that activation of aggrephagy is transcriptionally induced by the transcription factor NRF1 (NFE2L1) in response to proteasome dysfunction. Although NRF1 has been previously shown to induce the expression of proteasome genes after proteasome inhibition (i.e., the proteasome bounce-back response), our genome-wide transcriptome analyses identified autophagy-related p62/SQSTM1 and GABARAPL1 as genes directly targeted by NRF1. Intriguingly, NRF1 was also found to be indispensable for the formation of p62-positive puncta and their colocalization with ULK1 and TBK1, which play roles in p62 activation via phosphorylation. Consistently, NRF1 knockdown substantially reduced the phosphorylation rate of Ser403 in p62. Finally, NRF1 selectively upregulated the expression of GABARAPL1, an ATG8 family gene, to induce the clearance of ubiquitinated proteins. Our findings highlight the discovery of an activation mechanism underlying NRF1-mediated aggrephagy through gene regulation when proteasome activity is impaired.

Role of NFE2L1 in the Regulation of Proteostasis: Implications for Aging and Neurodegenerative Diseases.

A hallmark of aging and neurodegenerative diseases is a disruption of proteome homeostasis ("proteostasis") that is caused to a considerable extent by a decrease in the efficiency of protein degradation systems. The ubiquitin proteasome system (UPS) is the major cellular pathway involved in the clearance of small, short-lived proteins, including amyloidogenic proteins that form aggregates in neurodegenerative diseases. Age-dependent decreases in proteasome subunit expression coupled with the inhibition of proteasome function by aggregated UPS substrates result in a feedforward loop that accelerates disease progression. Nuclear factor erythroid 2- like 1 (NFE2L1) is a transcription factor primarily responsible for the proteasome inhibitor-induced "bounce-back effect" regulating the expression of proteasome subunits. NFE2L1 is localized to the endoplasmic reticulum (ER), where it is rapidly degraded under basal conditions by the ER-associated degradation (ERAD) pathway. Under conditions leading to proteasome impairment, NFE2L1 is cleaved and transported to the nucleus, where it binds to antioxidant response elements (AREs) in the promoter region of proteasome subunit genes, thereby stimulating their transcription. In this review, we summarize the role of UPS impairment in aging and neurodegenerative disease etiology and consider the potential benefit of enhancing NFE2L1 function as a strategy to upregulate proteasome function and alleviate pathology in neurodegenerative diseases.

High resolution structures define divergent and convergent mechanisms of archaeal proteasome activation

Considering the link between neurodegenerative diseases and impaired proteasome function, and the neuro-protective impact of enhanced proteasome activity in animal models, it’s crucial to understand proteasome activation mechanisms. A hydrophobic-tyrosine-any residue (HbYX) motif on the C-termini of proteasome-activating complexes independently triggers gate-opening of the 20S core particle for protein degradation; however, the causal allosteric mechanism remains unclear. Our study employs a structurally irreducible dipeptide HbYX mimetic to investigate the allosteric mechanism of gate-opening in the archaeal proteasome. High-resolution cryo-EM structures pinpoint vital residues and conformational changes in the proteasome α-subunit implicated in HbYX-dependent activation. Using point mutations, we simulated the HbYX-bound state, providing support for our mechanistic model. We discerned four main mechanistic elements triggering gate-opening: 1) back-loop rearrangement adjacent to K66, 2) intra- and inter- α subunit conformational changes, 3) occupancy of the hydrophobic pocket, and 4) a highly conserved isoleucine-threonine pair in the 20S channel stabilizing the open and closed states, termed the "IT switch." Comparison of different complexes unveiled convergent and divergent mechanism of 20S gate-opening among HbYX-dependent and independent activators. This study delivers a detailed molecular model for HbYX-dependent 20S gate-opening, enabling the development of small molecule proteasome activators that hold promise to treat neurodegenerative diseases.

