Proteasome

Proteasome: function, genes and structure

Good overview here - (R21).

Extracellular proteasome

Proteasomes were also found in the extracellular CSF, which raises questions of their role.

In all patients, extracellular proteasome was found in the CSF. The mean concentration was 24.6 ng/ml. Enzymatic activity of the 20S subunits of proteasomes was positively identified by the fluorescenic subtrate cleavage at a mean of 8.5 fkat/ml.

Concentrations of extracellular proteasomes in the CSF, total protein content and Il-6 were uncorrelated. Immunoelectron microscopy revealed merging vesicles of proteasomes with the outer cell membrane suggestive of an exozytic transport mechanism. For the first time, extracellular circulating 20S proteasome in the CSF of healthy individuals is identified and its enzymatic activity detected. A possible exozytic vesicle-bond transportation mechanism is suggested by immunoelectron microscopy. (R30)

Types: 20S and 26S

Activities of 26S

chymotrypsin-like

trypsin-like

peptidylglutamyl-peptide hydrolase protease activity

Regulation of proteasome

NRF1 is a master regulator and O-GlcNAcylation is required

The ability to sense proteasome insufficiency and respond by directing the transcriptional synthesis of de novo proteasomes is a trait that is conserved in evolution and is found in organisms ranging from yeast to humans. This homeostatic mechanism in mammalian cells is driven by the transcription factor NRF1.

Interestingly, NRF1 is synthesized as an endoplasmic reticulum (ER) membrane protein and when cellular proteasome activity is sufficient, it is retrotranslocated into the cytosol and targeted for destruction by the ER-­associated degradation pathway (ERAD).

However, when proteasome capacity is diminished, retrotranslocated NRF1 escapes ERAD and is activated into a mature transcription factor that traverses to the nucleus to induce proteasome genes. (R32)

A major cause of this resistance (to proteasome inhibitors) is the proteasome bounce-back response mediated by NRF1, a transcription factor that coordinately activates proteasome subunit genes.

To identify new targets for efficient suppression of UPS, we explored, using immunoprecipitation and mass spectrometry, the possible existence of nuclear proteins that cooperate with NRF1 and identified O-linked N-acetylglucosamine transferase (OGT) and host cell factor C1 (HCF-1) as two proteins capable of forming a complex with NRF1.

O-GlcNAcylation catalyzed by OGT was essential for NRF1 stabilization and consequent upregulation of proteasome subunit genes. (R31)

Neural tissue-specific Nrf1 knockout mice exhibit abnormal accumulation of polyubiquitinated proteins in the brain, supporting an essential role of NRF1 in the maintenance of proteasome function (R31)

Activators

Linoleic acid

The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controls. (R11)

Retinoic acid

We demonstrate that RA directs proteasome-mediated degradation of RARα, thereby accounting for the degradation of APL-associated fusion proteins. The domains required for transcriptional activation are also required for receptor catabolism, providing a striking illustration of how transcription factors are turned off following activation. Our findings demonstrate that RA degrades PML/RARα by two distinct pathways: caspase activation and direct proteasome targeting, accounting for the biphasic degradation in Fig. ​Fig.11A and also for the fact that both caspase and proteasome inhibitors were previously shown to antagonize PML/RARα catabolism.

Both RA and AS induce the activation of caspases (not shown), accounting for the fact that z-VAD partially inhibited both RA- and AS-induced PML/RARα degradation. Inhibitor studies suggest that arsenic also targets PML/RARα through an as yet unidentified proteasome-dependent pathway (Fig. ​(Fig.11D). This unidentified pathway may be the one responsible for AS-induced PML degradation, because PML/RARα, like PML, is targeted onto nuclear bodies (NBs) after AS treatment.

Alternatively, arsenic appears to alter RARα phosphorylation and induces its progressive depletion. Such AS-induced RARα alterations may also be implicated in PML/RARα catabolism. (R17)

In the present study, we demonstrate that all-trans-retinoic acid (RA) pretreatment of SH-SY5Y cells protects them from PCD death after subsequent epoxomicin treatment which causes proteasome inhibition.

