A novel acidic pH-dependent metacaspase governs defense-response against pathogens in tomato.

The importance of metacaspases in programmed cell death and tissue differentiation is known, but their significance in disease stress response, particularly in a crop plant, remained enigmatic. We show the tomato metacaspase expression landscape undergoes differential reprogramming during biotrophic and necrotrophic modes of pathogenesis; also, the metacaspase activity dynamics correlate with the disease progression. These stresses have contrasting effects on the expression pattern of SlMC8, a Type II metacaspase, indicating that SlMC8 is crucial for stress response. In accordance, selected biotic stress-related transcription factors repress SlMC8 promoter activity. Interestingly, SlMC8 exhibits maximum proteolysis at an acidic pH range of 5-6. Molecular dynamics simulation identified the low pH-driven protonation event of Glu246 as critical to stabilize the interaction of SlMC8 with its substrate. Mutagenesis of Glu246 to charge-neutral glutamine suppressed SlMC8's proteolytic activity, corroborating the importance of the amino acid in SlMC8 activation. The glutamic acid residue is found in an equivalent position in metacaspases having acidic pH dependence. SlMC8 overexpression leads to heightened ROS levels, cell death, and tolerance to PstDC3000, and SlMC8 repression reversed the phenomena. However, the overexpression of SlMC8 increases tomato susceptibility to necrotrophic Alternaria solani. We propose that SlMC8 activation due to concurrent changes in cellular pH during infection contributes to the basal resistance of the plant by promoting cell death at the site of infection, and the low pH dependence acts as a guard against unwarranted cell death. Our study confirms the essentiality of a low pH-driven Type II metacaspase in tomato biotic stress-response regulation.

Physico-chemical features and functional relevance of tomato rhomboid proteases.

In plants, regulated intramembrane proteolysis (RIP) is crucial for proper growth, development, and stress management. Rhomboid proteases (RPs) residing in the membrane play a vital role in orchestrating RIP. Although RPs can be found in most sequenced genomes, tomato rhomboids (SlRPs) have not yet been studied. Using alternative and comprehensive strategies, we found ten SlRPs encoded in the tomato genome. These SlRPs possess signature motifs and transmembrane domains, showing structural similarity to other members of the RP family. Also, SlRPs are genetically related to other known RPs of the Solanaceae family. Seven of the SlRPs retain serine-histidine catalytic dyads, making them proteolytically active, while three iRhoms lack the dyad and other structural motifs. Although SlRPs could have functional redundancy, their distribution and expression pattern indicate tissue specificity and responsiveness to specific external stimuli. The presence of development and stress-response-related cis-elements in the promoters of SlRPs supports this view. Furthermore, our strategically designed substrate-reporter assay shows that SlRPs have proteolytic activity similar to that of known RPs. This study provides a detailed understanding of all SlRPs and their physico-chemical features, shedding light on their involvement in physiological processes.

Disruption of tomato TGS machinery by ToLCNDV causes reprogramming of vascular tissue-specific TORNADO1 gene expression.

MAIN CONCLUSION: Vascular development-related TRN1 transcription is suppressed by cytosine methylation in fully developed leaves of tomato. ToLCNDV infection disrupts methylation machinery and reactivates TRN1 expression - likely causing abnormal leaf growth pattern. Leaf curl disease of tomato caused by tomato leaf curl New Delhi virus (ToLCNDV) inflicts huge economical loss. Disease symptoms resemble leaf developmental defects including abnormal vein architecture. Leaf vein patterning-related TORNADO1 gene's (SlTRN1) transcript level is augmented in virus-infected leaves. To elucidate the molecular mechanism of the upregulation of SlTRN1 in vivo, we have deployed SlTRN1 promoter-reporter transgenic tomato plants and investigated the gene's dynamic expression pattern in leaf growth stages and infection. Expression of the gene was delimited in the vascular tissues and suppressed in fully developed leaves. WRKY16 transcription factor readily activated SlTRN1 promoter in varied sized leaves and upon virus infection, while silencing of WRKY16 gene resulted in dampened promoter activity. Methylation-sensitive PCR analyses confirmed the accumulation of CHH methylation at multiple locations in the SlTRN1 promoter in older leaves. However, ToLCNDV infection reverses the methylation status and restores expression level in the leaf vascular bundle. The virus dampens the level of key maintenance and de novo DNA methyltransferases SlDRM5, SlMET1, SlCMT2 with concomitant augmentation of two DNA demethylases, SlDML1 and SlDML2 levels in SlTRN1 promoter-reporter transgenics. Transient overexpression of SlDML2 mimics the virus-induced hypomethylation state of the SlTRN1 promoter in mature leaves, while silencing of SlDML2 lessens promoter activity. Furthermore, in line with the previous studies, we confirm the crucial role of viral suppressors of RNA silencing AC2 and AC4 proteins in promoting DNA demethylation and directing it to restore activated transcription of SlTRN1. Unusually elevated expression of SlTRN1 may negatively impact normal growth of leaves.

