Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Alimentary prion infections: Touchdown in the intestine

Neurodegenerative diseases are caused by proteinaceous aggregates, usually consisting of misfolded proteins which are often typified by a high proportion of β-sheets that accumulate in the central nervous system. These diseases, including Morbus Alzheimer, Parkinson disease and Transmissible Spongiform Encephalopathies (TSEs)—also termed prion disorders—afflict a substantial proportion of the human population and, as such, the etiology and pathogenesis of these diseases has been the focus of mounting research. Although many of these diseases arise from genetic mutations or are sporadic in nature, the possible horizontal transmissibility of neurodegenerative diseases poses a great threat to population health. In this article we discuss recent studies that suggest that the “non-transmissible” status bestowed upon Alzheimer and Parkinson diseases may need to be revised as these diseases have been successfully induced through tissue transplants. Furthermore, we highlight the importance of investigating the “natural” mechanism of prion transmission including peroral and perenteral transmission, proposed routes of gastrointestinal uptake and neuroinvasion of ingested infectious prion proteins. We examine the multitude of factors which may influence oral transmissibility and discuss the zoonotic threats that Chronic Wasting disease (CWD), Bovine Spongiform Encephalopathy (BSE) and Scrapie may pose resulting in vCJD or related disorders. In addition, we suggest that the 37 kDa/67 kDa laminin receptor on the cell surface of enterocytes, a major cell population in the intestine, may play an important role in the intestinal pathophysiology of alimentary prion infections.Key words: prion, 37 kDa/67 kDa laminin receptor, CJD, BSE, CWD, scrapie, Alzheimer disease, Parkinson disease, intestine, enterocytesMany different mechanisms exist which underlie the etiology of the numerous neurodegenerative diseases affecting the human population. Amongst the most prominent are Morbus Alzheimer, prion disorders, Parkinson disease, Chorea Huntington, frontotemporal dementia and amylotrophic lateral sclerosis. The molecular mechanisms underlying these diseases vary; however, all neurodegenerative diseases share a common feature: they are caused by protein aggregation. The only neurodegenerative diseases proven to be transmissible are prion disorders. In contrast to frontotemporal dementia, recent evidence suggests that Alzheimer and Parkinson diseases may also be transmissible. Pre-symptomatic Alzheimer disease (APP23) mice exhibited an increase in the Alzheimer phenotype when brain homogenate of autopsied human Alzheimer disease patients and older, amyloid beta- (Aβ-) laden APP23 mice was injected into their hippocampi.¹ These findings suggest that the Aβ-abundant brain homogenate of Alzheimer disease patients may possess the ability to induce or supplement the overproduction of Aβ, possibly leading to the onset of Alzheimer disease.The pathological feature associated with Parkinson disease is the formation of Lewy bodies in cell bodies and neuronal processes in the brain.² The main component of these protein aggregates is α-synuclein (reviewed in ref. ²). Autopsies of Parkinson disease patients revealed that Lewy bodies had formed on healthy embryonic neurons that had been grafted onto the brain tissue of the patients several years before (prior to said examination).^3–5 It may thus be proposed that α-synuclein transmission is possible from diseased to healthy neurons, suggesting that Parkinson disease may be transmissible from a Parkinson disease patient to a healthy individual. These findings imply that Alzheimer and Parkinson diseases may be transmissible through tissue transplants and the use of contaminated surgical tools.⁶Prion disorders, also termed Transmissible Spongiform Encephalopathies (TSEs), are fatal neurodegenerative diseases that affect the central nervous system (CNS) of multiple animal species. In lieu of the social, economic and political ramifications of such infections, as well as the possible intra- and interspecies transmissibility of such disorders, various routes of experimental transmission have been investigated including intracerebral, intraperitoneal, intraventricular, intraocular, intraspinal and subcutaneous injections (reviewed in ref. ^7–9). However, such routes of transmission are not representative of the “natural” mechanism as the majority of prion disorders are contracted through ingestion of infectious prion (PrP^Sc) containing material. Thus, the peroral and perenteral prion transmission is of greatest consequence with respect to TSE disease establishment. Moreover, the presence of PrP^Sc in the buccal cavity of scrapie-infected sheep¹⁰ (reviewed in ref. ¹¹) and the possible horizontal transfer as a result hereof, as may be similarly proposed for animals suffering from other TSEs, may further contribute to the oral transmissibility of TSEs.A number of model systems have been employed to study TSE transmissibility. Owing to ethical constraints, TSE transmissibility to humans via the oral route may not be directly investigated and as a result hereof, alternative model systems are needed. These may include the use of transgenic mice, cell lines which are permissive to infection¹² and experimental animals such as sheep, calves, goats, minks, ferrets and non-human primates (reviewed in ref. ⁹).Intestinal entry of PrP^Sc has been proposed to occur via two pathways, the membranous (M) cell-dependent and M cell-independent pathways (Fig. 1).^13,14 The former involves endocytic M (microfold)-cells, which cover the intestinal lymphoid follicles (Peyer''s patches)¹⁴ and may take up prions and thereby facilitate the translocation of these proteins across the intestinal epithelium into the lymphoid tissues (reviewed in ref. ⁹) as has been demonstrated in a cellular model.¹³ Following such uptake by the M cells, the prions may subsequently pass to the dendritic cells and follicular dendritic cells (FDCs) (Fig. 1), which allow for prion transport to the mesenteric lymph nodes and replication, respectively.¹⁵ The prion proteins may subsequently gain access to the enteric nervous system (ENS) and ultimately the central nervous system (CNS).¹⁵Open in a separate windowFigure 1Proposed routes of gastrointestinal entry of ingested infectious prions (PrP^Sc) as well as possible pathways of amplification and transport to the central nervous system.However, prion intestinal translocation has been observed in the absence of M cells and has been demonstrated to be as a result of the action of polar, 37 kDa/67 kDa LRP/LR (non-integrin laminin receptor; reviewed in ref. ^16–18) expressing enterocytes. Enterocytes are the major cell population of the intestinal epithelium and due to their ability to endocytose pathogens, nutrients and macromolecules,¹⁹ it has been proposed that these cells may represent a major entry site for alimentary prions (Fig. 1).Since enterocyte prion uptake has been demonstrated to be dependent on the presence of LRP/LR on the apical brush border of the cells,^14,20 the interaction between varying prion protein strains and the receptor^21–23 may be employed as a model system to study possible oral transmissibility of prion disorders across species as well as the intestinal pathophysiology of alimentary prion infections.