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.     

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.     

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.     

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

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.     

Activity‐based profiling of the proteasome pathway during hepatitis C virus infection

Hepatitis C virus (HCV) infection often leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The stability of the HCV proteins is controlled by ubiquitin‐dependent and ubiquitin‐independent proteasome pathways. Many viruses modulate proteasome function for their propagation. To examine the interrelationship between HCV and the proteasome pathways we employed a quantitative activity‐based protein profiling method. Using this approach we were able to quantify the changes in the activity of several proteasome subunits and found that proteasome activity is drastically reduced by HCV replication. The results imply a link between the direct downregulation of the activity of this pathway and chronic HCV infection.     

Proteasome activity imaging and profiling characterizes bacterial effector syringolin A

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: β1, β2, and β5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets β2 and β5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to β2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.     

Polyglycine hydrolases: Fungal β‐lactamase‐like endoproteases that cleave polyglycine regions within plant class IV chitinases

Polyglycine hydrolases are secreted fungal proteases that cleave glycine–glycine peptide bonds in the inter‐domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es‐cmp) and Cochliobolus carbonum (Bz‐cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. Peptides from a tryptic digest of Es‐cmp were analyzed by LC‐MS/MS and the spectra obtained were matched to a draft genome sequence of E. sorghi. From this analysis, a 642 amino acid protein containing a predicted β‐lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS‐PAGE and MALDI‐MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es‐cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full‐length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β‐lactamase‐like region. This is the first report of a predicted β‐lactamase that is an endoprotease.     

Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell‐based high‐throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell‐based reporters, detection of global ubiquitination status, and proteasome‐mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell‐based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG‐132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

Increased 4‐hydroxy‐2‐nonenal‐induced proteasome dysfunction is correlated with cardiac damage in streptozotocin‐injected rats with isoproterenol infusion

Increase in 4‐hydroxy‐2‐nonenal (4HNE) due to oxidative stress has been observed in a variety of cardiac diseases such as diabetic cardiomyopathy. 4HNE exerts a damaging effect in the myocardium by interfering with subcellular organelles like mitochondria by forming adducts. Therefore, we hypothesized that increased 4HNE adduct formation in the heart results in proteasome inactivation in isoproterenol (ISO)‐infused type 1 diabetes mellitus (DM) rats. Eight‐week‐old male Sprague Dawley rats were injected with streptozotocin (STZ, 65 mg kg^?1). The rats were infused with ISO (5 mg kg^?1) for 2 weeks by mini pumps, after 8 weeks of STZ injection. We studied normal control (n = 8) and DM + ISO (n = 10) groups. Cardiac performance was assessed by echocardiography and Millar catheter at the end of the protocol at 20 weeks. Initially, we found an increase in 4HNE adducts in the hearts of the DM + ISO group. There was also a decrease in myocardial proteasomal peptidase (chymotrypsin and trypsin‐like) activity. Increases in cardiomyocyte area (446 ± 32·7 vs 221 ± 10·83) (µm²), per cent area of cardiac fibrosis (7·4 ± 0·7 vs 2·7 ± 0·5) and cardiac dysfunction were also found in DM + ISO (P < 0·05) relative to controls. We also found increased 4HNE adduct formation on proteasomal subunits. Furthermore, reduced aldehyde dehydrogenase 2 activity was observed in the myocardium of the DM + ISO group. Treatment with 4HNE (100 μM) for 4 h on cultured H9c2 cardiomyocytes attenuated proteasome activity. Therefore, we conclude that the 4HNE‐induced decrease in proteasome activity may be involved in the cardiac pathology in STZ‐injected rats infused with ISO. Copyright © 2016 John Wiley & Sons, Ltd.     

