Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.     

Membrane localization diversity of TPK channels and their physiological role

Potassium (K) is one of the major nutrients that is essential for plant growth and development. The majority of cellular K⁺ resides in the vacuole and tonoplast K⁺ channels of the TPK (Two Pore K) family are main players in cellular K⁺ homeostasis. All TPK channels were previously reported to be expressed in the tonoplast of the large central lytic vacuole (LV) except for one isoform in Arabidopsis that resides in the plasma membrane. However, plant cells often contain more than one type of vacuole that coexist in the same cell. We recently showed that two TPK isoforms (OsTPKa and OsTPKb) from Oryza sativa localize to different vacuoles with OsTPKa predominantly found in the LV tonoplast and OsTPKb primarily in smaller compartments that resemble small vacuoles (SVs). Our study further revealed that it is the C-terminal domain that determines differential targeting of OsTPKa and OsTPKb. Three C-terminal amino acids were particularly relevant for targeting TPKs to their respective endomembranes. In this addendum we further evaluate how the different localization of TPKa and TPKb impact on their physiological role and how TPKs provide a potential tool to study the physiology of different types of vacuole.Key words: TPK channels, small vacuoles, vacuolar targeting, potassiumThe roles of plant vacuolar K⁺ channels are diverse and include potassium homeostasis, turgor regulation and responses to abiotic stress. Vacuolar K⁺-selective channels belong to two-pore K⁺ (TPK) channel families which have been found in genomes of many plant species such as Arabidopsis, poplar, Physcomitrella, Eucalyptus, barley, potato, rice and tobacco (Fig. 1). TPKs have structural similarity to mammalian “tandem P domain” channels with a secondary structure that contains four transmembrane domains and two pore regions (Fig. 2).^1–5 TPK channels have pore regions with a GYGD signature that endows K⁺ selectivity and a variable number of Ca²⁺ binding EF domains in the C terminus.^3–8 One of the best characterized members of the TPK family is AtTPK1 from Arabidopsis thaliana. AtTPK1 activity is voltage independent but sensitive to cytosolic Ca²⁺, cytosolic pH and N-terminal phosphorylation by 14-3-3 proteins.^5,6,8,9 In Arabidopsis, AtTPK1 expresses in the large lytic vacuole (LV) and plays roles in cellular K⁺ homeostasis, K⁺-release during stomatal closure and seed germination.^4,5 Other members of the Arabidopsis TPK family (AtTPK2, AtTPK3, AtTPK5) have been shown to localize to the LV but also showed some expression in smaller, vesicle-like, compartments.⁴ However, none of these isoforms appears to form functional channels in planta although our experiments with heterologous expression of AtTPK3 and AtTPK5 in the K⁺ uptake deficient E. coli LB2003 demonstrates complementation of bacterial growth phenotype (Isayenkov S, et al. unpublished results). Equally intriguing, is the plasma membrane localization of the Arabidopsis TPK4 isoform, in spite of its sequence being very similar to that of other TPKs.¹⁰Open in a separate windowFigure 1Phylogenetic tree of plant TPKs. The three main clusters of TPKs comprise: Cluster 1 with AtTPK1-like channels; Cluster 2 with AtTPK3/TPK5-like channels; Cluster 3 with barley HvTPKb. Bootstrap analysis was performed using ‘Molecular Evolutionary Genetics Analysis, MEGA4’ software available at www.megasoftware.net/mega4/megaOpen in a separate windowFigure 2Two-pore potassium channel secondary structure. TPK channels comprise four transmembrane domains (1–4) and two pore regions (P) per subunit. Functional channels are formed from two subunits. In most TPKs, both P regions contain a K⁺ selectivity signature, GYGD. However, the tobacco NtTPKa isoform has different motifs in the second P domain. In the N terminal region, TPKs have a 14-3-3 binding domain that impact on channel activity, with the binding of 14-3-3 protein leading to channel activation. C-termini of TPKs show a varying number of putative Ca²⁺ binding “EF hands” which may vary from zero to two.     

Increased incidence of pregnancy complications in women who later develop scleroderma: a case control study

Introduction  

Studies have shown that fetal progenitor cells persist in maternal blood or bone marrow for more than 30 years after delivery. Increased trafficking of fetal cells occurs during pregnancy complications, such as hypertension, preeclampsia, miscarriage and intra-uterine growth restriction (IUGR). Women with these pregnancy complications are significantly more often HLA-class II compatible with their spouses. Women who later develop scleroderma also give birth to an HLA-class II child more often. From these prior studies we hypothesized that preeclampsia and other pregnancy complications could be associated with increased levels of fetal cell trafficking, and later be involved in the development of scleroderma.     

RETRACTED ARTICLE: Mast cells are the main interleukin 17-positive cells in anticitrullinated protein antibody-positive and -negative rheumatoid arthritis and osteoarthritis synovium

Introduction  

Mast cells have been implicated to play a functional role in arthritis, especially in autoantibody-positive disease. Among the cytokines involved in rheumatoid arthritis (RA), IL-17 is an important inflammatory mediator. Recent data suggest that the synovial mast cell is a main producer of IL-17, although T cells have also been implicated as prominent IL-17 producers as well. We aimed to identify IL-17 expression by mast cells and T cells in synovium of arthritis patients.     

Phytase enzymology, applications, and biotechnology

Phytases are phosphohydrolases that initiate the step-wise removal of phosphate from phytate. These enzymes have been widely used in animal feeding to improve phosphorus nutrition and to reduce phosphorus pollution of animal waste. The potential of phytases in improving human nutrition of essential trace minerals in plant-derived foods is being explored. This review covers the basic biochemistry and application of phytases, and emphasizes the emerging biotechnology used for developing new effective phytases with improved properties.     

Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis

Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3⁺ T cells, CD38⁺ plasma cells and CD68⁺ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland–Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials.