Proteasome activity profiling: a simple,robust and versatile method revealing subunit‐selective inhibitors and cytoplasmic,defense‐induced proteasome activities

The proteasome plays essential roles in nearly all biological processes in plant defense and development, yet simple methods for displaying proteasome activities in extracts and living tissues are not available to plant science. Here, we introduce an easy and robust method to simultaneously display the activities of all three catalytic proteasome subunits in plant extracts or living plant tissues. The method is based on a membrane‐permeable, small‐molecule fluorescent probe that irreversibly reacts with the catalytic site of the proteasome catalytic subunits in an activity‐dependent manner. Activities can be quantified from fluorescent protein gels and used to study proteasome activities in vitro and in vivo. We demonstrate that proteasome catalytic subunits can be selectively inhibited by aldehyde‐based inhibitors, including the notorious caspase‐3 inhibitor DEVD. Furthermore, we show that the proteasome activity, but not its abundance, is significantly increased in Arabidopsis upon treatment with benzothiadiazole (BTH). This upregulation of proteasome activity depends on NPR1, and occurs mostly in the cytoplasm. The simplicity, robustness and versatility of this method will make this method widely applicable in plant science.     

The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma

Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor.     

Subunit‐selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections

The proteasome is a nuclear‐cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit‐selective inhibitors and dual‐color fluorescent activity‐based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant–microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1‐1 [PtoDC3000(ΔhQ)] whilst the activity profile of the β1 subunit changes. Infection with wild‐type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species.     

Proteasome dysfunction in Drosophila signals to an Nrf2‐dependent regulatory circuit aiming to restore proteostasis and prevent premature aging

The ubiquitin–proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi‐mediated knockdown of 20S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose‐dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several ‘old‐age’ phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2‐dependent upregulation of the proteasome subunits. RNAi‐mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress‐related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2‐dependent tissue‐ and age‐specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging.     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Synthesis and proteasome inhibition of N‐allyl vinyl ester‐based peptides

Inhibition of the proteasome, the multicatalytic protease complex responsible for the turnover of many cellular proteins, represents an attractive target in the development of new drug therapies, proteasome inhibitors being potentially useful tools for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Based on our previous development of a new class of inhibitors bearing a C‐terminal VE cluster able to interact with catalytic threonine, we report herein the synthesis and activity of new VE‐based peptide analogs bearing an additional allyl pharmacophore unit at the C‐ or N‐terminal position of the pseudotripeptide sequence. In the new series, the structural modification carried out to the prototype determine a decrease of proteasome inhibition. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of protein kinase C and calmodulin by the geometric isomers cis- and trans-tamoxifen

The triphenylethylene antiestrogen trans-tamoxifen is an effective antitumor agent used in the treatment of human breast cancer. While the antiestrogenic activity of trans-tamoxifen clearly plays an important role in its tumoricidal action, some of the biological effects of trans-tamoxifen are independent of estrogen. Therapeutic concentrations of trans-tamoxifen inhibit protein kinase C (PKC) and calmodulin-dependent enzymes. PKC and calmodulin play critical roles in growth regulation, and there is evidence that inhibition of PKC and calmodulin by trans-tamoxifen may contribute to the antiumor activity of the drug in vivo. The geometric isomers cis- and trans-tamoxifen have a number of opposing biological activities that have been attributed to their interactions with the estrogen receptor, Cis-tamoxifen is generally estrogenic, whereas trans-tamoxifen is generally antiestrogenic. In this report, we compared the effects of cis- and trans-tamoxifen on PKC activity and on calmodulin-dependent cAMP phosphodiesterase activity. Cis- and trans-tamoxifen inhibited the Ca²⁺- and phosphatidylserine- (PS-) dependent activity of purified rat brain PKC with indistinguishable potencies, but cis-tamoxifen was somewhat more potent than the trans isomer in the inhibition of the Ca²⁺- and PS-independent activity of PKC. In addition, cis-tamoxifen was the more potent isomer in the inhibition of T lymphocyte activation, an event that entails a PKC-requiring signal transduction pathway. A modest preference of the cis isomer was also observed in the inhibition of a calmodulin-dependent cAMP phosphodiesterase. These results suggest a congruence between triphenylethylene binding sites on PKC and on the activated calmodulin–cAMP phosphodiesterase complex. We conclude that the interactions of cis- and trans-tamoxifen with PKC and the activated calmodulin–cAMP phosphodiesterase complex offer a criterion for distinguishing biological effects of triphenylethylenes that are due to interactions with the estrogen receptor from the biological effects resulting from their inhibitory activities against PKC and calmodulin-dependent processes.     

