Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC–MS

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20–30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.     

Characterization of 26S proteasome α- and β-type and ATPase subunits from spinach and their expression during early stages of seedling development

Three kinds of cDNAs encoding 26S proteasome subunits have been cloned from spinach (Spinacia oleracea L.). These genes, designated as SOPSC8, SOPSC1 and SOPRS7, encode an -type and a -type subunit of the 20S catalytic core, and an ATPase subunit of the 19/22S regulatory complex, respectively. The deduced protein sequences showed high sequence similarities to other proteasome - and -type and ATPase subunit proteins. Southern blot analysis indicates that there are additional members of these dispersed proteasome families in the spinach genome. These three subunit genes are expressed simultaneously during germination and reach a maximum one day after sowing followed by a decline. The expression of these genes also increases during cotyledon senescence.     

Phenoxypropanolamine derivatives as selective inhibitors of the 20S proteasome β1 and β5 subunits

New series of thiophene-containing phenoxypropanolamines were synthesized and evaluated for their potency to inhibit the three proteolytic activities of the mammalian 20S proteasome. Noticeable inhibition of both ChT-L and PA activities was obtained with three compounds: one with unsubstituted phenoxypropanolamine group (7) and the two others with a p-Cl-substituted group (4 and 9). For three other compounds (3, 8 and 10), ChT-L activity alone was significantly inhibited. In silico docking performed on the β5 and β1 subunits bearing the respective ChT-L and PA catalytic sites showed features common to poses associated with active compounds. These features may constitute a selectivity criterion for structure-guided inhibitor design.     

Activation of NFκB and Ub-proteasome pathway during apoptosis induced by a serum factor is mediated through the upregulation of the 26S proteasome subunits

We have been investigating differential gene expression associated with apoptosis in AK-5 cells (a spontaneously regressing rat histiocytoma) and have observed catalytic subunits beta 7 and alpha 5 of the 26S proteasome and ubiquitin to be upregulated during apoptosis induced by a variety of agents. The observed elevation in gene expression was parallel to a comparable increase in the cytosolic protein expression of the proteasome and ubiquitin and a markedly amplified increase in the proteasome activity. Inhibition of the increase in gene expression resulted in the inhibition of the rise in the proteasome activity subsequently leading to an inhibition of apoptosis. Similarly, pretreatment with proteasome inhibitors, MG132 and lactacystin, resulted in a significant inhibition of apoptosis pointing to the requirement of a highly active protein degradation machinery during apoptosis. The apoptosis inhibitory effect of the proteasome inhibitors involved an inhibition of the activation of various initiator and effector caspases but was independent of any changes in the mitochondrial membrane depolarization and cytochrome c release associated with apoptosis. Inhibition of proteasome activity or its upstream PI3 kinase activity inhibited NFκB translocation thereby suppressing apoptosis, which highlights the requirement of NFκB activation for completion of apoptosis in AK-5 cells. Hence, the apoptosis associated induction of the Ub-proteasome pathway components and the proteasome activity suggests that the proteasome, in its capacity as an efficient protein degradation complex, plays an important role in the successful execution of apoptosis.     

