Fungal-contaminated grass and well water and sporadic amyotrophic lateral sclerosis.

Fungi are important infectious disease-causing agents, but are often overlooked as environmental factors in disease. We review several lines of evidence that point to a potential fungal origin of sporadic amyotrophic lateral sclerosis (ALS), the most common form of motor neurone disease. Approximately 90% cases of ALS are sporadic, and the aetiology of sporadic ALS is still unknown. We have previously postulated that grass or soil-associated fungal infections may be a leading cause of sporadic ALS. Herein we extend this proposal to water-associated fungi. A wide variety of fungi have been reported in drinking water including Acremonium, Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium and Trichoderma. Some of these are known to produce neurotoxic mycotoxins. Despite this, drinking water is not routinely monitored for fungal contamination. Fungal contamination could explain the close correlation between distribution of well water and cases of sporadic ALS in the United States. We propose several mechanisms by which an opportunistic fungal infection from environmental exposure (to water, soil or plants) can lead to long term neuronal degradation resulting in the hallmarks of ALS. If confirmed, the association between fungal infection and sporadic ALS could lead to novel treatment strategies for this progressive and fatal disease.

Fungal Neurotoxins and Sporadic Amyotrophic Lateral Sclerosis.

We review several lines of evidence that point to a potential fungal origin of sporadic amyotrophic lateral sclerosis (ALS). ALS is the most common form of motor neuron disease (MND) in adults. It is a progressive and fatal disease. Approximately 90% cases of ALS are sporadic, and 5-10% are due to genetic mutations (familial). About 25 genes implicated in familial ALS have been identified so far, including SOD1 and TARDBP, the gene encoding 43 kDa transactive response (TAR) DNA-binding protein (TDP-43). Despite intensive research over many decades, the aetiology of sporadic ALS is still unknown. An environmental cause, including grass or soil-associated fungal infections, is suggested from a range of widely diverse lines of evidence. Clusters of ALS have been reported in soccer players, natives of Guam and farmers. Grass-associated fungi are known to produce a range of neurotoxins and, in symbiotic associations, high levels of fungal SOD1. Exposure of neurons to fungal neurotoxins elicits a significant increase in glutamate production. High levels of glutamate stimulate TDP-43 translocation and modification, providing a link between fungal infection and one of the molecular and histologic hallmarks of sporadic ALS. A recent study provided evidence of a variety of fungi in the cerebrospinal fluid and brain tissue of ALS patients. This review provides a rational explanation for this observation. If a fungal infection could be confirmed as a potential cause of ALS, this could provide a straightforward treatment strategy for this fatal and incurable disease.

Protein phosphatase 2A carboxymethylation and regulatory B subunits differentially regulate mast cell degranulation.

Asthma is characterised by antigen-mediated mast cell degranulation resulting in secretion of inflammatory mediators. Protein phosphatase 2A (PP2A) is a serine/threonine protein phosphatase composed of a catalytic (PP2A-C) subunit together with a core scaffold (PP2A-A) subunit and a variable, regulatory (PP2A-B) subunit. Previous studies utilising pharmacological inhibition of protein phosphatases have suggested a positive regulatory role for PP2A in mast cell degranulation. In support of this we find that a high okadaic acid concentration (1µM) inhibits mast cell degranulation. Strikingly, we now show that a low concentration of okadaic acid (0.1µM) has the opposite effect, resulting in enhanced degranulation. Selective downregulation of the PP2A-Cα subunit by short hairpin RNA also enhanced degranulation of RBL-2H3 mast cells, suggesting that the primary role of PP2A is to negatively regulate degranulation. PP2A-B subunits are responsible for substrate specificity, and carboxymethylation of the PP2A-C subunit alters B subunit binding. We show here that carboxymethylation of PP2A-C is dynamically altered during degranulation and inhibition of methylation decreases degranulation. Moreover downregulation of the PP2A-Bα subunit resulted in decreased MK2 phosphorylation and degranulation, whilst downregulation of the PP2A-B'δ subunit enhanced p38 MAPK phosphorylation and degranulation. Taken together these data show that PP2A is both a positive and negative regulator of mast cell degranulation, and this differential role is regulated by carboxymethylation and specific PP2A-B subunit binding.

Discovery and identification of serine and threonine phosphorylated proteins in activated mast cells: implications for regulation of protein synthesis in the rat basophilic leukemia mast cell line RBL-2H3.