Antioxidative Activity of Resveratrol and Its Derivatives Isolated from Seeds of Paeonia lactiflora

Seven stilbenes, a new cis-ε-viniferin and the six known stilbenes, trans-resveratrol, trans-resveratrol-4′-O-β-D-glucopyranoside, trans-ε-viniferin, gnetin H, and suffruticosol A and B, were isolated and identified from seeds of Paeonia lactiflora. The antioxidative activity of these stilbene derivatives was evaluated against the 2-deoxyribose degradation and rat liver microsomal lipid peroxidation induced by the hydroxyl radical generated via a Fenton-type reaction. Among these stilbenes, trans-ε-viniferin and gnetin H significantly inhibited 2-deoxyribose degradation and lipid peroxidation in rat liver microsomes. In addition, cis-ε-viniferin, and suffruticosol A and B also exhibited moderate antioxidative activity. These results suggest that resveratrol dimers and trimers, together with resveratrol from seeds of Paeonia lactiflora may be useful as potential sources of natural antioxidants.     

White muscle 20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minor

The effect of temperature and mass on specific growth rate (G) was examined in spotted wolffish Anarhichas minor of different size classes (ranging from 60 to 1500 g) acclimated at different temperatures (4, 8 and 12° C). The relationship between G and 20S proteasome activity in heart ventricle, liver and white muscle tissue was then assessed in fish acclimated at 4 and 12° C to determine if protein degradation via the proteasome pathway could be imposing a limitation on somatic growth. Cardiac 20S proteasome activity was not affected by acclimation temperature nor fish mass and had no correlation with G. Hepatic 20S proteasome activity was higher at 12° C but did not show any relationship with G. Partial correlation analysis showed that white muscle 20S proteasome activity was negatively correlated to G (partial Pearson's r = ?0·609) but only at cold acclimation temperature (4° C). It is suggested that acclimation to cold temperature involves compensation of the mitochondrial oxidative capacity which would in turn lead to increased production of oxidatively damaged proteins that are degraded by the proteasome pathway and ultimately negatively affects G at cold temperature.     

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the rans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horseradish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.     

Mouse FKBP23 mediates conformer-specific functions of BiP by catalyzing Procis/trans isomerization

FK506-binding proteins (FKBPs) are cellular receptors for the immunosuppressant FK506 and rapamycin. They belong to the ubiquitous peptidyl-prolyl cis/trans isomerases (PPIases) family, which can catalyze the cis/trans isomerization of peptidyl-prolyl bond in peptides and proteins. In previous work, we revealed that mouse FKBP23 binds immunoglobulin binding protein (BiP), the major heat shock protein (Hsp) 70 chaperone in the ER, and the binding is interrelated with [Ca²⁺]. Furthermore, the binding can suppress the ATPase activity of BiP through the PPIase activity of FKBP23. In this work, FKBP23 is demonstrated to mediate functions of BiP by catalyzing the Pro¹¹⁷cis/trans conformational interconversion in the ATPase domain of BiP. This result may provide new understanding to the novel role of PPIase as a molecular switch.     

Structural investigation of poly-L,D-proline, a synthetic ion channel across bilayer membranes: crystal structure and molecular conformation of two tetra-D,L-proline derivatives

The crystal structures and molecular conformations of two tetraproline derivatives with alternating configurations Boc(D -Pro,L -Pro)₂OH and Boc(D -Pro,L -Pro)₂OCH₃ are investigated in connection with the ability of the homologous polymer to selectively increase (as an ion channel) the ion permeability across bilayer membranes. Both tetramers are characterized by the cis-trans alternating conformation of the peptide bonds, which formally transforms in a turn of the poly-D ,L -proline channel after a cis-trans change of the central peptide residue.     

Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on constitutive androstane receptor activation

2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective species-specific inducer of CYP2В in rats. Several analogs of TPD were synthesized to verify a hypothesis that minor changes in the inducer structure can cause changes in induction abilities (R = H, cisTPD and transTPD; R = N(CH₃)₂, transpDMA; R = NO₂, transpNO₂; R = F, transpF; R = OCH₃, transpMeO). Five of six compounds were able to activate CAR in rat liver. Results of Western-blot and ChIP showed that cisTPD and transTPD, transpDMA, transpNO₂, transpF treatment stimulated nuclear accumulation of CAR and evoked CAR receptor PBREM-binding activity in rat liver. cisTPD, transTPD, transpDMA, transpNO₂ and transpF administration significantly increased total CYP content (1.3–2.5 fold) and the level of PROD (12–20 fold), CYP2B specific activity, whereas transpMeO did not have any effects. Western blot and real-time RT-PCR showed that the increase of PROD in liver is related to the high content of CYP2B proteins and paralleled the increase of CYP2B1 (10–43 fold) and CYP2B2 (8–26 fold) mRNAs. At the same time content of CYP2B proteins and CYP2B1 and CYP2B2 mRNA levels were unchanged in rat liver after transpMeO treatment. The dose–response studies have shown that cisTPD, transpDMA, transpF and transpNO₂ have similar potency, and transTPD is less potent derivative. Moreover, it is likely transTPD act as a partial CAR activator. Thus, our results provide evidence to support the conclusion that the differences of TPD analogs ability to activate CYP2B gene expression can be explained by various interactions with CAR.     