²⁴ Moreover, the blockage of such interactions through the use of anti-LRP/LR specific antibodies has been reported to reduce PrP^Sc endocytosis¹⁹ and thus these antibodies may serve as potential therapeutics to prevent infectious prion internalization and thereby prevent prion infections. It must be emphasized that the adhesion of prion proteins to cells is not solely dependent on the LRP/LR-PrP^Sc interactions;²⁴ however, this interaction is of importance with regards to internalization and subsequent pathogenesis.We applied the aforementioned cell model to study the possible oral transmission of PrP^BSE, PrP^CWD and ovine PrP^Sc to cervids, cattle, swine and humans.²⁴ The direct transmission of the aforementioned animal prion disorders to humans as a result of dietary exposure and the possible establishment of zoonotic diseases is of great public concern. It must however be emphasized that the study investigated the co-localization of LRP/LR and various prion strains and not the actual internalization process.PrP^BSE was shown to co-localize with LRP/LR on human enterocytes²⁴, thereby suggesting that PrP^BSE is transmissible to humans via the oral route which is widely accepted as the manner by which variant CJD originated. This suspicion was previously investigated using a macaque model, which was successfully perorally infected by BSE-contaminated material and subsequently lead to the development of a prion disorder that resembles vCJD.²⁵ These results, due to the evolutionary relatedness between macaques and humans, allowed researchers to confirm the oral transmissibility of PrP^BSE to humans. PrP^BSE may also potentially lead to prion disorder establishment in swine,²⁴ livestock of great economic and social importance.The prion disorder affecting elk, mule deer and white-tailed deer is termed CWD. Cases of the disease are most prevalent in the US but are also evident in Canada and South Korea.^26,27 As the infectious prion isoform is reported to be present in the blood²⁸ and skeletal muscle,²⁹ hunting, consumption of wild venison and contact with other animal products derived from CWD-infected elk and deer may thereby pose a public health risk. Our studies demonstrate that PrP^CWD co-localizes with LRP/LR on human enterocytes²⁴ thereby suggesting a possible oral transmissibilty of this TSE to humans. This is, however, inconsistent with results obtained during intra-cerebral inoculation of the brains and spinal cords of transgenic mice overexpressing the human cellular prion protein (PrP^c),^26,27 which is essential for TSE disease establishment and progression. Further, discrepancies have also been reported with respect to non-human primates, as squirrel monkeys have been successfully intracerebrally inoculated with mule-deer prion homogenates,³⁰ while cynolmolgus macaques were resistant to infection.³¹ CWD has been transmitted to ferrets, minks and goats³² and as these animals may serve as domestic animals or livestock, secondary transmission from such animals to humans, through direct contact or ingestion of infected material, may be an additional risk factor that merits further scientific investigation.Ovine PrP^Sc co-localization with LRP/LR on human and bovine enterocytes may be indicative of the infectious agents'' ability to effect cross-species infections. The oral transmissibility of Scrapie has been confirmed in hamsters fed with sheep-scrapie-infected material.³³The discrepancies with regards to the transmissibility of certain infectious prion proteins when assessed by different model systems may be due to the experimental transmission route employed. Oral exposure often results in significantly prolonged incubation times when compared to intracerebral inoculation techniques and thus failure of transgenic mice and normal experimental animals to develop disease phenotypes after being fed TSE-contaminated material may not necessarily indicate that the infection process failed.¹⁴ Apart from the route of infection, numerous other factors may influence transmission between species, including dose, PrP polymorphisms and genetic factors, the prion strain employed as well as the efficacy of prion transport to the CNS.³⁴ The degree of homology between the PrP^c protein in the animals serving as the infectious prion source and recipient has also been described as a feature limiting cross-species transmission.³⁴ The negative results, as referred to above, obtained upon prion-protein inoculation of animal models may have resulted due to the slow rate at which the infectious prion induces conformational conversion of the endogenous PrP^c in the animal cells and this in turn results in low levels of infectious prion replication and symptom development.²⁷Furthermore, even in the event that certain prion disorders are not directly transmissible to humans, most are transmissible to at least a single species of domestic animal or livestock. The infectious agents properties may be altered in the secondary host such that it becomes transmissible to humans (reviewed in ref. ³⁵). Thus, interspecies transmission between animals may indirectly influence human health.It is noteworthy to add that although the oral route of PrP^Sc transmission may result in prolonged incubation times, it may broaden the range of susceptible hosts. A common constituent of food is ferritin, a protein that is resistant to digestive enzyme hydrolysis and, due to its homology across species, it may serve as co-transporter of PrP^Sc and facilitate enterocyte internalization of the infectious prion.³⁶ It may thus be proposed that prion internalization may occur via a ferritin-PrP^Sc complex even in the absence of co-localization between the infectious agent and LRP/LR such that many more cross-species infections (provided that the other infection factors are favorable) may be probable.³⁷ In addition, digestive enzymes in the gastrointestinal tract facilitate PrP^Sc binding to the intestinal epithelium and subsequent intestinal uptake³⁶ and thus depending on the individuals'' digestive processes, the susceptibility to infection and the rate of disease development may vary accordingly. As a result hereof, though laboratory experiments in cell-culture and animal models may render a particular prion disorder non-infectious to humans, this may not be true for all individuals.In lieu of the above statements, with particular reference to inconsistencies in reported results and the multiple factors influencing oral transmissibility of TSEs, further transmission studies are required to evaluate the zoonotic threat which CWD, BSE and Scrapie may pose through ingestion.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Disulfide Bond Formation Significantly Accelerates the Assembly of Ure2p Fibrils because of the Proximity of a Potential Amyloid Stretch