Even though ubiquitinated protein aggregates are present, there is no evidence to suggest that autophagy is involved. We conclude that protection by RA is likely by mechanisms that interfere with cell stress-PCD pathway that otherwise would result from protein accumulation after proteasome inhibition. (R18)

Oleuropein

Continuous treatment with oleuropein has been shown to increase the degradation rates of proteasome derivatives in human embryonic fibroblast cultures. This process could result from conformational changes in the proteasome with a decrease in the number of oxidized proteins. In addition, oleuropein-treated cells maintained proteasome function during replicative senescence.

Cultures showed a delay in the onset of senescence morphology and an extension of mean lifespan by approximately 15% (R24)

We demonstrate that oleuropein, the major constituent of Olea europea leaf extract, olive oil and olives, enhances the proteasome activities in vitro stronger than other known chemical activators, possibly through conformational changes of the proteasome. Moreover, continuous treatment of early passage human embryonic fibroblasts with oleuropein decreases the intracellular levels of reactive oxygen species (ROS), reduces the amount of oxidized proteins through increased proteasome-mediated degradation rates (R25)

Oleic acid amide derivatives

Here in we report here the conjugation of oleic acid with a variety of amines, Fig. 1B, and evaluated their ability to increase the ability of the 20S CP to degrade a fluorescent peptide reporter and alpha-synuclein, an intrinsically disordered protein that has been associated with Parkinson’s disease. When compared with AM-404, our best molecule, 17, exhibited a similar in cellulostimulatory activity of the 20S CP, as well as toxicity profile. (R26)

AM-404

AM-404, a derivative of arachidonic acid, was discovered in 2017 as a 20S CP stimulator following a high throughput screen, and then being validated in a cell-based assay. Previously, we investigated the structure activity relationship between the aliphatic chain of AM-404 and its 20S stimulatory activity. In this study, it was concluded that molecules with a cis-alkene and an extended saturated chain produced the most potent stimulators. (R26)

Inhibitors

Curcumin

Most notably, curcumin (diferuloylmethane), a naturally-occurring polyphenolic compound extracted from turmeric root, has been shown to significantly inhibit UPS activity in both in vitro and in vivo animal studies. Moreover, it has been reported high doses of curcumin supplementation in humans are well tolerated without significant side effects (R6)

There have been multiple proposed mechanisms to explain the observed dysregulation in UPS activity. Curcumin has been reported to induce proteasomal malfunction through directly binding to the 20S subunit (likely due to curcumin’s carbonyl carbons susceptible to nucleophilic attack by Thr 1 within the β5 subunit of the proteasome) inhibiting chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolase protease activity, or indirectly by inhibiting DUB activity, inducing oxidative stress, increasing misfolded and/or oxidized proteins, or suppressing ubiquitin gene/protein expression. (R6)

Lithium Chloride

Several studies show that LiCl inhibits 26S:

LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor α and markedly reduced chymotryptic-like activity of WEHI-3B D+ 20 S and 26 S proteasome enzymes. (R1)

LiCl interacts synergistically with all-trans-retinoic acid, promoting the terminal differentiation of WEHI-3B D(+) cells, a phenomenon partially due to the ability of the monovalent lithium cation to inhibit the proteasome-dependent degradation of retinoic acid receptor alpha protein. In this report, the 20S proteasome was purified from WEHI-3B D(+) cells and the effects of LiCl on chymotrypsin-like (Chtl) activity and peptidyl-glutamyl peptide hydrolyzing (PGPH) activity were determined. LiCl functions to inactivate both proteasomal activities in a time-dependent manner, without affecting non-proteasomal proteases. The half-lives for inactivation of Chtl and PGPH hydrolyzing activities were approximately 23 and 36min, respectively, at 10mM LiCl. … The findings suggest that the inactivation of Chtl and PGPH activities by LiCl occurs through a proteasomal conformational change. (R2)

However another study reports somewhat conflicting (or confusing) finding:

Lithium is also a potent inhibitor of glycogen synthase kinase-3β (GSK3β) activity, which is linked to Alzheimer’s disease (AD). In experiments with cultured HEK293T cells, we show here that GSK3β stabilizes synaptic acetylcholinesterase (AChE-S), a critical component of AD development.

