SMXL5 attenuates strigolactone signaling in Arabidopsis thaliana by inhibiting SMXL7 degradation.

Hormone-activated proteolysis is a recurring theme of plant hormone signaling mechanisms. In strigolactone signaling, the enzyme receptor DWARF14 (D14) and an F-box protein, MORE AXILLARY GROWTH2 (MAX2), mark SUPPRESSOR OF MAX2 1-LIKE (SMXL) family proteins SMXL6, SMXL7, and SMXL8 for rapid degradation. Removal of these transcriptional corepressors initiates downstream growth responses. The homologous proteins SMXL3, SMXL4, and SMXL5, however, are resistant to MAX2-mediated degradation. We discovered that the smxl4 smxl5 mutant has enhanced responses to strigolactone. SMXL5 attenuates strigolactone signaling by interfering with AtD14-SMXL7 interactions. SMXL5 interacts with AtD14 and SMXL7, providing two possible ways to inhibit SMXL7 degradation. SMXL5 function is partially dependent on an ethylene-responsive-element binding-factor-associated amphiphilic repression (EAR) motif, which typically mediates interactions with the TOPLESS family of transcriptional corepressors. However, we found that loss of the EAR motif reduces SMXL5-SMXL7 interactions and the attenuation of strigolactone signaling by SMXL5. We hypothesize that integration of SMXL5 into heteromeric SMXL complexes reduces the susceptibility of SMXL6/7/8 proteins to strigolactone-activated degradation and that the EAR motif promotes the formation or stability of these complexes. This mechanism may provide a way to spatially or temporally fine-tune strigolactone signaling through the regulation of SMXL5 expression or translation.

Detection of Cytomegalovirus Interleukin 10 (cmvIL-10) by Enzyme-Linked Immunosorbent Assay (ELISA).

Since its introduction in 1971, the enzyme-linked immunosorbent assay (ELISA) has revolutionized medicine by enabling detection of both antigens and antibodies in a variety of samples. We describe here a customized sandwich ELISA developed for the detection of Human Cytomegalovirus interleukin-10 (cmvIL-10). CmvIL-10 is a virally encoded cytokine and ortholog of human interleukin 10 (hIL-10). While cmvIL-10 and hIL-10 are similar in structure and function, overall amino acid sequence identity is only 27%, resulting in antigenically distinct proteins. The cmvIL-10 ELISA is specific and does not detect hIL-10. The assay is sensitive enough to detect cmvIL-10 in both culture supernatants and patient serum. The ability to quantify cmvIL-10 levels during HCMV infection could provide valuable information about immune evasion strategies and viral control of host signaling pathways.

Control of Cytokines in Latent Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) has evolved a number of mechanisms for long-term co-existence within its host. HCMV infects a wide range of cell types, including fibroblasts, epithelial cells, monocytes, macrophages, dendritic cells, and myeloid progenitor cells. Lytic infection, with the production of infectious progeny virions, occurs in differentiated cell types, while undifferentiated myeloid precursor cells are the primary site of latent infection. The outcome of HCMV infection depends partly on the cell type and differentiation state but is also influenced by the composition of the immune environment. In this review, we discuss the role of early interactions between HCMV and the host immune system, particularly cytokine and chemokine networks, that facilitate the establishment of lifelong latent infection. A better understanding of these cytokine signaling pathways could lead to novel therapeutic targets that might prevent latency or eradicate latently infected cells.

Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2.

Karrikins (KARs) are butenolides found in smoke that can influence germination and seedling development of many plants. The KAR signaling mechanism is hypothesized to be very similar to that of the plant hormone strigolactone (SL). Both pathways require the F-box protein MORE AXILLARY GROWTH2 (MAX2), and other core signaling components have shared ancestry. Putatively, KAR activates the receptor KARRIKIN INSENSITIVE2 (KAI2), triggering its association with the E3 ubiquitin ligase complex SCFMAX2 and downstream targets SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2). Polyubiquitination and proteolysis of SMAX1 and SMXL2 then enable growth responses to KAR. However, many of the assumptions of this model have not been demonstrated. Therefore, we investigated the posttranslational regulation of SMAX1 from the model plant Arabidopsis (Arabidopsis thaliana). We find evidence that SMAX1 is degraded by KAI2-SCFMAX2 but is also subject to MAX2-independent turnover. We identify SMAX1 domains that are responsible for its nuclear localization, KAR-induced degradation, association with KAI2, and ability to interact with other SMXL proteins. KAI2 undergoes MAX2-independent degradation after KAR treatment, which we propose results from its association with SMAX1 and SMXL2. Finally, we discover an SMXL domain that mediates receptor-target interaction preferences in KAR and SL signaling, laying the foundation for understanding how these highly similar pathways evolved to fulfill different roles.