Aggregation of the Ure2 protein is at the origin of the [URE3] prion trait in the yeast Saccharomyces cerevisiae. The N-terminal region of Ure2p is necessary and sufficient to induce the [URE3] phenotype in vivo and to polymerize into amyloid-like fibrils in vitro. However, as the N-terminal region is poorly ordered in the native state, making it difficult to detect structural changes in this region by spectroscopic methods, detailed information about the fibril assembly process is therefore lacking. Short fibril-forming peptide regions (4–7 residues) have been identified in a number of prion and other amyloid-related proteins, but such short regions have not yet been identified in Ure2p. In this study, we identify a unique cysteine mutant (R17C) that can greatly accelerate the fibril assembly kinetics of Ure2p under oxidizing conditions. We found that the segment QVNI, corresponding to residues 18–21 in Ure2p, plays a critical role in the fast assembly properties of R17C, suggesting that this segment represents a potential amyloid-forming region. A series of peptides containing the QVNI segment were found to form fibrils in vitro. Furthermore, the peptide fibrils could seed fibril formation for wild-type Ure2p. Preceding the QVNI segment with a cysteine or a hydrophobic residue, instead of a charged residue, caused the rate of assembly into fibrils to increase greatly for both peptides and full-length Ure2p. Our results indicate that the potential amyloid stretch and its preceding residue can modulate the fibril assembly of Ure2p to control the initiation of prion formation.The [URE3] phenotype of Saccharomyces cerevisiae arises because of conversion of the Ure2 protein to an aggregated propagatable prion state (1, 2). Ure2p contains two regions: a poorly structured N-terminal region and a compactly folded C-terminal region (3, 4). The N-terminal region is rich in Asn and Gln residues, is highly flexible, and is without any detectable ordered secondary structure (4–6). This region is necessary and sufficient for prion behavior in vivo (2) and amyloid-forming capacity in vitro (5, 7), so it is referred to as the prion domain (PrD).² The C-terminal region has a fold similar to the glutathione S-transferase superfamily (8, 9) and possesses glutathione-dependent peroxidase activity (10). Upon fibril formation, the N-terminal region undergoes a significant conformational change from an unfolded to a thermally resistant conformation (11), whereas the glutathione S-transferase-like C-terminal domain retains its enzymatic activity, suggesting that little conformational change occurs (10, 12). Ure2p fibrils show various morphologies, including variations in thickness and the presence or absence of a periodic twist (13–16). The overall structure of the fibrils imaged by cryoelectron microscopy suggests that the intact fibrils contain a 4-nm amyloid filament backbone surrounded by C-terminal globular domains (17).It is widely accepted that disulfide bonds play a critical role in maintaining protein stability (18–21) and also affect the process of protein folding by influencing the folding pathway (22–25). A recent study shows that the presence of a disulfide bond in a protein can markedly accelerate the folding process (26). Therefore, a disulfide bond is a useful tool to study protein folding. In the study of prion and other amyloid-related proteins, cysteine scanning has been widely used to study the structure of amyloid fibrils, the driving force of amyloid formation, and the plasticity of amyloid fibrils (13, 27–31).Short segments from amyloid-related proteins, including IAPP (islet amyloid polypeptide), β₂-microglobulin, insulin, and the amyloid-β peptide, show amyloid-forming capacity (32–34). Hence, the amyloid stretch hypothesis has been proposed, which suggests that a short amino acid stretch bearing a highly amyloidogenic motif might supply most of the driving force needed to trigger the self-catalytic assembly process of a protein to form fibrils (35, 36). In support of this hypothesis, it was found that the insertion of an amyloidogenic stretch into a non-amyloid-related protein can trigger the amyloidosis of the protein (36). At the same time, the structural information obtained from microcrystals formed by amyloidogenic stretches and bearing cross-β-structure has contributed significantly to our understanding of the structure of intact fibrils at the atomic level (34, 37). However, no amyloidogenic stretches <10 amino acids have so far been identified in the yeast prion protein Ure2.In this study, we performed a cysteine scan within the N-terminal PrD of Ure2p and found a unique cysteine mutant (R17C) that eliminates the lag phase of the Ure2p fibril assembly reaction upon the addition of oxidizing agents. Furthermore, we identified a 4-residue region adjacent to Arg¹⁷ as a potential amyloid stretch in Ure2p.     

Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

The role of the prion protein membrane anchor in prion infection

Normal cellular and abnormal disease-associated forms of prion protein (PrP) contain a C-terminal glycophosphatidyl-inositol (GPI) membrane anchor. The importance of the GPI membrane anchor in prion diseases is unclear but there are data to suggest that it both is and is not required for abnormal prion protein formation and prion infection. Utilizing an in vitro model of prion infection we have recently demonstrated that, while the GPI anchor is not essential for the formation of abnormal prion protein in a cell, it is necessary for the establishment of persistent prion infection. In combination with previously published data, our results suggest that GPI anchored PrP is important in the amplification and spread of prion infectivity from cell to cell.Key words: prion, GPI anchor, PrP, prion spread, scrapieIn transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrP^C) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrP^Sc. PrP-res/PrP^Sc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site.¹ Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.In tissue culture cells infected with mouse scrapie, PrP-res formation occurs at the cell surface and/or along the endocytic pathway^2–4 and may be dependent upon the membrane environment of PrP-sen. For example, localization via the GPI anchor to caveolae-like domains favors PrP-res formation⁵ while substitution of the GPI anchor addition site with carboxy termini favoring transmembrane anchored PrP-sen inhibits formation of PrP-res.^5,6 Other studies have shown that localization of both PrP-sen and PrP-res to lipid rafts, cholesterol and sphingolipid rich membrane microdomains where GPI anchored proteins can be located, is important in PrP-res formation.^6–9However, there are also data which suggest that such localization is not necessarily essential for PrP-res formation. Anchorless PrP-sen isolated from cells by immunoprecipitation or wild-type PrP-sen purified by immunoaffinity column followed by cation exchange chromatography are efficiently converted into PrP-res in cell-free systems.^10,11 Furthermore, recombinant PrP-sen derived from E. coli, which has no membrane anchor or glycosylation, can be induced to form protease-resistant PrP in vitro when reacted with prion-infected brain homogenates.^12–14 Finally, in at least one instance, protease-resistant recombinant PrP-res generated in the absence of infected brain homogenate was reported to cause disease when inoculated into transgenic mice.¹⁵The data concerning the role of the PrP-sen GPI anchor in susceptibility to TSE infection are similarly contradictory. Transgenic mice expressing anchorless mouse PrP-sen are susceptible to infection with mouse scrapie and accumulate both PrP-res and prion infectivity.