Heightened miR6024-NLR interactions facilitate necrotrophic pathogenesis in tomato.

KEY MESSAGE: miR6024 acts as a negative regulator of R genes, hence of Tomato plant immunity, and facilitates disease by the necrotrophic pathogen A. solani. Plant resistance genes or Nucleotide-binding leucine-rich repeat (NLR) genes, integral components of plant disease stress-signaling are targeted by variable groups of miRNAs. However, the significance of miRNA-mediated regulation of NLRs during a pathogen stress response, specifically for necrotrophic fungus, is poorly understood. A thorough examination of Tomato NLRs and miRNAs could map substantial interactions of which half the annotated NLRs were targets of Solanaceae-specific and conserved miRNAs, at the NB subdomain. The Solanaceae-specific miR6024 and its NLR targets analysed in different phytopathogenic stresses revealed differential and mutually antagonistic regulation. Interestingly, miR6024-targeted cleavage of a target NLR also triggered the generation of secondary phased siRNAs which could potentially amplify the defense signal. RNA-seq analysis of leaf tissues from miR6024 overexpressing Tomato plants evidenced a perturbation in the defense transcriptome with the transgenics showing unwarranted immune response-related genes' expression with or without infection with necrotrophic Alternaria solani, though no adverse effect could be observed in the growth and development of the transgenic plants. Transgenic plants exhibited constitutive downregulation of the target NLRs, aggravated disease phenotype with an enhanced lesion, greater ROS generation and hypersusceptibility to A. solani infection, thus establishing that miR6024 negatively impacts plant immune response during necrotrophic pathogenesis. Limited knowledge about the outcome of NLR-miRNA interaction during necrotrophic pathogenesis is a hindrance to the deployment of miRNAs in crop improvement programs. With the elucidation of the necrotrophic disease-synergistic role played by miR6024, it becomes a potent candidate for biotechnological manipulation for the rapid development of pathogen-tolerant solanaceous plants.

Plant metacaspases: Decoding their dynamics in development and disease.

Plant metacaspases were evolved in parallel to well-characterized animal counterpart caspases and retained the similar histidine-cysteine catalytic dyad, leading to functional congruity between these endopeptidases. Although phylogenetic relatedness of the catalytic domain and functional commonality placed these proteases in the caspase family, credible counterarguments predominantly about their distinct substrate specificity raised doubts about the classification. Metacaspases are involved in regulating the PCD during development as well as in senescence. Balancing acts of metacaspase activity also dictate cell fate during defense upon the perception of adverse environmental cues. Accordingly, their activity is tightly regulated, while suppressing spurious activation, by a combination of genetic and post-translational modifications. Structural insights from recent studies provided vital clues on the functionality. This comprehensive review aims to explore the origin of plant metacaspases, and their regulatory and functional diversity in different plants while discussing their analogy to mammalian caspases. Besides, we have presented various modern methodologies for analyzing the proteolytic activity of these indispensable molecules in the healthy or stressed life of a plant. The review would serve as a repository of all the available pieces of evidence indicating metacaspases as the key regulator of PCD across the plant kingdom and highlight the prospect of studying metacaspases for their inclusion in a crop improvement program.

Inhibition of Bemisia tabaci vectored, GroEL mediated transmission of tomato leaf curl New Delhi virus by garlic leaf lectin (Allium sativum leaf agglutinin).

