Enhancement Of Proteasome Activity Research Articles

Proteasome Activation as a New Therapeutic Approach To Target Proteotoxic Disorders.

Proteasomes are multienzyme complexes that maintain protein homeostasis (proteostasis) and important cellular functions through the degradation of misfolded, redundant, and damaged proteins. It is well established that aging is associated with the accumulation of damaged and misfolded proteins. This phenomenon is paralleled by declined proteasome activity. When the accumulation of redundant proteins exceed degradation, undesirable signaling and/or aggregation occurs and are the hallmarks of neurodegenerative diseases and many cancers. Thus, increasing proteasome activity has been recognized as a new approach to delay the onset or ameliorate the symptoms of neurodegenerative and other proteotoxic disorders. Enhancement of proteasome activity has many therapeutic potentials but is still a relatively unexplored field. In this perspective, we review current approaches, genetic manipulation, posttranslational modification, and small molecule proteasome agonists used to increase proteasome activity, challenges facing the field, and applications beyond aging and neurodegenerative diseases.

Resveratrol Induces Brain Resilience Against Alzheimer Neurodegeneration Through Proteostasis Enhancement.

Resveratrol is a natural compound that mimics the antioxidant and antiaging effects of caloric restriction, mainly mediated through SIRT1, a deacetylase that induces longevity and neuroprotection. We aimed to analyze the effects of resveratrol on the brain status of control non-transgenic (NoTg) and AD transgenic (3xTg-AD) mice to discern the mechanisms involved in a potential inducement of resilience against age-related neurodegeneration and Alzheimer's disease (AD). Mice were fed with a diet supplemented with 100mg/kg of resveratrol from 2months of age during 10months. Resveratrol administration induced complete protection against memory loss and brain pathology in 3xTg-AD mice, and also induced cognitive enhancement in healthy NoTg mice. Resveratrol improved exploration and reduced anxiety in both mouse strains, indicative of well-being. Resveratrol reduced the presence of Aβ and p-tau pathology in the hippocampus of the 3xTg-AD mouse. Proteostasis analysis showed the following in both NoTg and 3xTg-AD mice: (i) increased levels of the amyloid-degrading enzyme neprilysin, (ii) reduction of the amyloidogenic secretase BACE1, and (iii) increase of proteasome protein levels and enhancement of proteasome activity. Resveratrol also increased AMPK protein levels, then upregulating the SIRT1 pathway, as shown by the activation of PGC-1α and CREB in both mice, resulting in further beneficial changes. Our data demonstrated that resveratrol induces cognitive enhancement and neuroprotection against amyloid and tau pathologies. Improvement of proteostasis by resveratrol, in both healthy and AD mice, suggests that it is a mechanism of brain resilience and defense against neurodegeneration caused by the accumulation of aberrant proteins.

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.

Motor Neuron-specific Disruption of Proteasomes, but Not Autophagy, Replicates Amyotrophic Lateral Sclerosis

Evidence suggests that protein misfolding is crucially involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, controversy still exists regarding the involvement of proteasomes or autophagy in ALS due to previous conflicting results. Here, we show that impairment of the ubiquitin-proteasome system, but not the autophagy-lysosome system in motor neurons replicates ALS in mice. Conditional knock-out mice of the proteasome subunit Rpt3 in a motor neuron-specific manner (Rpt3-CKO) showed locomotor dysfunction accompanied by progressive motor neuron loss and gliosis. Moreover, diverse ALS-linked proteins, including TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), ubiquilin 2, and optineurin were mislocalized or accumulated in motor neurons, together with other typical ALS hallmarks such as basophilic inclusion bodies. On the other hand, motor neuron-specific knock-out of Atg7, a crucial component for the induction of autophagy (Atg7-CKO), only resulted in cytosolic accumulation of ubiquitin and p62, and no TDP-43 or FUS pathologies or motor dysfunction was observed. These results strongly suggest that proteasomes, but not autophagy, fundamentally govern the development of ALS in which TDP-43 and FUS proteinopathy may play a crucial role. Enhancement of proteasome activity may be a promising strategy for the treatment of ALS.