Tissue specific increase of the catalytic subunits of the 26S proteasome by indirect antioxidant dithiolethione in mice: enhanced activity for degradation of abnormal protein

Decreases in the 26S proteasome are related to the toxicities of abnormal protein aggregates and may contribute to pathogenesis of degenerative diseases. Therefore, maintenance of proteasome function can be a novel strategy to protect cells against abnormal protein-mediated toxicity. In the present study, we have demonstrated the tissue specific increase of the catalytic subunits of the proteasome in mice following oral administration of 3H-1,2-dithiole-3-thione (D3T, 0.5 mmol/kg), which functions as a cancer preventive agent in animal and human studies. Expression of the 20S catalytic core subunits PSMB5, PSMB6, and PSMB7 were increased in liver, lung, small intestine, and colon of mice at 24 h after D3T treatment. Elevated expression of proteasome catalytic subunits led to increases in proteasomal peptidase activities in these tissues. Oral administration of D3T also exerted a pharmacodynamic action in some brain regions of these mice and proteasomal peptidase activities were significantly elevated in the cerebral cortex–hippocampus. Moreover, tissue extracts from D3T-treated mice and cell lysates obtained from D3T-incubated murine neuroblastoma cells exhibited the enhanced capacity to degrade mutant human SOD1G93A protein. These results indicate that the catalytic subunits of the 26S proteasome are inducible in multiple tissues of mouse including brain by exogenous chemical treatment. Increased proteasome expression by inducers may have a role in protection/attenuation of protein aggregate-mediated disorders.     

Dissecting protein‐protein interactions in proteasome assembly: Implication to its self‐assembly

The 26S proteasome is a multi‐catalytic ATP‐dependent protease complex that recognizes and cleaves damaged or misfolded proteins to maintain cellular homeostasis. The 26S subunit consists of 20S core and 19S regulatory particles. 20S core particle consists of a stack of heptameric alpha and beta subunits. To elucidate the structure‐function relationship, we have dissected protein‐protein interfaces of 20S core particle and analyzed structural and physiochemical properties of intra‐alpha, intra‐beta, inter‐beta, and alpha‐beta interfaces. Furthermore, we have studied the evolutionary conservation of 20S core particle. We find the size of intra‐alpha interfaces is significantly larger and is more hydrophobic compared with other interfaces. Inter‐beta interfaces are well packed, more polar, and have higher salt‐bridge density than other interfaces. In proteasome assembly, residues in beta subunits are better conserved than alpha subunits, while multi‐interface residues are the most conserved. Among all the residues at the interfaces of both alpha and beta subunits, Gly is highly conserved. The largest size of intra‐alpha interfaces complies with the hypothesis that large interfaces form first during the 20S assembly. The tight packing of inter‐beta interfaces makes the core particle impenetrable from outer wall of the cylinder. Comparing the three domains, eukaryotes have large and well‐packed interfaces followed by archaea and bacteria. Our findings provide a structural basis of assembly of 20S core particle in all the three domains of life.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Laserpitium zernyi Hayek Flower and Herb Extracts: Phenolic Compounds,and Anti‐edematous,Antioxidant, and Antimicrobial Activities

Phenolic compounds and different biological activities of the dry MeOH extracts of the flowers and the herb (aerial parts without flowers) of Laserpitium zernyi Hayek (Apiaceae) were investigated. The total phenolic contents in the extracts were determined spectrophotometrically using Folin–Ciocalteu reagent. In both extracts, apigenin, luteolin, their 7‐O ‐glucosides, and chlorogenic acid were detected by HPLC . Identified phenolics were quantified in both extracts, except luteolin in L . zernyi herb extract. The extracts (p.o .) were tested for anti‐edematous activity in a model of carrageenan (i.pl .) induced rat paw edema. Antioxidant activity of the extracts was assessed by FRAP assay and DPPH and ^?OH radicals scavenging tests. Antimicrobial activity was investigated using broth microdilution test against five Gram ‐positive and three Gram ‐negative bacteria, as well as against two strains of Candida albicans . The polyphenol‐richer flower extract exerted higher anti‐edematous and antioxidant activities. The herb extract exhibited better antimicrobial effect against Micrococcus luteus , Enterococcus faecalis , Bacillus subtilis , and Pseudomonas aeruginosa , while against other tested microorganisms, the activity of both extracts was identical. Demonstrated biological activities of L . zernyi flower and herb extracts represent a good basis for their further investigation as potential new herbal medicinal raw materials.