Minimal mechanistic component of HbYX-dependent proteasome activation that reverses impairment by neurodegenerative-associated oligomers

The implication of reduced proteasomal function in neurodegenerative diseases combined with studies showing the protective effects of increasing proteasome activity in animal models highlight the need to understand the capacity for proteasome activation by small molecules. The C-terminal HbYX motif is present on many proteasome binding proteins and functions to tether activators to the 20S core particle. Previous studies have shown that peptides with a HbYX motif can autonomously activate 20S gate-opening to allow protein degradation. In this study, through an iterative process of peptide synthesis, we design a HbYX-like dipeptide mimetic that represents only the fundamental components of the HbYX motif. The mimetic robustly induces gate-opening in archaeal, yeast, and mammalian proteasomes. We identify multiple proteasome α subunit residues in the archaeal proteasome involved in HbYX-dependent activation. When stimulated by the mimetic, the mammalian 20S can degrade unfolded proteins such as tau. Findings using our peptide mimetic suggest the HbYX-dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Most significantly, our peptide mimetic reverses proteasome impairment by neurodegenerative disease-associated oligomers. Collectively, these results validate HbYX-like molecules as having robust potential to stimulate proteasome function, which are potentially useful for treating neurodegenerative diseases.

P2X7 receptor inhibition ameliorates ubiquitin–proteasome system dysfunction associated with Alzheimer’s disease

BackgroundOver recent years, increasing evidence suggests a causal relationship between neurofibrillary tangles (NFTs) formation, the main histopathological hallmark of tauopathies, including Alzheimer’s disease (AD), and the ubiquitin–proteasome system (UPS) dysfunction detected in these patients. Nevertheless, the mechanisms underlying UPS failure and the factors involved remain poorly understood. Given that AD and tauopathies are associated with chronic neuroinflammation, here, we explore if ATP, one of the danger-associated molecules patterns (DAMPs) associated with neuroinflammation, impacts on AD-associated UPS dysfunction.MethodsTo evaluate if ATP may modulate the UPS via its selective P2X7 receptor, we combined in vitro and in vivo approaches using both pharmacological and genetic tools. We analyze postmortem samples from human AD patients and P301S mice, a mouse model that mimics pathology observed in AD patients, and those from the new transgenic mouse lines generated, such as P301S mice expressing the UPS reporter UbG76V-YFP or P301S deficient of P2X7R.ResultsWe describe for the first time that extracellular ATP-induced activation of the purinergic P2X7 receptor (P2X7R) downregulates the transcription of β5 and β1 proteasomal catalytic subunits via the PI3K/Akt/GSK3/Nfr2 pathway, leading to their deficient assembly into the 20S core proteasomal complex, resulting in a reduced proteasomal chymotrypsin-like and postglutamyl-like activities. Using UPS-reported mice (UbGFP mice), we identified neurons and microglial cells as the most sensitive cell linages to a P2X7R-mediated UPS regulation. In vivo pharmacological or genetic P2X7R blockade reverted the proteasomal impairment developed by P301S mice, which mimics that were detected in AD patients. Finally, the generation of P301S;UbGFP mice allowed us to identify those hippocampal cells more sensitive to UPS impairment and demonstrate that the pharmacological or genetic blockade of P2X7R promotes their survival.ConclusionsOur work demonstrates the sustained and aberrant activation of P2X7R caused by Tau-induced neuroinflammation contributes to the UPS dysfunction and subsequent neuronal death associated with AD, especially in the hippocampus.

Activity-dependent tau cleavage by caspase-3 promotes neuronal dysfunction and synaptotoxicity

Tau-mediated toxicity is associated with cognitive decline and Alzheimer's disease (AD) progression. In particular, tau post-translational modifications (PTMs) are thought to generate aberrant tau species resulting in neuronal dysfunction. Despite being well characterized in postmortem AD brain, it is unclear how caspase-mediated C-terminal tau cleavage promotes neurodegeneration, as few studies have developed the models to dissect this pathogenic mechanism. Here, we show that proteasome impairment results in cleaved tau accumulation at the post-synaptic density (PSD), a process that is modulated by neuronal activity. Cleaved tau (at residue D421) impairs neuronal firing and causes inefficient initiation of network bursts, consistent with reduced excitatory drive. We propose that reduced neuronal activity, or silencing, is coupled to proteasome dysfunction, which drives cleaved tau accumulation at the PSD and subsequent synaptotoxicity. Our study connects three common themes in the progression of AD: impaired proteostasis, caspase-mediated tau cleavage, and synapse degeneration.