Plant–soil feedbacks and the coexistence of competing plants

Plant–soil feedbacks can have important implications for the interactions among plants. Understanding these effects is a major challenge since it is inherently difficult to measure and manipulate highly diverse soil communities. Mathematical models may advance this understanding by making the interplay of the various processes affecting plant–soil interaction explicit and by quantifying the relative importance of the factors involved. The aim of this paper is to provide a complete analysis of a pioneering plant–soil feedback model developed by Bever and colleagues (J Ecol 85: 561–573, 1997; Ecol Lett 2: 52–62, 1999; New Phytol 157: 465–473, 2003) to fully understand the range of possible impacts of plant–soil feedbacks on plant communities within this framework. We analyze this model by means of a new graphical method that provides a complete classification of the potential effects of soil communities on plant competition. Due to the graphical character of the method, the results are relatively easy to obtain and understand. We show that plant diversity depends crucially on two key parameters that may be viewed as measures of the intensity of plant competition and the direction and strength of plant–soil feedback, respectively. Our analysis provides a formal underpinning of earlier claims that plant–soil feedbacks, especially when they are negative, may enhance the diversity of plant communities. In particular, negative plant–soil feedbacks can enhance the range of plant coexistence by inducing competitive oscillations. However, these oscillations can also destabilize plant coexistence, leading to low population densities and extinctions. In addition, positive feedbacks can allow locally stable forms of plant coexistence by inducing alternative stable states. Our findings highlight that the inclusion of plant–soil interactions may fundamentally alter the predictions on the structure and functioning of above-ground ecosystems. The scenarios presented in this study can be used to formulate hypotheses about the ways soil community effects may influence plant competition that can be tested with empirical studies. This will advance our understanding of the role of plant–soil feedback in ecological communities.     

The polyphyly of Neotragus – Results from genetic and morphometric analyses

Dwarf antelope species were commonly united in the tribe “Neotragini” (Bovidae, Mammalia) due to their general morphological appearance. However, phylogenetic analyses have shown that not all dwarf antelopes are closely related, so it was suggested to restrict the name Neotragini to the type genus Neotragus. In our study we use mitochondrial cytochrome b sequences and linear skull measurements to further investigate the similarity of all three Neotragus species. Our analyses support the close relationship of N. moschatus and N. batesi. However, N. pygmaeus – the type species, which was never before included in phylogenetic analyses – is not closely related. It might share a most recent common ancestor with another “dwarf antelope”, the Klipspringer Oreotragus oreotragus, and the duikers in the taxon Cephalophini. Hence, we suggest resurrecting the genus Nesotragus von Dueben, 1846 for Nesotragus moschatus and N. batesi.     

Comparison of human COP9 signalosome and 26S proteasome ‘lid’

The human core COP9 signalosome consists of eight subunits which have been identified, cloned and sequenced. The components of COP9 signalosome possess homologies with eight non-ATPase regulatory subunits of the 26S proteasome. These polypeptides of the 19S regulator form a reversibly binding subcomplex called the lid. We isolated the lid from human red blood cells and compared it with the COP9 signalosome complex. In addition to the non-ATPase regulatory polypeptides, we found a high molecular mass ATPase copurifying with the human lid. The COP9 signalosome-associated kinase activity is either not at all or only weakly affected by common kinase inhibitors such as 1-(5-Isoquinolinesulfonyl)-2-methyl-piperazine (H7), 5,6-dichloro-1--D-ribofuranosyl-benzimidazole (DRB) or Wortmannin. Curcumin, a tumor suppressor and effector of AP-1 activation, is a potent inhibitor of the COP9 signalosome kinase activity with a Ki of about 10 M. Since curcumin is known as an inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway acting upstream of the MAP kinase kinase kinase level, one site of action of the COP9 signalosome might be proximal to regulators on that level.     

Interferon-gamma inducible exchanges of 20S proteasome active site subunits: why?

When cells are stimulated with the cytokines IFN-gamma or TNF-alpha, the synthesis of three proteasome subunits LMP2 (beta1i), LMP7 (beta5i), and MECL-1 (beta2i) is induced. These subunits replace the three subunits delta (beta1), MB1 (beta5), and Z (beta2), which bear the catalytically active sites of the proteasome, during proteasome neosynthesis. The cytokine-induced exchanges of three active site subunits of a complex protease is unprecedented in biology and one may expect a strong functional driving force for this system to evolve. These cytokine-induced replacements of proteasome subunits are believed to favour the production of peptide ligands of major histocompatibility complex (MHC) class I molecules for the stimulation of cytotoxic T cells. Although the peptide production by constitutive proteasomes is able to maintain peptide-dependent MHC class I cell surface expression in the absence of LMP2 and LMP7, these subunits were recently shown to be pivotal for the generation or destruction of several unique epitopes. In this review we discuss the recent data on LMP2/LMP7/MECL-1-dependent epitope generation and the functions of each of these subunit exchanges. We propose that these subunit exchanges have evolved not only to optimize class I peptide loading but also to generate LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites which are not proteolytically generated in uninflamed tissues. This difference in epitope generation may serve to better stimulate T cells in the sites of an ongoing immune response and to avoid autoimmunity in uninflamed tissues.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Genetic and functional diversity among root-associated psychrotrophic Pseudomonad’s isolated from the Himalayan plants