LPIAT, a lyso-Phosphatidylinositol Acyltransferase, Modulates Seed Germination in Arabidopsis thaliana through PIP Signalling Pathways and is Involved in Hyperosmotic Response.

Lyso-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from Arabidopsis thaliana (LPIAT). In vitro, this protein, expressed in Escherichia coli membrane, displayed a 2-lyso-phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. In planta, LPIAT gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the lpiat mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for lpiat line. In addition, germination of lpiat mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination.

Feedback regulation of BMP signaling by Caenorhabditis elegans cuticle collagens.

Cellular responsiveness to environment, including changes in extracellular matrix (ECM), is critical for normal processes such as development and wound healing, but can go awry, as in oncogenesis and fibrosis. One type of molecular pathway contributing to this responsiveness is the BMP signaling pathway. Owing to their broad and potent functions, BMPs and their pathways are regulated at multiple levels. In Caenorhabditis elegans, the BMP ligand DBL-1 is a regulator of body size. We previously showed that DBL-1/BMP signaling determines body size through transcriptional regulation of cuticle collagen genes. We now identify feedback regulation of DBL-1/BMP through analysis of four DBL-1-regulated collagen genes. Inactivation of any of these genes reduces DBL-1/BMP signaling, measured by a pathway activity reporter. Furthermore, depletion of these collagens reduces GFP::DBL-1 fluorescence and acts unexpectedly at the level of dbl-1 transcription. We conclude that cuticle, a specialized ECM, impinges on DBL-1/BMP expression and signaling. Interestingly, the feedback regulation of DBL-1/BMP signaling by collagens is likely to be contact independent due to physical separation of the cuticle from DBL-1-expressing cells in the ventral nerve cord. Our results provide an entry point into a novel regulatory mechanism for BMP signaling, with broader implications for mechanical regulation of gene expression.

Genetic interactions between the DBL-1/BMP-like pathway and dpy body size-associated genes in Caenorhabditis elegans.

Bone morphogenetic protein (BMP) signaling pathways control many developmental and homeostatic processes, including cell size and extracellular matrix remodeling. An understanding of how this pathway itself is controlled remains incomplete. To identify novel regulators of BMP signaling, we performed a forward genetic screen in Caenorhabditis elegans for genes involved in body size regulation, a trait under the control of BMP member DBL-1. We isolated mutations that suppress the long phenotype of lon-2, a gene that encodes a negative regulator that sequesters DBL-1. This screen was effective because we isolated alleles of several core components of the DBL-1 pathway, demonstrating the efficacy of the screen. We found additional alleles of previously identified but uncloned body size genes. Our screen also identified widespread involvement of extracellular matrix proteins in DBL-1 regulation of body size. We characterized interactions between the DBL-1 pathway and extracellular matrix and other genes that affect body morphology. We discovered that loss of some of these genes affects the DBL-1 pathway, and we provide evidence that DBL-1 signaling affects many molecular and cellular processes associated with body size. We propose a model in which multiple body size factors are controlled by signaling through the DBL-1 pathway and by DBL-1-independent processes.

A chemical genetic screen uncovers a small molecule enhancer of the N-acylethanolamine degrading enzyme, fatty acid amide hydrolase, in Arabidopsis.

N-Acylethanolamines (NAEs) are a group of fatty acid amides that play signaling roles in diverse physiological processes in eukaryotes. Fatty acid amide hydrolase (FAAH) degrades NAE into ethanolamine and free fatty acid to terminate its signaling function. In animals, chemical inhibitors of FAAH have been used for therapeutic treatment of pain and as tools to probe deeper into biochemical properties of FAAH. In a chemical genetic screen for small molecules that dampened the inhibitory effect of N-lauroylethanolamine (NAE 12:0) on Arabidopsis thaliana seedling growth, we identified 6-(2-methoxyphenyl)-1,3-dimethyl-5-phenyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3 H,6 H)-dione (or MDPD). MDPD alleviated the growth inhibitory effects of NAE 12:0, in part by enhancing the enzymatic activity of Arabidopsis FAAH (AtFAAH). In vitro, biochemical assays showed that MDPD enhanced the apparent Vmax of AtFAAH but did not alter the affinity of AtFAAH for its NAE substrates. Structural analogs of MDPD did not affect AtFAAH activity or dampen the inhibitory effect of NAE 12:0 on seedling growth indicating that MDPD is a specific synthetic chemical activator of AtFAAH. Collectively, our study demonstrates the feasibility of using an unbiased chemical genetic approach to identify new pharmacological tools for manipulating FAAH- and NAE-mediated physiological processes in plants.

Smoke and Hormone Mirrors: Action and Evolution of Karrikin and Strigolactone Signaling.