¹⁶ Thus, the GPI anchor is clearly not needed for PrP-res formation or productive TSE infection in vivo. However, we recently published data demonstrating that, in vitro, anchored PrP-sen is in fact required to persistently infect cells.¹⁷ Given that anchorless PrP-sen is not present on the cell surface but is released into the cell medium, we speculated that the differences between the in vitro and in vivo data were related to the location of PrP-res formation. In the mice expressing anchorless PrP-sen, environments conducive to PrP-res formation are present in certain areas of the complex extracellular milieu of the brain where anchorless, secreted PrP-sen can accumulate and come into contact with PrP-res from the infectious inoculum. Since similar environments are missing in vitro, any PrP-res formation in cells expressing anchorless PrP-sen must be cell-associated. While this explanation addresses how extracellular PrP-res could be generated in an unusual transgenic mouse model of TSE infection, it does not really help to define how the GPI anchor is involved in normal prion infection of a cell.As with other infectious organisms such as viruses, TSE infection can be roughly divided into three steps: uptake, replication and spread. Over the last several years, data derived from new techniques as well as new cell lines susceptible to prion infection have increased our knowledge of some of the basic events that occur during each of these steps. In order to try to tease out the role of the GPI anchor in normal TSE pathogenesis, it is therefore useful to consider the process of TSE infection of a cell and how the GPI anchor might be involved in each stage.In a conventional viral infection, binding and uptake of the virus is essential to establish infection. Studying PrP-res uptake has been complicated by the lack of an antibody that can specifically distinguish PrP-res from PrP-sen in live cells and by the difficulty of detecting the input PrP-res from the PrP-res made de novo by the cell. Recently, however, several groups have been able to study PrP-res uptake using input PrP-res that was either fluorescently labeled^18–20 or tagged with the epitope to the monoclonal antibody 3F4,²¹ or cell lines that express little or no PrP-sen.^19,21–23 The data show that PrP-res uptake is independent of scrapie strain or cell type but is influenced by the PrP-res microenvironment as well as PrP-res aggregate size.²¹ Importantly, these studies demonstrated that PrP-sen expression was not required.^19,21–23 Given these data, it is clear that GPI anchored PrP-sen is not involved in the initial uptake of PrP-res into the cell.The next stage of prion infection involves replication of infectivity which is typically assayed by following cellular PrP-res formation. Once again, however, the issue of how to distinguish PrP-res in the inoculum from newly formed PrP-res in the cells has made it difficult to study the early stages of prion replication. To overcome this difficulty, we developed a murine tissue culture system that utilizes cells expressing mouse PrP-sen tagged with the epitope to the 3F4 antibody (Mo3F4 PrP-sen).²⁴ Wild-type mouse PrP does not have this epitope. As a result, following exposure to an infected mouse brain homogenate, de novo PrP-res formation can be followed by assaying for 3F4 positive PrP-res. Our studies showed that there were two stages of PrP-res formation: (1) an initial acute burst within the first 96 hours post-infection that was cell-type and scrapie strain independent and, (2) persistent PrP-res formation (i.e., formation of PrP-res over multiple cell passages) that was dependent on cell-type and scrapie strain and associated with long-term infection.²⁴ Acute PrP-res formation did not necessarily lead to persistent PrP-res formation suggesting that other cell-specific factors or processes are needed for PrP-res formation to persist.²⁴When cells expressing Mo3F4 PrP-sen without the GPI anchor (Mo3F4 GPI-PrP-sen) were exposed to mouse scrapie infected brain homogenates, GPI negative, 3F4 positive PrP-res (Mo3F4 GPI-PrP-res) was detected within 96 hours indicating that acute PrP-res formation had occurred.¹⁷ Thus, despite the fact that Mo3F4 GPI-PrP-sen is not expressed on the cell surface¹⁶ (Fig. 1A), it was still available for conversion to PrP-res. These results are consistent with data from cell-free systems and demonstrate that, at least acutely, membrane anchored PrP is not necessary for PrP-res formation in a cell.Open in a separate windowFigure 1Persistent infection of cells in vitro requires the expression of GPI-anchored cell surface PrP-sen. PrP knockout cells (CF10)²¹ were transduced with 3F4 epitope tagged mouse PrP-sen (Mo3F4), 3F4 epitope tagged mouse PrP-sen without the GPI anchor (Mo3F4 GPI-), or Mo3F4 GPI-PrP-sen plus wild-type, GPI anchored mouse PrP-sen (MoPrP). The cells were then exposed to the mouse scrapie strain 22L and passaged. (A) The presence of 3F4 epitope tagged, cell surface mouse PrP-sen was assayed by FACS analysis of fixed, non-permeabilized cells. CF10 cells expressing the following mouse PrP-sen molecules were assayed: Mo3F4 (solid line); Mo3F4 GPI⁻ (dashed line); Mo3F4 GPI⁻ + MoPrP (dotted and dashed line); Mo3F4 GPI⁻ + MoPrP infected with 22L scrapie (dotted line). Only cells expressing Mo3F4 PrP-sen were positive for cell surface, 3F4 epitope tagged PrP. (B) Persistent infection was analyzed by inoculating the cells intracranially into transgenic mice overexpressing MoPrP (Tga20 mice). Only cells expressing anchored mouse PrP-sen were susceptible to scrapie infection. Cells expressing anchorless mouse PrP-sen did not contain detectable infectivity in either the cells or the cellular supernatant (data not shown). Data in (B) are adapted from McNally 2009.¹⁷In terms of persistent PrP-res formation, however, our data suggest that the GPI anchor is important. Despite an initial burst of PrP-res formation within the first 96 hours post-infection, Mo3F4 GPI-PrP-res was not observed following passage of the cells nor did the cells become infected. This effect was not due either to resistance of the cells to scrapie infection or to an inability of the scrapie strain used to infect cells. When the same cells expressed anchored Mo3F4 PrP-sen and were exposed to the same mouse scrapie strain, both acute and persistent PrP-res formation were detected and the cells were persistently infected with scrapie (Fig. 1B).¹⁷ Taken together, these data demonstrate that cells expressing anchorless PrP-sen do not support persistent PrP-res formation. Furthermore, the data strongly suggest that GPI-anchored PrP-sen is required during the transition from acute to persistent scrapie infection. In support of this hypothesis, the resistance of cells expressing Mo3F4 GPI-PrP-sen to persistent prion infection could be overcome if wild-type GPI anchored PrP-sen was co-expressed in the same cell. When both forms of PrP-sen were expressed, anchored and anchorless forms of PrP-res were made and the cells became persistently infected (Fig. 1B).