Co‐ and post‐translational modifications of the 26S proteasome in yeast

The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co‐ and post‐translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N‐terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N‐terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho‐amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with λ phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N‐ and O‐linked oligosaccharides nor O‐linked β‐N‐acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co‐ and post‐translational modifications, including N^α‐acetylation, N^α‐myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.     

PURIFICATION AND CHARACTERIZATION OF THE 20S PROTEASOME FROM THEALGA CHARA CORALLINA (CHAROPHYCEAE)1

We purified the 20S proteasome from the alga Chara corallina Willd with DEAE–ion‐exchange column chromatography and preparative nondenaturing PAGE. The analysis of the purified enzyme bynondenaturing PAGE gave a single band whose molecular mass was estimated to be about 600,000 Da by gel permeation chromatography and whose isoelectric point was at pH 5.5. Two‐dimensional gel electrophoresis gave at least 12 spots with molecular masses from 26,000 to 32,000 Da in a wide range of isoelectric points. The 20S proteasome hydrolyzed three types of artificial substrates used to differentiate chymotrypsin‐like, trypsin‐like, and peptidyl glutamyl peptidase activities. Both the chymotrypsin‐like and the peptidyl glutamyl peptidase activities were enhanced by SDS. In the presence of 0.03% SDS, the optimal pH for both activities was 8.5. Trypsin‐like activity of the 20S proteasome had a broad pH optimum in an alkaline region and was not activated but inhibited by SDS. Its chymotrypsin‐like activity was inhibited by N‐ethylmaleimide, p‐chloromercuribenzoic acid, and chymostatin. In contrast, its peptidyl glutamyl peptidase activity was not inhibited by chymostatin. Moreover, proteasome inhibitors MG 115 and MG 135 were effective against the chymotrypsin‐like activity and less so against the peptidyl glutamyl peptidase activity. These properties were very similar to those of the proteasomes of mammalian, yeast, and spinach cells. The large size of Chara cells will make in vivo manipulations and investigations of the proteasome proteolytic system possible.     

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo-RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (α5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (α6) and PSMA3 (α7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.     

Cytokinin activity of zeatin allylic phosphate, a natural compound

Zeatin allylic phosphate (ZAP) retarded chlorophyll loss in the barleyleaf senescence assay at a concentration 20 times higher than for6-benzyladenine (BA): the effective concentrations for ZAP and BA were 10 and 0.5 , respectively. Sodium molybdate,an inhibitor of phosphatases, decreased the ZAP effective concentration to 0.5 without affecting leaf senescence andtrans-zeatin activity in the control. This demonstrates theimportance of the phosphate group for ZAP activity or its penetration into leafcells. ZAP up-regulated the protein kinase activity of the barley leaf chromatinwith concentration dependence similar to that oftrans-zeatin. Conversely, ZAP was 1000 times less activethan trans-zeatin in the competition with anti-idiotypeantibodies (raised against antibody to zeatin) for binding with atrans-zeatin-binding site oftrans-zeatin-binding protein ZBP67 isolated from barleyleaves. In contrast to trans-zeatin, ZAP did not activateRNA synthesis in the presence of ZBP in the in vitro systemcontaining chromatin and RNA polymerase I isolated from barley leaves. Insummary, data presented show that ZAP possesses cytokinin activity asdemonstrated by the retardation of barley leaf senescence, but moleculartarget(s) for ZAP in barley leaf cells differs, at least partially, from thesefor trans-zeatin. It seems possible that the cytokininactivity of ZAP results from its hydrolysis while producing zeatin.     

Kinetic regulations of dihydroquercetin oxidation with horseradish peroxide

The steady-state kinetics of horseradish peroxidase-catalyzed dihydroquercetin oxidation was studied. Dihydroquercetin was shown to be a slowly oxidized substrate of horseradish peroxidase. Two dihydroquercetin isoforms (cis and trans forms) that were selectively involved in peroxidase-induced oxidation were found in water-alcohol and buffer solutions. The k _cat and K _m were determined in the pH range of 4.5–8.0.     

The proteasome subunit RPN10 functions as a specific receptor for degradation of the 26S proteasome by macroautophagy in Arabidopsis

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are 2 main degradative routes, which are important for cellular homeostasis. In a study conducted by Marshall et al., the authors demonstrated that the UPS and autophagy converge in Arabidopsis (see the punctum in issue #11–10). In particular, they found that the 26S proteasome is degraded by autophagy, either nonselectively (induced by nitrogen starvation) or selectively (induced by proteasome inhibition). The selective phenotype is mediated through the proteasome subunit RPN10, which can bind both ubiquitin and ATG8. This newly identified autophagic degradation of the proteasome is termed “proteaphagy,” and the process reveals an interesting relationship between these degradative systems.