GroEL or symbionin synthesized by the endosymbionts of whitefly (Bemisia tabaci)/ aphids play a cardinal role in the persistent, circulative transmission of plant viruses by binding to viral coat protein/ read-through protein. Allium sativum leaf agglutinin (ASAL), a Galanthus nivalis agglutinin (GNA)- related mannose-binding lectin from garlic leaf has been reported as a potent controlling agent against hemipteran insects including whitefly and aphids. GroEL related chaperonin- symbionin was previously identified as a receptor of ASAL by the present group in the brush border membrane vesicle (BBMV) of mustard aphid. In the present study similar GroEL receptor of ASAL has been identified through LC-MS/MS in the BBMV of B. tabaci which serves as a vector for several plant viruses including tomato leaf curl New Delhi virus (ToLCNDV). Ligand blot analysis of ASAL-fed B. tabaci showed that when GroEL is pre-occupied by ASAL, it completely blocks its further binding to ToLCNDV coat protein (ToLCNDV-CP). Prior feeding of ASAL hindered the co-localization of ToLCNDV-CP and GroEL in the midgut of B. tabaci. Immunoprecipitation followed by western blot with ASAL-fed B. tabaci yielded similar result. Moreover, ASAL feeding inhibited viral transmission by B. tabaci. Together, these results confirmed that the interaction of ASAL with GroEL interferes with the binding of ToLCNDV-CP and inhibits further B. tabaci mediated viral transmission.

Method to Study Dynamics of Membrane-Bound Plant Transcription Factors During Biotic Interactions in Tomato.

Sequestration of a transcription factor in a cellular membrane and releasing it on demand is an additional layer of gene regulation that is considered a rapid mode to reprogram a gene expression cascade when a plasma membrane stress signal is perceived. Better understanding of the dynamic exchange of membrane-bound transcription factors (MTFs) during biotic stress requires the development of a simple, efficient, and quick assay system. Here we report an Agrobacterium-based transient transformation method to assay the localization of fluorescent protein-tagged MTFs in tomato leaf epidermal peels that are subsequently infected with a pathogenic fungus. Essentially, our method mimics natural infection and facilitates the realistic monitoring of MTF movement during activation of a signaling event.

Distinct transcriptional and processing regulations control miR167a level in tomato during stress.

Besides their definite role in plant developmental processes miR167 also serve as mediator of stress response. Although differential expression of miR167 occurs during stresses, the regulatory-mechanism of biogenesis remained elusive. Therefore, using tomato as the model plant we have explored the mechanism of regulation of miR167a expression during stresses. Fungus or virus infections and exposure to cold stress raised the level of miR167a expression. Whereas, salt, drought and heat treatments resulted in the downregulation, indicating different stresses activated alternative mechanisms for miR167a regulation. Interestingly, the relative expression level of precursors in control versus temperature stressed plants differed from the pattern observed in the mature miR167a expression, suggesting that both transcriptional and processing regulation were important for biogenesis. The promoter-regulatory sequence of the major isoform MIR167a harbours several development and stress-related regulatory sites. Accordingly, promoter assays using transient transformation and transgenic tobacco plants proved stress-dependent regulation of the promoter. Further analyses corroborated the role of tomato DREB2A protein in the transcriptional regulation during temperature stress. Finally, in vitro assays established the importance of processing factors in cold-stress dependent efficient processing of MIR167a precursors. These data confirm distinct role of transcriptional and processing machinery in stress-influenced regulation of tomato miR167a biogenesis.

Integrated miRNA and mRNA expression profiling reveals the response regulators of a susceptible tomato cultivar to early blight disease.

Early blight, caused by the fungus Alternaria solani, is a devastating foliar disease of tomatoes, causes massive yield loss each year worldwide. Molecular basis of the compatible host-pathogen interaction was elusive. We adopted next generation sequencing approach to decipher miRNAs and mRNAs that are differentially expressed during Alternaria-stress in tomato. Some of the interesting findings were also validated by alternative techniques. Our analysis revealed 181 known-miRNAs, belonging to 121 miRNA families, of which 67 miRNAs showed at least 2-fold change in expression level with the majority being downregulated. Concomitantly, 5,450 mRNAs were significantly regulated in the same diseased tissues. Differentially expressed genes were most significantly associated with response to stimulus process, photosynthesis, biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone signal transduction pathways. GO term enrichment-based categorization of gene-functions further supported this observation, as terms related to pathogen perception, disease signal transduction, cellular metabolic processes including oxidoreductase and kinase activity were over represented. In addition, we have discovered 102 miRNA-mRNA pairs which were regulated antagonistically, and careful study of the targeted mRNAs depicted that multiple transcription factors, nucleotide-binding site leucine-rich repeats, receptor-like proteins and enzymes related to cellular ROS management were profoundly affected. These studies have identified key regulators of Alternaria-stress response in tomato and the subset of genes that are likely to be post-transcriptionally silenced during the infection.