¹⁷ Thus, the data suggest that GPI anchored PrP is necessary to establish prion infection within a cell.How could GPI membrane anchored PrP be involved in the establishment and maintenance of persistent prion infection? Several studies have suggested that the GPI anchor is needed to localize PrP-sen to specific membrane environments where PrP-res formation is favored.^5–8 However, if this localization was essential for PrP-res formation, GPI-PrP-sen would presumably never form PrP-res. Lacking the GPI anchor, it would not be in the correct membrane environment to support conversion. As a result, neither acute nor persistent prion infection could occur. This is obviously not the case. Transgenic mice expressing only anchorless PrP-sen generate PrP-res and can be infected with scrapie even though (1) flotation gradients showed that anchorless PrP-sen was not in the same membrane environment as anchored PrP-sen and, (2) flow cytometry analysis demonstrated that anchorless PrP-sen was not present on the cell surface.¹⁶ Thus, the GPI anchor is not needed to target PrP-sen to a conversion friendly membrane environment.Consistent with the idea that the GPI anchor is not essential for PrP-res formation, in our studies anchorless PrP-sen could form PrP-res in cells acutely infected with scrapie despite the fact that it is processed differently than anchored PrP-sen, is not present on the cell surface (Fig. 1A), and is secreted.¹⁷ Persistent formation of anchorless PrP-res only occurred when both anchored and anchorless forms of PrP were expressed in the same cell.¹⁷ For this to happen both types of PrP must share a cellular compartment where PrP-res formation occurs, presumably either on the cell surface or in a specific location along the endocytic pathway^2,3 such as the endosomal recycling compartment.⁴ Analysis of infected and uninfected cells co-expressing Mo3F4 GPI-PrP-sen and wild-type PrP-sen demonstrated that Mo3F4 GPI-PrP-sen was not present on the cell surface (Fig. 1A). Thus, it is unlikely that GPI-PrP-res formation is occurring on the cell surface. We speculate that the anchored form of PrP-res encounters anchorless PrP-sen along either a secretory or endocytic pathway, allowing for the formation of anchorless PrP-res. Regardless of the precise location, the in vitro and in vivo data strongly suggest that the role of the anchor in persistent prion infection is not simply to localize PrP-sen to an environment compatible with PrP-res formation.However, the data are consistent with the idea that GPI anchored PrP is absolutely essential for the establishment of persistent infection in vitro. This is likely related to the spread of infectivity within a culture that is necessary for maintaining a persistent infection over time. Evidence suggests that PrP-res can be transferred between cells in a variety of ways including mother-daughter cell division,²⁵ cell-to-cell contact^26,27 and exosomes.²⁸ Tunneling nanotubes have also been hypothesized to be involved in intercellular prion spread¹⁹ and recent data suggest that spread can occur via these structures.²⁰ Any of these processes could involve the cell-to-cell transfer of PrP-res in membrane containing particles as has been observed in cell-free⁷ and cell-based systems.²⁹ If cell-to-cell contact were required, for example via simple physical proximity or perhaps tunneling nanotubes,^19,20 then the conversion of cell surface PrP-sen on the naïve cell by cell surface PrP-res on the infected cell would transfer infection to the naïve cell. In this instance, GPI membrane anchored, cell surface PrP-sen would be essential as it would allow for PrP-res formation on the cell surface. If spread is via cell division, then GPI-anchored, cell surface PrP-sen would be important for its role as a precursor to PrP-res formation.² In this instance, cell surface PrP-sen would be an essential intermediate in the continuous formation of PrP-res necessary for the accumulation and amplification of PrP-res within the cell. It would also help to cycle PrP between the cell surface and intracellular compartments where PrP-res can be formed.⁴ In either case, GPI-anchored PrP-sen would facilitate the accumulation of intracellular PrP-res to high enough levels to maintain both persistent infection in the mother cell and enable the transfer of organelles containing sufficient PrP-res to initiate infection in the daughter cell. Thus, we would suggest that efficient spread of infectivity requires not just the passive transfer of PrP-res from cell-to-cell but the concurrent initiation of conversion and amplification of PrP-res via cell surface, GPI anchored PrP-sen.In vivo, several lines of evidence suggest that the spread of scrapie infectivity also requires de novo PrP-res formation in the recipient cell and not simply transfer of PrP-res from one cell to another. For example, when neurografts from PrP expressing mice were placed in the brains of PrP knockout mice and the mice were challenged intracranially with scrapie, the graft showed scrapie pathology, but the surrounding tissue did not.³⁰ Furthermore, PrP-res from the graft migrated to the host tissue demonstrating that simple transfer of PrP-res was not sufficient and that PrP-sen expression was required for the spread of scrapie pathology.³⁰ In fact, these mice did not develop scrapie pathology following peripheral infection even when peripheral lymphoid tissues were reconstituted with PrP-sen expressing cells.³¹ Even though PrP-sen expressing cells were present in both the brain and spleen, in order for infectivity to spread from the lymphoreticular system to the central nervous system PrP-sen expression was also required in an intermediate tissue such as peripheral nerve.^31,32 Given that PrP-res uptake and trafficking do not require PrP-sen, the most obvious explanation for the requirement of PrP-sen in contiguous tissues is that de novo PrP-res formation in naïve cells is necessary for (1) infectivity to move from cell to cell within a tissue and, (2) infectivity to move from tissue to tissue.Another study demonstrated that peripheral expression of heterologous mouse PrP significantly increased the incubation time and actually prevented clinical disease in the majority of transgenic mice expressing hamster PrP in neurons of the brain.³³ Once again, if simple transfer and uptake of PrP-res were sufficient for spread, the presence of heterologous PrP molecules should not interfere because cellular uptake of PrP-res is independent of PrP-sen expression.^19,21–23 Clinical disease in these mice was likely prevented by the heterologous PrP molecule interfering with conversion of PrP-sen to PrP-res suggesting that prevention of de novo PrP-res formation inhibits spread of PrP-res and infectivity. These in vivo data, when combined with our recent in vitro data,¹⁷ provide evidence to support the importance of cell surface, and by extension GPI-anchored, PrP in the spread of prion infection.Our data demonstrate that the GPI anchor plays a role in the establishment of persistent scrapie infection in vitro. In our tissue culture system,²¹ as well as others where spread of infectivity by cell to cell contact appears to be limited,^25,34 the role of GPI anchored PrP-sen would be to amplify PrP-res to enable the efficient transfer of infectivity from mother to daughter cell. In cell systems where spread of prion infectivity may require cell to cell contact,^26,27 we propose that the role of GPI anchored PrP-sen is to facilitate the spread of prion infection via a chain of conversion from cell-to-cell, a “domino” type spread of infection that has been previously hypothesized.