Mast cells are important in allergic inflammation and innate immunity. Antigen-induced activation via cell-surface receptors initiates a series of intracellular signaling events, leading to the secretion of inflammatory mediators. While many of the kinases involved in this process have been defined, their substrates are generally unknown. This study aimed to identify proteins phosphorylated by serine or threonine kinases in the early stages of mast cell activation, using the rat basophilic leukemia cell line RBL-2H3 as a model system. Cells were activated via FcepsilonRI cross-linking, and lysed at different time points between 1-10 min. A novel, specific mixture of serine and threonine phospho-specific antibodies was utilized, and was shown to selectively detect proteins that were phosphorylated upon cell activation. The mixture of antibodies was used to immunoprecipitate such regulated phosphoproteins from cell lysates enriched in phosphoproteins by phospho-affinity chromatography. Immunoprecipitated proteins were analyzed by SDS-PAGE, Western blotting and liquid chromatography/mass spectrometry. With this approach, we highlighted a number of phosphoproteins, demonstrated differences in the phosphorylation/dephosphorylation rates among the regulated proteins, and identified eleven serine- or threonine-phosphorylated proteins that are substrates of kinases involved in mast cell intracellular signaling. Among these were proteins with functions in protein metabolism, including elongation factor 2, calnexin and heat shock proteins; and cell structure, including moesin, tubulin and actin. The novel approach applied in this study proved useful for the identification of kinase substrates, and can readily be extended for use in similar phosphoproteomic studies.

Phosphorylation of nonmuscle myosin heavy chain IIA on Ser1917 is mediated by protein kinase C beta II and coincides with the onset of stimulated degranulation of RBL-2H3 mast cells.

Dynamic remodeling of the actinomyosin cytoskeleton is integral to many biological processes. It is regulated, in part, by myosin phosphorylation. Nonmuscle myosin H chain IIA is phosphorylated by protein kinase C (PKC) on Ser(1917). Our aim was to determine the PKC isoform specificity of this phosphorylation event and to evaluate its potential role in regulated secretion. Using an Ab against the phosphorylated form of Ser(1917), we show that this site is not phosphorylated in unstimulated RBL-2H3 mast cells. The physiological stimulus, Ag, or the pharmacological activators, PMA plus A23187, induced Ser(1917) phosphorylation with a time course coincident with the onset of granule mediator secretion. Dephosphorylation at this site occurred as Ag-stimulated secretion declined from its peak, but dephosphorylation was delayed in cells activated with PMA plus A23187. Phosphate incorporation was also enhanced by PMA alone and by inhibition of protein phosphatase 2A. Gö6976, an inhibitor of conventional PKC isoforms, abolished secretion and Ser(1917) phosphorylation with similar dose dependencies consistent with involvement of either PKCalpha or PKCbeta. Phorbol ester-stimulated Ser(1917) phosphorylation was reconstituted in HEK-293 cells (which lack endogenous PKCbeta) by overexpression of both wild-type and constitutively active PKCbetaII but not the corresponding PKCbetaI or PKCalpha constructs. A similar selectivity for PKCbetaII overexpression was also observed in MIN6 insulinoma cells infected with recombinant PKC wild-type adenoviruses. Our results implicate PKC-dependent phosphorylation of myosin H chain IIA in the regulation of secretion in mast cells and suggest that Ser(1917) phosphorylation might be a marker of PKCbetaII activation in diverse cell types.

Mast cell function: regulation of degranulation by serine/threonine phosphatases.

Mast cells play both effector and modulatory roles in a range of allergic and immune responses. The principal function of these cells is the release of inflammatory mediators from mast cells by degranulation, which involves a complex interplay of signalling molecules. Understanding the molecular architecture underlying mast cell signalling has attracted renewed interest as the capacity for therapeutic intervention through controlling mast cell degranulation is now accepted as a viable proposition. The dynamic regulation of signalling by protein phosphorylation is a well-established phenomenon and many of the early events involved in mast cell activation are well understood. Less well understood however are the events further downstream of receptor activation that allow movement of granules through the cytoskeletal barrier and docking and fusion of granules with the plasma membrane. Whilst a potential role for the protein phosphatase family of signalling enzymes in mast cell function has been accepted for some time, the evidence has largely been derived from the use of broad specificity pharmacological inhibitors and results often depend upon the experimental conditions, leading to conflicting views. In this review, we present and discuss the pharmacological and recent molecular evidence that protein phosphatases, and in particular the protein phosphatase serine/threonine phosphatase type 2A (PP2A), have major regulatory roles to play and may be potential targets for the design of new therapeutic agents.

The role of serine/threonine protein phosphatases in exocytosis.