Out of 534 psychrotrophic bacteria, 12 bacteria were selected on the basis of plant growth promoting activities at 4 °C and identified as Pseudomonas genus. These strains showed high level of genetic polymorphisms based on RAPD and rep-PCR fingerprinting. This genetic variability revealed that isolates belonging to same species were as high as the variability among different species. Further inoculation of these Pseudomonas strains significantly improves root/shoot biomass and nutrients uptake of lentil plant as compared to non-bacterized control after 40 days of seed showing. Agglomerative hierarchical clustering analysis of pot assay results revealed that genetically diverse strains showing the same prototype in functional parameter and representing diverse blueprint of plant growth promoting attributes. Results of present findings explain the huge beneficial microbial resources from root zone of hilly crops of Himalayan region that could be effectively exploited as bio-inoculums for cold climatic condition.     

Response of bird assemblages to windstorm and salvage logging — Insights from analyses of functional guild and indicator species

Natural disturbances, such as fire, windstorms and insect outbreaks, are important drivers of biodiversity in forest ecosystems, but at the same time cause large economic losses. Among the natural disturbances in Europe, windstorms cause the highest economic loss. After such storms, damaged forest stands are commonly salvage logged to restore economic value. However, such interventions could affect species assemblages of various taxonomic groups, including breeding birds. Despite these potential effects, investigations of the impacts of post-storm logging are largely lacking. We thus investigated assemblages of breeding birds in 21 logged and 21 unlogged windstorm-disturbed forest plots and 18 undisturbed, control forest plots using fixed-radius point-stop counts three, five and seven years after a windstorm within the Bavarian Forest National Park as part of the European Long-Term Ecosystem Research Network. We recorded 2100 bird individuals of 55 bird species. Bird assemblages were predominantly altered by the consequences of the windstorm and affected only to a minor degree by subsequent logging of storm-felled trees. Nevertheless, bird species richness was significantly reduced by post-storm logging within the first season. In general, the windstorm led to a shift in bird assemblage composition from typical forest species towards open- and shrub-land species. Assemblages of logged and unlogged disturbed plots consisted mainly of long-distance migrants and ground-foraging bird species, whereas assemblages of undisturbed control plots consisted of resident species that forage within vegetation. Both unlogged and logged storm-felled coniferous forest stands were inhabited by endangered or declining bird species, such as Water Pipit (Anthus spinoletta) on logged plots and Eurasian Redstart (Phoenicurus phoenicurus) on unlogged plots. Indicator species analyses suggested that species of unlogged storm disturbed plots depended on storm-created legacies, such as pits and snags, for foraging and nesting. Hence, we recommend reducing post-storm logging of these legacies to support species restricted to unlogged disturbed forest. To increase the diversity of breeding birds on the landscape scale, diverse logged and unlogged post-disturbance stands should be provided, which could be gained by a partial benign neglect strategy of storm disturbed forest stands.     

Region 1112–1123 in the central domain of 18S rRNA in 40S subunits of plant ribosomes: Accessibility for complementary interactions and the functional role

The binding of the 18S rRNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112–1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5′-untranslated region upstream of the reporter sequence coding for β-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5′ end allowed for the demonstration that the 1112–1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112–1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.     

What curves alpha-solenoids? Evidence for an alpha-helical toroid structure of Rpn1 and Rpn2 proteins of the 26 S proteasome

The alpha-helical solenoid proteins adopt a variety of elongated curved structures. They have been examined to identify the interactions that determine their curvature. A sequence pattern characteristic for strongly curved alpha-helical solenoids has been constructed and was found to match protein sequences containing the proteasome/cyclosome repeats. Based on this, a structural model of the repeat-containing domains of the Rpn1/S2 and Rpn2/S1 proteins, which represent the largest subunits of the 26 S proteasome, has been proposed. The model has a novel architecture resembling an alpha-helical toroid. Molecular modeling shows that these toroids have a central pore that would allow passage of an unfolded protein substrate through it. This implies that the Rpn1 and Rpn2 toroids are aligned along the common axial pores of the ATPase hexamer and form an "antechamber" of the 26 S proteasome. The proposed quaternary structure agrees with the available experimental data. It is suggested that the function of this antechamber is assistance to the ATPases in the unfolding of protein substrates prior to proteolysis. An evolutionary link between the PC repeat-containing proteins and tetratricopeptide repeat proteins is proposed.     

The amino acid sequences of the α subunits of the lectins from the seeds of Lathyrus hirsutus and Lathyrus tingitanus

The amino acid sequences of the α₁ and α₂ subunits of the isolectins (LhL1 and LhL2) from seeds of Lathyrus hirsutus and the α subunit of the lectin from L. tingitanus were determined by analysis of peptides derived from the proteins by separate digestions with chymotrypsin and the protease from S. aureus V8. The α₁ subunit of the L. hirsutus LhL1 isolectin differed from the α₂ form in LhL2 only in having an extra lysine inserted near the C-terminus. The L. tingitanus α subunit differed from the L. hirsutus α₁ in three positions and from L. hirsutus α₂ in four.     

Analyzing the structural and functional roles of residues from the ‘black’ and ‘gray’ clusters of human S100P protein

Two highly conserved structural motifs observed in members of the EF-hand family of calcium binding proteins. The motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif represents a cluster of three amino acids called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster). Cluster I is more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II is noticeably less conserved and includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. In the human calcium binding S100 P protein, these ‘black’ and ‘gray’ clusters include residues F15, F71, and F74 and L33, L58, and K30, respectively. To evaluate the effects of these clusters on structure and functionality of human S100 P, we have performed Ala scanning. The resulting mutants were studied by a multiparametric approach that included circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probes. Spectrofluorimetric Ca2+-titration of wild type S100 P showed that S100 P dimer has 1–2 strong calcium binding sites (K₁ = 4 × 106 M⁻¹) and two cooperative low affinity (K₂ = 4 × 104 M⁻¹) binding sites. Similarly, the S100 P mutants possess two types of calcium binding sites. This analysis revealed that the alanine substitutions in the clusters I and II caused comparable changes in the S100 P functional properties. However, analysis of heat- or GuHCl-induced unfolding of these proteins showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Opposite to literature data, the F15 A substitution did not cause the S100 P dimer dissociation, indicating that F15 is not crucial for dimer stability. Overall, similar to parvalbumins, the S100 P cluster I is more important for protein conformational stability than the cluster II.     

The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes

Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. We demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by 'pre-treatment' with very low concentrations of H2O2, and tested by measuring inducible resistance to a subsequent much higher 'challenge' dose of H2O2. Following an initial direct physical activation of pre-existing proteasomes, the 20S proteasome, immunoproteasome and PA28αβ regulator all exhibited substantially increased de novo synthesis during adaptation over 24?h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and PA28αβ synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and PA28 siRNA (short interfering RNA) knockdown treatments. Additionally, PA28αβ-knockout mutants achieved only half of the H2O2-induced adaptive increase in proteolytic capacity of wild-type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased, and oxidized proteins accumulated, during high H2O2 challenge, but prior H2O2 adaptation was protective. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or PA28αβ regulator blocked 50-100% of these adaptive increases in cell division and DNA replication, and immunoproteasome knockdown largely abolished protection against protein oxidation.