Enhancement of proteasome activity by a small-molecule inhibitor of USP14

Proteasomes, the primary mediators of ubiquitin-protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that Usp14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin-protein conjugates, in vivo and in vitro. A catalytically inactive variant of Usp14 has reduced inhibitory activity, suggesting that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human Usp14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. Usp14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of Usp14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

The flavonoid fisetin promotes nerve cell survival from trophic factor withdrawal by enhancement of proteasome activity

To explore the possibility that specific flavonoids can substitute for neurotrophic factors, we examined the ability of the flavonol fisetin and several related flavonoids to support the survival of low density, serum-free cultures of rat cortical neurons. Normally these cells die within 24 h in the absence of trophic factors but in the presence of fisetin and several related flavonoids the cells survive and produce long neurites. While the survival-promoting effect of several of the fisetin-related flavonoids was partially dependent on ERK activation, the effect of fisetin was not. Fisetin can enhance glutathione synthesis but the survival-promoting effect of fisetin was also not dependent on glutathione. However, proteasome inhibitors almost completely blocked the ability of fisetin to promote survival. Consistent with this observation, fisetin increased proteasome activity. Together these results demonstrate a new activity for fisetin and tie this activity to its neurotrophic effects.

Ubiquitin-Proteasome Pathway Components as Therapeutic Targets for CNS Maladies

In the central nervous system (CNS), abnormal deposition of insoluble protein aggregates or inclusion bodies within nerve cells is commonly observed in association with several neurodegenerative diseases. The ubiquitinated protein aggregates are believed to result from malfunction or overload of the ubiquitin-proteasome pathway or from structural changes in the protein substrates which prevent their recognition and degradation by the ubiquitin-proteasome pathway. Impaired proteolysis might also contribute to the synaptic dysfunction seen early in neurodegenerative diseases because the ubiquitin-proteasome pathway is known to play a role in normal functioning of synapses. Because specificity of the ubiquitin proteasome mediated proteolysis is determined by specific ubiquitin ligases (E3s), identification of specific E3s and their allosteric modulators are likely to provide effective therapeutic targets for the treatment of several CNS disorders. Another unexplored area for the discovery of drug targets is the proteasome. Although many inhibitors of the proteasome are available, no effective drugs exist that can stimulate the proteasome. Since abnormal protein aggregation is a common feature of different neurodegenerative diseases, enhancement of proteasome activity might be an efficient way to remove the aggregates that accumulate in the brain. In this review, we discuss how the components of the ubiquitin-proteasome pathway could be potential targets for therapy of CNS diseases and disorders.

A proteasomal stress response: pre‐treatment with proteasome inhibitors increases proteasome activity and reduces neuronal vulnerability to oxidative injury

We report here that exposure to low concentrations of proteasome inhibitors (e.g. 10-100 nm MG-132, 0.1-3 nm epoxomicin or 10-30 nm clasto-lactacystin beta-lactone) resulted in an enhancement, rather than an inhibition, of proteasome activity in cultured neocortical neurons. Size-fractionation chromatography confirmed that the enhanced peptide cleavage activity was associated with proteasome-sized complexes. This sub toxic exposure reduced neuronal death caused by subsequent exposure to oxidative stress (100-200 microm H(2)O(2) for 30 min, 24-h exposure to 100 microm paraquat or 7.5 microm menadione), but did not alter vulnerability to excitotoxicity (5-min exposure to 30-100 microm NMDA or 24 exposure to 12 microm NMDA). Sub toxic proteasome inhibitor exposure caused an increase in levels of proteasome core subunit proteins and mRNAs, but not in levels of potentially cytoprotective heat shock proteins (hsp70, hsp90 and hsp40). The neuroprotective effects of proteasome inhibitor pre-treatment were blocked by coapplication of proteasome inhibitors during the oxidative insult. These findings support a model in which sublethal proteasome inhibition induces neurons to increase proteasome activity and promotes resistance to oxidative injury and suggests that enhancement of proteasome activity is a potential therapeutic target for diseases in which oxidative stress has been implicated.