Karrikins and strigolactones are two classes of butenolide molecules that have diverse effects on plant growth. Karrikins are found in smoke and strigolactones are plant hormones, yet both molecules are likely recognized through highly similar signaling mechanisms. Here we review the most recent discoveries of karrikin and strigolactone perception and signal transduction. Two paralogous α/ß hydrolases, KAI2 and D14, are respectively karrikin and strigolactone receptors. D14 acts with an F-box protein, MAX2, to target SMXL/D53 family proteins for proteasomal degradation, and genetic data suggest that KAI2 acts similarly. There are striking parallels in the signaling mechanisms of karrikins, strigolactones, and other plant hormones, including auxins, jasmonates, and gibberellins. Recent investigations of host perception in parasitic plants have demonstrated that strigolactone recognition can evolve following gene duplication of KAI2.

Fatty Acid Amide Hydrolase Regulates Peripheral B Cell Receptor Revision, Polyreactivity, and B1 Cells in Lupus.

C57BL/6 mice bearing the Sle2(z) lupus-susceptibility congenic interval on chromosome 4 display high titers of polyclonal autoantibodies with generalized B cell hyperactivity, hallmarks of systemic lupus erythematosus. In B6.Sle2(z)HEL(Ig).sHEL BCR-transgenic mice, Sle2(z) did not breach central tolerance, but it led to heightened expression of endogenous Ig H and L chains in splenic B cells, upregulation of RAG, and serological polyreactivity, suggestive of excessive receptor revision. Fatty acid amide hydrolase (FAAH), a gene in the minimal subcongenic interval generated through recombinant mapping, was found to be upregulated in Sle2(z) B cells by microarray analysis, Western blot, and functional assays. Pharmacological inhibition of FAAH reversed the increase in receptor revision, RAG expression, and polyreactive autoantibodies in lupus-prone mice. These studies indicate that increased peripheral BCR revision, or selective peripheral expansion of BCR-revised B cells, may lead to systemic autoimmunity and that FAAH is a lupus-susceptibility gene that might regulate this process.

Effects of synthetic alkamides on Arabidopsis fatty acid amide hydrolase activity and plant development.

Alkamides and N-acylethanolamines (NAEs) are bioactive, amide-linked lipids that influence plant development. Alkamides are restricted to several families of higher plants and some fungi, whereas NAEs are widespread signaling molecules in both plants and animals. Fatty acid amide hydrolase (FAAH) has been described as a key contributor to NAE hydrolysis; however, no enzyme has been associated with alkamide degradation in plants. Herein reported is synthesis of 12 compounds structurally similar to a naturally occurring alkamide (N-isobutyl-(2E,6Z,8E)decatrienamide or affinin) with different acyl compositions more similar to plant NAEs and various amino alkyl head groups. These "hybrid" synthetic alkamides were tested for activity toward recombinant Arabidopsis FAAH and for their effects on plant development (i.e., cotyledon expansion and primary root length). A substantial increase in FAAH activity was discovered toward NAEs in vitro in the presence of some of these synthetic alkamides, such as N-ethyllauroylamide (4). This "enhancement" effect was found to be due, at least in part, to relief from product inhibition of FAAH by ethanolamine, and not due to an alteration in the oligomerization state of the FAAH enzyme. For several of these alkamides, an inhibition of seedling growth was observed with greater results in FAAH knockouts and less in FAAH over-expressing plants, suggesting that these alkamides could be hydrolyzed by FAAH in planta. The tight regulation of NAE levels in vivo appears to be important for proper seedling establishment, and as such, some of these synthetic alkamides may be useful pharmacological tools to manipulate the effects of NAEs in situ.

Synthesis of phenoxyacyl-ethanolamides and their effects on fatty acid amide hydrolase activity.

N-Acylethanolamines (NAEs) are involved in numerous biological activities in plant and animal systems. The metabolism of these lipids by fatty acid amide hydrolase (FAAH) is a key regulatory point in NAE signaling activity. Several active site-directed inhibitors of FAAH have been identified, but few compounds have been described that enhance FAAH activity. Here we synthesized two sets of phenoxyacyl-ethanolamides from natural products, 3-n-pentadecylphenolethanolamide and cardanolethanolamide, with structural similarity to NAEs and characterized their effects on the hydrolytic activity of FAAH. Both compounds increased the apparent Vmax of recombinant FAAH proteins from both plant (Arabidopsis) and mammalian (Rattus) sources. These NAE-like compounds appeared to act by reducing the negative feedback regulation of FAAH activity by free ethanolamine. Both compounds added to seedlings relieved, in part, the negative growth effects of exogenous NAE12:0. Cardanolethanolamide reduced neuronal viability and exacerbated oxidative stress-mediated cell death in primary cultured neurons at nanomolar concentrations. This was reversed by FAAH inhibitors or exogenous NAE substrate. Collectively, our data suggest that these phenoxyacyl-ethanolamides act to enhance the activity of FAAH and may stimulate the turnover of NAEs in vivo. Hence, these compounds might be useful pharmacological tools for manipulating FAAH-mediated regulation of NAE signaling in plants or animals.

N-Acylethanolamines: lipid metabolites with functions in plant growth and development.

Twenty years ago, N-acylethanolamines (NAEs) were considered by many lipid chemists to be biological 'artifacts' of tissue damage, and were, at best, thought to be minor lipohilic constituents of various organisms. However, that changed dramatically in 1993, when anandamide, an NAE of arachidonic acid (N-arachidonylethanolamine), was shown to bind to the human cannabinoid receptor (CB1) and activate intracellular signal cascades in mammalian neurons. Now NAEs of various types have been identified in diverse multicellular organisms, in which they display profound biological effects. Although targets of NAEs are still being uncovered, and probably vary among eukaryotic species, there appears to be remarkable conservation of the machinery that metabolizes these bioactive fatty acid conjugates of ethanolamine. This review focuses on the metabolism and functions of NAEs in higher plants, with specific reference to the formation, hydrolysis and oxidation of these potent lipid mediators. The discussion centers mostly on early seedling growth and development, for which NAE metabolism has received the most attention, but also considers other areas of plant development in which NAE metabolism has been implicated. Where appropriate, we indicate cross-kingdom conservation in NAE metabolic pathways and metabolites, and suggest areas where opportunities for further investigation appear most pressing.

Analysis of fatty acid amide hydrolase activity in plants.

N-Acylethanolamines (NAEs) are fatty acid derivatives amide-linked to ethanolamine. NAEs vary in chain lengths and numbers of double bonds and generally reflect the fatty acids found in membrane lipids in the tissues in which they reside. NAEs are present naturally in trace amounts and occur in a wide range of organisms including plants, animals, and microbes. Some NAE types are known to be involved in the endocannabinoid signaling system of vertebrates, and in plants they may play important regulatory roles in several physiological processes, such as root growth, seedling development, stress responses, and pathogen interactions. The biological effects of NAEs are terminated through their hydrolysis into the ethanolamine and free fatty acid by a membrane enzyme known as the fatty acid amide hydrolase (FAAH). Thus, FAAH represents an important target to better understand the function of these lipid mediators in numerous cellular processes. FAAH has been extensively characterized in mammalian and plant systems, and they share a conserved Ser-Ser-Lys catalytic mechanism. Here we describe procedures and experimental conditions to assay and characterize recombinant and endogenous FAAH enzymatic activity derived from plant tissues.

N-Acylethanolamines and related compounds: aspects of metabolism and functions.

N-Acylethanolamines (NAE) are fatty acid derivates that are linked with an ethanolamine group via an amide bond. NAE can be characterized as lipid mediators in the plant and animal kingdoms owing to the diverse functions throughout the eukaryotic domain. The functions of NAE have been widely investigated in animal tissues in part due to their abilities to interact with the cannabinoid receptors, vanilloid receptors or peroxisome proliferator activated receptors. However, the interest of studying the functions of these lipids in plants is progressively becoming more apparent. The number of publications about the functions related to NAE and to structural analogs (homoserine lactone and alkamides) is greatly increasing, showing the importance of these lipids in various plant physiological processes. This review sheds light on their role in different processes such as seedling development, plant pathogen interaction, phospholipase D alpha inhibition and senescence of cut flowers, and underlines the interaction between NAE and NAE-related molecules with plant hormone signaling. The different metabolic pathways promoting the synthesis and degradation of NAE are also discussed, in particular the oxygenation of polyunsaturated N-acylethanolamines, which leads to NAE-oxylipins, a new family of bioactive lipids.

Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE).

N-acylphosphatidylethanolamine (NAPE) is a minor phospholipid resulting from the transfer of an acyl chain from an acyl donor to the primary amine of the ethanolamine moiety of phosphatidylethanolamine (PE). Occurring in plant and animal kingdoms as well as in prokaryotic cells, it is synthesized in higher amounts in membranes during cellular stresses and tissue damage, and it is widely thought to be the precursor of the lipid mediator, N-acylethanolamine (NAE), which modulates the endocannabinoid signaling pathway and therefore regulates various physiological processes. However, recent studies have shown that NAPE is also a bioactive molecule that is involved in several physiological functions. The present paper reviews the occurrence of NAPE in animals and plants and focuses on the various properties of NAPE observed in vitro and in vivo. The different metabolic pathways promoting the synthesis and degradation of NAPE are also discussed and the differences between animals and plants are underlined.

Discovery and characterization of an Arabidopsis thaliana N-acylphosphatidylethanolamine synthase.

N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D beta/gamma isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. beta-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.