^35,36In vivo, such a mechanism might explain why neuroinvasion does not necessarily require axonal transport^32,37,38 and can occur independently of the axonal neurofilament machinery.³⁹ It would likely vary with cell type²⁷ and be most important in areas where infectivity is transferred from the periphery to the nervous system as well as in areas where cell division may be limited. It is also possible, if the location of PrP-res formation differs for different scrapie strains,⁴⁰ that the relative importance of a domino-like spread of infectivity in vivo would vary with the scrapie strain.Of course, spread of infectivity via a “wave” of GPI anchored, PrP mediated conversion would not preclude the spread of infectivity by other intracellular means such as axonal transport (reviewed in ref. ⁴¹). Furthermore, spread of infectivity may still also occur extracellularly such as in the unique case of mice which express anchorless PrP-sen,¹⁶ where our in vitro data would suggest that the cells themselves are not infected. In such a case, spread would require neither GPI anchored PrP-sen nor amplification of PrP-res in cells but would likely occur via other means such as blood⁴¹ or interstitial fluid flow.⁴²     

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis

The process of epithelial lumenogenesis requires coordination of a network of signaling machinery communicated to each cell through subsequent cell divisions. Formation of a single hollow lumen has previously been shown to require Tuba, a Cdc42 GEF, for Cdc42 activation and correct spindle orientation. Using a Caco-2 model of lumenogenesis, we show that knockdown (KD) of the actin regulator N-WASP, causes a multilumen phenotype similar to Tuba KD. Defects in lumenogenesis in Tuba KD and N-WASP KD cells are observed at the two-cell stage with inappropriate marking of the pre-apical patch (PAP )—the precursor to lumen formation. Strikingly, both Tuba and N-WASP depend on each other for localization to the PAP. We conclude that N-WASP functions cooperatively with Tuba to facilitate lumenogenesis and this requires the polyproline region of N-WASP.Key words: lumen, N-WASP, tuba, E-cadherin, pre-apical patchMany epithelial tissues are organized as hollow tubes whose open lumina connect the body with its external environment.^1,2 These tubes consist of a monolayer of polarized cells that envelope the central lumen. Lumen formation is thus a key process in epithelial morphogenesis that depends upon cell polarity to establish three cell surface domains: a basal surface adherent to the extracellular matrix, a lateral surface between cells, and an apical surface that is exposed to the luminal fluids. Of note, the apical membrane is biochemically and morphologically distinct from the baso-lateral surfaces and effectively defines the luminal surface.^3,4For a lumen to form, cells must first mark the site at which apical membrane is to be inserted, something that is achieved at the first cell division.⁵ Targeted trafficking of apical membrane constituents defines a pre-apical patch (PAP), the precursor to the definitive lumen.⁵ Such insertion of apical membrane must presumably be coordinated with the assembly of apical junctions to segregate nascent apical from lateral membrane domains.² Subsequent cell divisions direct apical membrane and protein constituents to this point of initial apical membrane placement.⁶ Coordinated luminal positioning enables the initial formation of a single hollow lumen that subsequently expands through polarized fluid secretion to separate apical membranes, such as occurs in the embryonic gastrointestinal tract,⁷ or by apoptosis or autophagy of the central cells as is observed in mammary gland development.^8,9 Failure to establish initial luminal positioning causes defective lumenogenesis, often resulting in multiple, morphologically abnormal lumina.^5,6Crucial to lumenal morphogenesis is then the mechanism(s) that mark the site where the PAP will form. Cdc42 signaling is increasingly implicated in this process,^2,10 with downstream consequences that include control of mitotic spindle orientation,⁵ which itself influences PAP placement⁵ and potentially regulation of cell-cell junctions. Like other Rho family GTPases, the subcellular location of Cdc42 signaling is determined by the action of upstream proteins, notably guanine nucleotide exchange factors (GEFs).^11,12 Of these, Tuba, a Cdc42-specific GEF,¹³ has emerged as a regulator of lumenal morphogenesis that controls PAP placement through mitotic spindle orientation.¹⁰Tuba is also a scaffolding protein¹³ capable of linking the actin assembly machinery with trafficking pathways. Not only is Tuba required for Cdc42 activation to direct spindle orientation,⁵ it also has the potential to interact with phosphoinositides that define the PAP.¹⁴ Additionally, Tuba binds directly to the actin regulator N-WASP, a key molecule in the organization of actin and itself a Cdc42 effector.¹⁵ Further, Tuba and N-WASP cooperate in various forms of actin-driven cellular motility, such as vesicle propulsion and cell invasive behavior.¹⁶ Interestingly, in epithelial cells N-WASP is also found at cadherin-based cell-cell junctions.¹⁷ In fact it has been proposed that N-WASP functions downstream of Tuba in the maintenance of epithelial junctional homeostasis as N-WASP overexpression was capable of rescuing a Tuba KD phenotype.¹⁸ Therefore, Tuba has the potential to play a central role in coordinating the molecular complexes required for productive polarization of epithelial cells and placement of the PAP during lumenogenesis. However, whether other protein interactions contribute to the morphogenetic impact of Tuba remain to be assessed.Three-dimensional cell culture systems are being utilized to identify critical components in lumen formation. In particular, Madin-Darby canine kidney cells (MDCK) and Caco-2 gastrointestinal cells are commonly used to study cyst and/or tubule formation. MDCK cells undergo both cyst and tubule growth, apoptosis being primarily responsible for the final step in lumen formation,¹⁹ while Caco-2 cells primarily utilize fluid influx to expand cysts.⁵ Cyst culture systems replicate aspects of in vivo organogenesis²⁰ providing tangible, powerful models to analyze and dissect the coordinated cellular mechanisms and processes that occur during epithelial morphogenesis.In this study we examined the relationship between Tuba and N-WASP in early epithelial lumenogenesis using Caco-2 three dimensional cyst cultures. Both Tuba and N-WASP RNAi cell lines result in mature cysts with multiple lumina, and at the two-cell stage, formed multiple PAPs. Interestingly, N-WASP KD perturbed Tuba localization at the PAP, however, N-WASP localization to the PAP was not affected to the same extent by Tuba KD. Taken together, these results suggest a complex interrelationship between Tuba and N-WASP for the coordinated formation of a single hollow lumen.     

Aerosols: An underestimated vehicle for transmission of prion diseases?