Modulation of exocytosis is integral to the regulation of cellular signalling, and a variety of disorders (such as epilepsy, hypertension, diabetes and asthma) are closely associated with pathological modulation of exocytosis. Emerging evidence points to protein phosphatases as key regulators of exocytosis in many cells and, therefore, as potential targets for the design of novel therapies to treat these diseases. Diverse yet exquisite regulatory mechanisms have evolved to direct the specificity of these enzymes in controlling particular cell processes, and functionally driven studies have demonstrated differential regulation of exocytosis by individual protein phosphatases. This Review discusses the evidence for the regulation of exocytosis by protein phosphatases in three major secretory systems, (1) mast cells, in which the regulation of exocytosis of inflammatory mediators plays a major role in the respiratory response to antigens, (2) insulin-secreting cells in which regulation of exocytosis is essential for metabolic control, and (3) neurons, in which regulation of exocytosis is perhaps the most complex and is essential for effective neurotransmission.

Troponin I phosphorylation enhances crossbridge kinetics during beta-adrenergic stimulation in rat cardiac tissue.

Inotropic agents that increase the intracellular levels of cAMP have been shown to increase crossbridge turnover kinetics in intact rat ventricular muscle, as measured by the parameter f(min) (the frequency at which dynamic stiffness is minimum). These agents are also known to increase the level of phosphorylation of two candidate myofibrillar proteins: myosin binding protein C (MyBPC) and Troponin I (TnI), but have no effect on myosin light chain 2 phosphorylation (MyLC2). The aim of this study was to investigate whether the phosphorylation of TnI and/or MyBPC was responsible for the increase in crossbridge cycling kinetics (as captured by f(min)) seen with the elevation of cAMP within cardiac tissue. Using barium-activated intact rat papillary muscle, we investigated the actions of isobutylmethylxanthine (IBMX), an inhibitor of cAMP-dependent phosphatase, which simulates the action of beta-adrenergic agents, and the chemical phosphatase 2,3-butanedione monoxime (BDM), which has been shown to dephosphorylate a number of contractile proteins. The presence of 0.6 mM IBMX approximately doubled the f(min) value of intact rat papillary muscle. This action was unaffected by the addition of BDM. In the presence of IBMX and BDM, the level of phosphorylation of MyBPC was unchanged, that of MyLC2 was reduced to 60 % of control, yet that of TnI was markedly increased (to 30 % above control levels). We conclude that TnI phosphorylation, mediated by cAMP-dependent protein kinase A, is the molecular basis for the enhanced crossbridge cycling seen during beta-adrenergic stimulation of the heart.

Protein phosphatases 1 and 2A transiently associate with myosin during the peak rate of secretion from mast cells.

Mast cells undergo cytoskeletal restructuring to allow secretory granules passage through the cortical actomyosin barrier to fuse with the plasma membrane and release inflammatory mediators. Protein phosphorylation is believed to regulate these rearrangements. Although some of the protein kinases implicated in this phosphorylation are known, the relevant protein phosphatases are not. At the peak rate of antigen-induced granule mediator release (2.5 min), protein phosphatases PP1 and PP2A, along with actin and myosin II, are transiently relocated to ruffles on the apical surface and a band at the peripheral edge of the cell. This leaves an area between the nucleus and the peripheral edge significantly depleted (3-5-fold) in these proteins. Phorbol 12-myristate 13-acetate (PMA) plus A23187 induces the same changes, at a time coincident with its slower rate of secretion. Coimmunoprecipitation experiments demonstrated a significantly increased association of myosin with PP1 and PP2A at the time of peak mediator release, with levels of association decreasing by 5 min. Jasplakinolide, an inhibitor of actin assembly, inhibits secretion and the cytoskeletal rearrangements. Surprisingly, jasplakinolide also affects myosin, inducing the formation of short rods throughout the cytoplasm. Inhibition of PP2A inhibited secretion, the cytoskeletal rearrangements, and led to increased phosphorylation of the myosin heavy and light chains at protein kinase C-specific sites. These findings indicate that a dynamic actomyosin cytoskeleton, partially regulated by both PP1 and PP2A, is required for mast cell secretion.

The complex nature of protein phosphatases.

Protein phosphatases are integrally associated with the regulation of cellular signaling. The mechanisms underlying the specific regulatory roles are likely to be unique to each cell system. Nevertheless, analysis of phosphatase regulation in a number of systems has identified phosphatase targeting through association with a wide range of binding partners to be a fundamental mechanism of regulation. Using protein phosphatase 2A (PP2A) as an example, this snapshot summarizes these fundamental mechanisms of protein phosphatase regulation.