Cells treated with lithium exhibited rapid proteasomal degradation of AChE-S. Furthermore treatment of the cells with MG132, an inhibitor of the 26S proteasome, prevented the destabilizing effect of lithium on AChE-S. (R3)

Another study shows that LiCl increases both content and activation of Glycogen Synthase:

Incubation of rat hepatocytes with LiCl resulted in an overall increase in the activity ratio of glycogen synthase (GS), concomitantly with a decrease in active GS kinase-3 levels. GS total activity was also increased in a dose- and time-dependent manner. This latter effect correlated with the amount of immunoreactive enzyme determined by immunoblotting. … Our results indicate that LiCl increases hepatocyte GS activity through increasing both the activation state of the enzyme and its cellular content. This latter increase is mediated through a modification of the proteasome-regulated proteolytic pathway of the enzyme. (R4)

MG132

MG132 is a potent, reversible, and cell-permeable 20S proteasome inhibitor and it is derived from a Chinese medicinal plant. … Our results showed that MG132 downregulated the expression of antiapoptotic proteins, including CDK2, CDK4, Bcl-xL, and Bcl-2, whereas it upregulated the expression of proapoptotic proteins, including p21, p27, p53, p-p53 (ser15, ser20, and ser46), cleaved forms of caspase-3, caspase-7, caspase-9, and PARP, and FOXO3 in U2OS cells.

These results demonstrated that MG132 activated apoptotic signaling pathways in U2OS cells.

Interestingly, MG132 downregulated the phosphorylation of Akt and Erk. (R7)

Manganese Chloride

In our research, manganese chloride exposure inhibited the activity of proteasome and induced oxidative stress. Both can be reversed by antioxidant agent N-acetylcysteine. N-acetylcysteine also inhibited the cytotoxicity induced by manganese chloride. In conclusion, our results imply that proteasome inhibition may be associated with manganese-induced cytotoxicity in dopaminergic neurons, which may be connected with oxidative damage. (R8)

The research performed shows that single and repeated Mn treatment of SN56 cholinergic neurons from BF induces P20S inhibition, increases Aβ and pTau protein levels, produces HSP90 and HSP70 proteins expression alteration, and oxidative stress generation, being the last two effects mediated by NRF2 pathway alteration. The increment of Aβ and pTau protein levels was mediated by HSPs and proteasome dysfunction. (R9)

Heme

The most significant signals specific to heme toxicity were consistent with oxidative stress and impaired protein degradation by the proteasome. This ultimately led to an activation of the response to unfolded proteins. These observations were explained mechanistically by demonstrating binding of heme to the proteasome that was linked to impaired proteasome function.

Oxidative heme reactions and proteasome inhibition could be differentiated as synergistic activities of the porphyrin. Based on the present data a novel model of cellular heme toxicity is proposed, whereby proteasome inhibition by heme sustains a cycle of oxidative stress, protein modification, accumulation of damaged proteins and cell death. (R10)

Palmitic acid

The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controls. … Furthermore, confocal immunomicroscopy showed that the colocalization of ubiquitin and tyrosinase was facilitated by linoleic acid and diminished by palmitic acid. Taken together, these data support the view that fatty acids regulate the ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation of tyrosinase. In broader terms, the function of the ubiquitin-proteasome pathway might be regulated physiologically, at least in part, by fatty acids within cellular membranes. (R11)

Based on this, it seems that

• Linoleic acid increases proteasomal degradation of the proteins
• Palmitic acid lowers proteasomal degradation

This effect could be related to altered ubiquitination, not only proteasomal activity.

EPA

When compared with a vehicle control group (olive oil) there was a significant decrease in proteolysis of the soleus muscles of mice treated with EPA after starvation for 24 h, together with an attenuation of the proteasome “chymotryptic-like” enzyme activity and the induction of the expression of the 20S proteasome alpha-subunits, the 19S regulator and p42, an ATPase subunit of the 19S regulator in gastrocnemius muscle, and the ubiquitin-conjugating enzyme E2(14k). … These results suggest that protein catabolism in starvation and cancer cachexia is mediated through a common pathway, which is inhibited by EPA and is likely to involve a lipoxygenase metabolite as a signal transducer. (R16)

Tocotrienols, but not Tocopherols

• Tocopherols don’t inhibit proteasome.
• Tocotrienols inhibit proteasome.

In the absence of any proteasome inhibitor, intrinsic cellular proteasome activity was not modulated by alpha-, beta-, and gamma-tocopherols; however, delta-tocopherol, alpha-tocotrienol, and alpha-tocopheryl phosphate could significantly inhibit cellular proteasome activity and increased the level of p27(Kip1) and p53. (R27)

Hypoxia

The exposure to hypoxia leads to dissociation of 19S and 20S subunits, and inactivation of 26S proteasome. This prevented the degradation of MHC-II and, as a result, the MSCs became immunogenic. Furthermore, we found that hypoxia-induced decrease in the levels of a chaperon protein HSP90α is responsible for inactivation of 26S proteasome. Maintaining HSP90α levels in hypoxic MSCs preserved the immunoprivilege of MSCs. (R28)

We found that in normoxic MSCs, 26S proteasome degrades HLA-DRα and maintains immunoprivilege of MSCs. The exposure to hypoxia leads to inactivation of 26S proteasome and formation of immunoproteasome in MSCs, which is associated with upregulation and activation of HLA-DRα, and as a result, MSCs become immunogenic (R29)

Health implications

Deactivation

Muscle growth defects

we report that the muscle-specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in profound muscle growth defects and a decrease in force production in mice.

Specifically, developing muscles in conditional Rpt3-knockout animals showed dysregulated proteasomal activity. The autophagy pathway was upregulated, but the process of autophagosome formation was impaired. A microscopic analysis revealed the accumulation of basophilic inclusions and disorganization of the sarcomeres in young adult mice.

Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways. The deletion of a component of the proteasome complex contributed to myofiber degeneration and weakness in muscle disorders that are characterized by the accumulation of abnormal inclusions. (R5)

Improves dystrophic phenotype

Interestingly, proteasome inhibition using MG-132 significantly improved the dystrophic phenotype (Carmignac et al., 2011). MG-132 also improves the dystrophic phenotype in a model of dystrophin deficiency (Bonuccelli et al., 2003; Winder et al., 2011).

Therefore, the upregulation of proteasomal proteolysis likely leads to a reduction in skeletal muscle mass, which is in contrast to animal models of proteasomal dysfunction or downregulation in brain neurons that leads to degeneration. (R5)

Possible release of arachidonic acid from membranes

The proteasome inhibitors, epoxomicin, lactacystin and carbobenzoxy-leucyl-leucyl-leucinal, stimulate the release of arachidonic acid from rat glial, human colon carcinoma, human breast carcinoma and the rat liver cells. They also stimulate basal and induced prostacycin production in the rat liver cells. The stimulated arachidonic acid release and basal prostaglandin I2 production in rat liver cells is inhibited by actinomycin D.

Conclusions

Stimulation of arachidonic acid release and arachidonic acid metabolism may be associated with some of the biologic effects observed after proteasome inhibition, e.g. prevention of tumor growth, induction of apoptosis, stimulation of bone formation. (R12)

I have shown that inhibition of proteolysis by phenylmethylsulphonyl fluoride, the peptide aldehydes carbobenzoxy-leucyl-leucyl-norvalinal and carbobenzoxy-leucyl-leucyl-leucinal (ZLLL) and lactacystin stimulate induced prostaglandin (PGI2) production in rat liver cells.

Lactacystin stimulates arachidonic acid (AA) release from these cells. Others have reported that proteasome inhibition up-regulates cyclooxygenase-2 (COX-2) and stimulates PGE2 production in neuronal cells. (R12)

# Proteasome inhibition in neuronal cells induces a proinflammatory response

Two proteasome inhibitors, a peptidyl aldehyde and an epoxy ketone, which cause accumulation of ubiquitinated proteins, were found to enhance expression of stress-inducible genes, including HSP70i and the polyubiquitin genes UbB and UbC.

Under these conditions, mRNA and protein levels of the inducible form of cyclooxygenase (COX-2) were upregulated together with its product, PGE(2), a proinflammatory prostaglandin.

Proteasomal inhibition also led to stabilization of COX-2 as ubiquitin conjugates, suggesting that the ubiquitin/proteasome pathway contributes to the regulation of COX-2 protein levels.

Treatment with antioxidants known to inhibit NFkappaB and AP-1 transcriptional activation failed to abrogate COX-2 upregulation. Instead, these inhibitors exacerbated the stress response by potentiating HSP70i levels while eliciting a decrease in PGE(2) production.