We and others have recently reported that prions can be transmitted to mice via aerosols. These reports spurred a lively public discussion on the possible public-health threats represented by prion-containing aerosols. Here we offer our view on the context in which these findings should be placed. On the one hand, the fact that nebulized prions can transmit disease cannot be taken to signify that prions are airborne under natural circumstances. On the other hand, it appears important to underscore the fact that aerosols can originate very easily in a broad variety of experimental and natural environmental conditions. Aerosols are a virtually unavoidable consequence of the handling of fluids; complete prevention of the generation of aerosols is very difficult. While prions have never been found to be transmissible via aerosols under natural conditions, it appears prudent to strive to minimize exposure to potentially prion-infected aerosols whenever the latter may arise—for example in scientific and diagnostic laboratories handling brain matter, cerebrospinal fluids, and other potentially contaminated materials, as well as abattoirs. Equally important is that prion biosafety training be focused on the control of, and protection from, prion-infected aerosols.Key words: prion, prion transmission, scrapie, chronic wasting diseases, CWD, Creutzfeldt-Jacob-disease, CJD, TSE, aerosol, pathogens, allergensPrions, the causative agents of transmissible spongiform encephalopathies, can be undoubtedly propagated from one individual organism to another. The specific routes of prion transmission have been subjected to intensive studies over the past two decades. Incidental and iatrogenic transmission has occurred through the intracerebral route in the case of Dura mater implants¹ and the parenteral route in the case of contaminated pituitary hormones.² In addition, the Bovine Spongiform Encephalopathy (BSE) disaster has provided grim evidence that prion can be transmitted enterally as well. Experimental transmission of prions has been routinely achieved via intraperitoneal and intravenous injection^3,4 but also through more exotic routes such as intralingual,⁵ intranerval⁶ and conjunctival inoculation⁷ and via the nasal cavity.⁸In all prion disease paradigms studied so far the propagation, accumulation and dissemination of the prion protein has been mostly shown to depend on a functional immune system.^9–12 This dependence of prion pathogenesis on the lymphoid compartment, however, is only true for peripheral routes of infection—whereas direct inoculation into the brain does not require any components of the adaptive or innate immune system.B cells in secondary lymphoid organs have been shown to be of importance for the neuroinvasion of the prion protein; in contrast, B lymphocytes in the blood do not appear to play a crucial role.^13–15A special role in prion pathogenesis can be assigned to follicular dendritic cells (FDC). The generation, maturation and function of FDC are dependent on cytokines and chemokines predominantly synthesized and secreted by B lymphocytes. Consistently with this role of B cells in prion pathogenesis, B cell deficient mice show a significantly impaired prion replication due to severely impaired maturation of FDCs.¹⁶ Other soluble and membrane-bound immune mediators such as lymphotoxin heterotrimers and TNFalpha^17,18 as well as components of the complement system^19,20 play an important role in prion pathogenesis.While prions mostly reside in tissues, prion infectivity has also been detected in a variety of body fluids including cerebrospinal fluid,²¹ blood,²² saliva,²³ milk²⁴ and urine.²⁵ Although shedding of prions may occur constitutively from these secretions and excretions, many of the latter phenomena are enhanced by chronic inflammatory processes such as granulomas²⁶ and follicular infiltrates,²⁷ which trigger the maturation of lymphotoxin-dependent, prion-replicating cells.²⁶ The presence of prions in fluids begs the question whether nebulization, and subsequent inhalation, of such fluids may trigger prion infections.Aerosols are finely dispersed particles originating from solid material or liquid using air or other gases as carriers. Natural examples of aerosols include dust (e.g., volcano ashes), smoke, haze and sprays (e.g., sneezing or sea water sprays from breaking waves). Aerosols might be formally categorized as primary or secondary, with primary aerosols being generated in mechanical or thermal processes e.g., by whirling up, impact on surfaces, or burning, whereas secondary aerosols are generated during chemical reactions or by using condensation nuclei.Primary aerosols play an important role in microbiology since they can act as efficacious vehicles for pollen, spores, algae, fungi, bacteria and viruses. Of medical importance are also dandruff, fragments of fur, hairs or skin and mites, which can all function as allergens and trigger e.g., allergic asthma.Moreover, aerosols are excellent vehicles for the transportation of drugs into the respiratory tract. The size of the individual droplets is crucial in specifying the target organs of aerosol. Particle sized 3–10 µm are generally deposited in the nasal cavity and in the throat, whereas smaller particles (e.g., 1 µm) tend to deposit within the lower airways. In rodents pulmonary deposition can reach 10%.^28,29 In humans, particles of 5 µm may reach the lung if inhaled orally, but deposition in the alveolar compartment after inhaling via the nose is highly unlikely.^28,29 For the reasons discussed above, we have become interested in exploring the transmission potential of aerosol-borne prions. Indeed, we found that mouse scrapie can be efficiently transmitted via aerosols.³⁰ In addition to results obtained by exposure to aerosols, we found that mice developed prion infections when inoculated intranasally.Interestingly, this route of transmission was entirely independent on immune cells as shown by challenging various transgenic mouse strains lacking defined functions of the immune system.Well-known examples of transmission of pathogens via aerosols are infections by respiratory viruses (e.g., influenza viruses, adenoviruses, rhinoviruses, coronaviruses) and bacterial diseases (e.g., legionellosis, pneumonic plague by Yersinia pestis, Q-fever by Coxiella burnettii, anthrax) and fungal diseases (particularly aspergillosis and candidosis). In stark contrast, aerosols have historically never been regarded as potential vectors for prion diseases—although very little data existed in favor or against this possibility. This attitude goes along with the implicit “conventional wisdom” that prions are not airborne diseases. However, the concept of “airborne disease” in all the bacterial, fungal and viral examples quoted above, encompasses three distinct phases: (1) release of the infectious agent into aerosols by an infected donor, (2) uptake by a healthy recipient and (3) establishment of disease. It is self-evident that little or no natural transmission between individuals will be observed if any one of these three steps is inefficient. The epidemiological evidence from human prion diseases seems to indicate, albeit indirectly, that step #1 does not occur in CJD patients—inter alia because there is a dearth of evidence of proximity clustering of sCJD.³¹ In the case of CWD the situation may be different since saliva and droppings, which might plausibly give rise to powerful aerosols under a variety of conditions, were found to harbor infectivity. Finally, milk from sheep affected by mastitis can carry scrapie infectivity and—again—could conceivably give rise to aerosols. Since both CWD and sheep scrapie can efficiently spread horizontally within animal collectives, it is extremely appealing to speculate whether aerosols may play a role in said transmission.In natural scrapie in sheep horizontal transmission of prion diseases has been long thought to arise from placental contamination. However, in mice suffering from nephritis prion infectivity is shed with the urine.²⁵ Furthermore, sheep having a mastitis can transmit infectious prions with milk.³²In Chronic Wasting disease (CWD) of deer several careful studies have been performed that, together with our present finding, depose in favor of airborne transmission in this naturally occurring disease. Indeed, CWD prions can be transmitted experimentally via aerosol and the nasal route to transgenic cervidized mice.³³ Although no anecdotal or epidemiological evidence has come forward that airborne transmission may be important for the spread of CWD, several lines of thought suggest that this possibility is not implausible. In deer, prions have been detected in urine, saliva, feces and blood of diseased animals. Moreover, it was claimed that pathological prion protein could be recovered from the environmental water in an endemic area.³⁴ Since all fluids can act as sources for the generation of aerosols, any of the body fluids mentioned above may represent the point of origin for airborne transmission of CWD prions.In this context, also the presence of infectious prions in blood of patients should be mentioned which was demonstrated by the transmission of vCJD by blood transfusions.^35,36 The growing body of evidence that prion transmission can be airborne—at least under certain conditions—dictates that the release of potentially contaminated aerosols should be avoided under all circumstances. In this context it is mandatory that reliable precautions be defined and followed in scientific and diagnostic laboratories. In particular, it is self-evident that safety cabinets should be used while processing brain and nerve tissue (or any other potentially contaminated tissue) of man and animals suspected with prion disease. Our experience shows that this necessity is generally very well-understood by prion scientists.A further stone of contention relates to the biosafety level of the laboratory environment. Because prions were hitherto considered not be airborne, so far no specific regulations have been implemented. As a consequence, prion laboratories have been mostly required to adhere to the category “BSL3**.” While it is understood that the airborne transmission of prions has thus far only been observed under extreme conditions, we feel that it is in order to critically reassess biosafety regulations in the light of the recent discoveries. In particular, one might consider implementing more stringent measures towards protecting workers within diagnostic and scientific laboratories from aerosols.The situation in slaughterhouses and plants handling potentially contaminated offal may be even more problematic. Although regulations in slaughterhouses dictate the use of protecting glasses and masks or, alternatively, visors the use of personal protecting equipment should be rigorously controlled. In addition, high-pressure cleaning devices produce massive aerosols and should be strictly avoided in areas of slaughterhouses where prion-containing material may be processed. Regulations concerning cleaning of heads from slaughtered animals do pay attention to aerosol avoidance, e.g., by allowing only water hoses without pressure.A case in point is the severe neurological syndrome arising in swine abattoir workers.³⁷ Here, an immune-mediated polyradiculoneuropathy was reported to be related to a process using high-pressure fluids to remove the brains of swine.³⁷ During this process, high amounts of swine brain tissue became aerosolized and were inhaled and/or gained access to the respiratory tract mucosa of abattoir workers, resulting in immunization with myelin constituents akin to experimental autoimmune encephalitis (EAE). Although significant physiological differences exist concerning breathing, where humans are regarded as mouth breathers and mice as nose breathers, many people indeed show nose breathing under no or only moderate body burden. Therefore, results obtained in mouse experiments might also be extrapolated to a considerable extent to the situation in man.In this context it is of importance to stress again that aerosols might be generated under various conditions and represent a normal entity of the environment in a variety of daily life situations.In our studies of airborne transmission of prion protein in mice³⁰ we took advantage of the fact that mice breathe exclusively through their nostrils^38,39 and therefore could be exposed in groups to aerosolized brain suspensions. Using this system, it was possible to vary both time of exposure as well as concentration of the prion load in the aerosol. We were surprised to discover that exposure times as short as 1 min were sufficient to achieve high attack rates. By extending the time of exposure it became obvious that incubation times were shortened. A possible alternative route of infection via the cornea or the conjunctiva was extremely unlikely, since newborn mice, whose eyelids were still closed, could also be infected. These findings show that the aerogenic transmission of prions is very efficient.But how do prions spread from the airways to the brain? Peripheral replication of prions in the lymphoid system—a characteristic of most other peripheral routes of transmission—appeared to be dispensable. Instead, the results argue for a direct pathway of brain invasion. One anatomical peculiarity of the nasal cavity is the “area cribriformis” of the olfactory epithelium. Here the olfactory bulb sprouts axons of olfactory receptor neurons passing through the cribriform plate of the ethmoidal bone to reach the olfactory mucosa where olfactory cilia extend representing non-myelinated nerve endings. Thus, open nerve endings are located in the nasal cavity through which aerosolized infectious prions might get access to the brain. In this context it is noteworthy that pathological prion protein was found in the olfactory cilia and basal cells of the olfactory mucosa of sCJD patients, as well as in the olfactory bulb and olfactory tract.^40,41 However, it was hitherto never clearly documented that olfactory receptor neurons represent an entry site for infectious prions; this might also be due to the sensitivity threshold of detection assays.In conclusion, aerosols can infect mice with a surprisingly high efficiency. Just how important a role is played by this newly recognized pathway of spread in natural transmission is, as of now, unclear and in need of further studies. Although it was not identified as a route of infection in epidemiological studies thus far, the worryingly high attack rate suggests that we would be well-advised to carefully avoid the inhalation of aerosols from prion-containing materials.     

Protein interactors of acyl-CoA-binding protein ACBP2 mediate cadmium tolerance in Arabidopsis

In our recent paper in the Plant Journal, we reported that Arabidopsis thaliana lysophospholipase 2 (lysoPL2) binds acyl-CoA-binding protein 2 (ACBP2) to mediate cadmium [Cd(II)] tolerance in transgenic Arabidopsis. ACBP2 contains ankyrin repeats that have been previously shown to mediate protein-protein interactions with an ethylene-responsive element binding protein (AtEBP) and a farnesylated protein 6 (AtFP6). Transgenic Arabidopsis ACBP2-overexpressors, lysoPL2-overexpressors and AtFP6-overexpressors all display enhanced Cd(II) tolerance, in comparison to wild type, suggesting that ACBP2 and its protein partners work together to mediate Cd(II) tolerance. Given that recombinant ACBP2 and AtFP6 can independently bind Cd(II) in vitro, they may be able to participate in Cd(II) translocation. The binding of recombinant ACBP2 to [¹⁴C]linoleoyl-CoA and [¹⁴C]linolenoyl-CoA implies its role in phospholipid repair. In conclusion, ACBP2 can mediate tolerance to Cd(II)-induced oxidative stress by interacting with two protein partners, AtFP6 and lysoPL2. Observations that ACBP2 also binds lysophosphatidylcholine (lysoPC) in vitro and that recombinant lysoPL2 degrades lysoPC, further confirm an interactive role for ACBP2 and lysoPL2 in overcoming Cd(II)-induced stress.Key words: acyl-CoA-binding protein, cadmium, hydrogen peroxide, lysophospholipase, oxidative stressAcyl-CoA-binding proteins (ACBP1 to ACBP6) are encoded by a multigene family in Arabidopsis thaliana.¹ These ACBP proteins are well studied in Arabidopsis in comparison to other organisms,^1–4 and are located in various subcellular compartments.¹ Plasma membranelocalized ACBP1 and ACBP2 contain ankyrin repeats that have been shown to function in protein-protein interactions.^5,6 ACBP1 and ACBP2 which share 76.9% amino acid identity also confer tolerance in transgenic Arabidopsis to lead [Pb(II)] and Cd(II), respectively.^1,5,7 Since recombinant ACBP1 and ACBP2 bind linolenoyl-CoA and linoleoyl-CoA in vitro, they may possibly be involved in phospholipid repair in response to heavy metal stress at the plasma membrane.^5,7 In contrast, ACBP3 is an extracellularly-localized protein⁸ while ACBP4, ACBP5 and ACBP6 are localized to cytosol.^9,10 ACBP1 and ACBP6 have recently been shown to be involved in freezing stress.^9,11 ACBP4 and ACBP5 bind oleoyl-CoA ester and their mRNA expressions are lightregulated.^12,13 Besides acyl-CoA esters, some ACBPs also bind phospholipids.^9,11,13 To investigate the biological function of ACBP2, we have proceeded to establish its interactors at the ankyrin repeats, including AtFP6,⁵ AtEBP⁶ and now lysoPL2 in the Plant Journal paper. While the significance in the interaction of ACBP2 with AtEBP awaits further investigations, some parallels can be drawn between those of ACBP2 with AtFP6 and with lysoPL2.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