These findings suggest that the accumulation of ubiquitinated proteins resulting from proteasome inhibition in neuronal cells is associated with a proinflammatory response that may be an important contributor to neurodegeneration.

Schizophrenia

Among these studies, three reported ubiquitin carboxyl-terminal esterase L1 (UCHL1) as significantly down-regulated in SCZ. Two studies reported proteasome (prosome, macropain) 26S subunit, ATPase, 6 (PSMC6) as significantly down-regulated in SCZ as well as BPD. (R14)

the 22 schizophrenia patients in both groups revealed decreases in clusters of genes that encode for protein turnover (proteasome subunits and ubiquitin), mitochondrial oxidative energy metabolism (isocitrate, lactate, malate, nicotinamide adenine dinucleotide [NADH], and succinate dehydrogenases; cytochrome C oxidase; adenosine triphosphate [ATP] synthase), and genes associated with neurite outgrowth, cytoskeletal proteins, and synapse plasticity. (R15)

Upregulation of OAT3 (transporter)

Preliminary data in this study showed that classical proteasome inhibitors (e.g., MG132), but not lysosome inhibitors, significantly increased the OAT3 ubiquitination and OAT3-mediated transport of estrone sulfate (ES) in OAT3 stable expressing cells, indicating that proteasome rather than lysosome is involved in the intracellular fate of OAT3.

Next, bortezomib and carfilzomib, two FDA-approved and widely applied anticancer agents through selective targeting proteasome, were further used to define the role of inhibiting proteasome in OAT3 regulation and related molecular mechanisms.

The results showed that 20S proteasome activity in cell lysates was suppressed with bortezomib and carfilzomib treatment, leading to the increased OAT3 ubiquitination, stimulated transport activity of ES, enhanced OAT3 surface and total expression. (R19)

Deactivation of DIO2

Ubiquitin is a major regulator of thyroid hormone type 2 deiodinase (type 2 iodothyronine deiodinase) and hence a major regulator of central conversion of thyroxine (T4) to T3; activation and inactivation of type 2 deiodinase are regulated by ubiquitination and deubiquitination.

The ubiquitinated form of type 2 deiodinase is inactivated in proteasomes or activated by deubiquitination.

Deubiquitination in the proteasome is associated with enzymes in the proteasome lid and at the entrance to the Rpt ring (see Fig. 2).

Tissue type 2 deiodinase is substantially increased in hypothyroidism. Control of the deiodinase by the ubiquitination/deubiquitination switch contributes to a mechanism which maintains appropriate tissue-specific levels of T3 in a variety of organs and tissues, including the brain. (R20)

DIO2 converts T4 to T3 inside the tissues.

Cooperation with other enzymes

ZFAND5 / ZNF216

ZFAND5/ZNF216, a member of the ubiquitous ZFAND protein family, contains two zinc finger domains.

It is induced in skeletal muscle during atrophy and was shown to be essential for the resulting large loss of muscle mass. We show here that purified ZFAND5 stimulates the 26S proteasomes’ capacity to degrade peptides and ubiquitinated proteins. In cells, it promotes the degradation of endogenous cell proteins through its ubiquitin-binding zinc finger domain (A20).

Unlike several ZFAND proteins, it is not induced in proteotoxic stresses. Unlike other proteasome activators, ZFAND5 actually stimulates overall protein breakdown by the ubiquitin proteasome pathway. (R22)

Bind three Zinc ions (Uniprot)

Involved in protein degradation via the ubiquitin-proteasome system. May act by anchoring ubiquitinated proteins to the proteasome. Plays a role in ubiquitin-mediated protein degradation during muscle atrophy. Plays a role in the regulation of NF-kappa-B activation and apoptosis. Inhibits NF-kappa-B activation triggered by overexpression of RIPK1 and TRAF6 but not of RELA. Inhibits also tumor necrosis factor (TNF), IL-1 and TLR4-induced NF-kappa-B activation in a dose-dependent manner. Overexpression sensitizes cells to TNF-induced apoptosis. Is a potent inhibitory factor for osteoclast differentiation.

Upon proteasome binding, ZFAND5 widens the entrance of the substrate translocation channel, yet it associates only transiently with the proteasome. Dissociation of ZFAND5 then stimulates opening of the 20S proteasome gate.

Using single-molecule microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation.

These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis. (R22)