Chelerythrine down regulates expression of VEGFA, BCL2 and KRAS by arresting G-Quadruplex structures at their promoter regions.

A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.

Functional characterization of tomato membrane-bound NAC transcription factors.

KEY MESSAGE: Genome-wide analysis was carried out to identify and analyze differential expression pattern of tomato membrane bound NAC transcription factors (SlNACMTFs) during stresses. Two biotic-stress-related SlNACMTFs have been characterized to elucidate their regulatory function. NAC transcription factors are known regulators of stress-related gene expression. As Stresses are perceived and transmitted by membrane-bound proteins, functional characterization of membrane-associated NAC transcription factors in tomato can reveal valuable insight about membrane-mediated stress-signalling. Tomato genome encodes 13 NAC genes which have predicted transmembrane domain(s) (SlNACMTFs). mRNA of 12 SlNACMTFs were readily detected in multiple tissues, and also in polysome isolated from leaf, confirming active transcription and translation from these genes occur under normal physiological condition. Additionally, most of the SlNACMTFs were differentially regulated during stresses and stress-related transcription factor binding sites are prevalent in their promoters. SlNACMTF3 and 8 were majorly regulated in biotic and abiotic stresses. Like other MTFs, SlNACMTF3 was translocated to the plasma membrane, whereas the C-terminus truncated (ΔC) form localized in the cytoplasm and the nucleus. Accordingly, the ΔC forms significantly influenced the activity of promoters harbouring NAC binding sites (NACbs). Furthermore, the NAC domain of these transcription factors could directly interact with an NACbs, and the proteins failed to regulate a promoter lacking a crucial NACbs. Interestingly, the type of influence to an NACbs containing promoter was dependent on the context of the NACbs, as the same SlNACMTF showed an alternative mode of regulation on different promoters, as well as the same promoter activity was oppositely regulated by two different SlNACMTF. Finally, both SlNACMTFs demonstrated the differential regulatory effect on the expression of several stress-related genes by interacting with the putative NACbs in their promoter region, suggesting their direct role in plant stress response.

Designing potent antimicrobial peptides by disulphide linked dimerization and N-terminal lipidation to increase antimicrobial activity and membrane perturbation: Structural insights into lipopolysaccharide binding.

HYPOTHESIS: The remarkable rise in multi-drug resistant Gram-negative bacterial pathogens is a major concern to the well being of humans as well as susceptible plants. In recent years, diseases associated with inflammation and septicemia have already become a global health issue. Therefore, there is a rising demand for the development of novel "super" antibiotics. In this context, antimicrobial peptides offer an attractive, alternate therapeutic solution to conventional antibiotics. EXPERIMENTS: Microbroth dilution assay was performed to investigate the antimicrobial activities of the two designed peptides against Gram negative bacterial pathogens. Fluorescence studies including NPN dye uptake assay, Calcein entrapped vesicle leakage assay, quenching and anisotropy in presence of lipopolysaccharide (LPS) were performed to elucidate binding interactions and enhanced membrane permeabilisation. Hemolytic assay and endotoxin/LPS neutralisation assay were performed to study the hemolytic effects and LPS scavenging abilities of the peptides. High resolution NMR studies were performed to obtain insights into LPS-peptide interaction at the molecular level. FINDINGS: Here, we report more potent analogues of previously designed peptide VG16KRKP, designed through dimerization via Cys-Cys disulphide linkage and N-terminal lipidation. Similar to the parent peptide, VG16KRKP, the modified analogue peptides are non hemolytic in nature, but possessed, 2-10-fold increase in antibacterial activities against E. coli, human pathogen Pseudomonas aeruginosa and the devastating plant pathogen, Xanthomonas campestris pv. campestris as well as membrane permeabilization, and endotoxin neutralization. LPS bound solution structure of both analogues, as determined by NMR spectroscopy, reveal that the conserved hydrophobic triad motif, formed by Trp5, Leu11 and Phe12 is compactly organized and stabilized either by the acyl chain or disulphide bond. This structural constraint accounts for the separation of polar face from the hydrophobic face of the peptides. Our novel peptides designed through Cys-Cys dimerization and N-terminal lipidation, will serve as a template to develop more potent antimicrobials in future, to control plant and human diseases.

Mechanism of regulation of tomato TRN1 gene expression in late infection with tomato leaf curl New Delhi virus (ToLCNDV).

Tomato leaf curl disease caused by geminiviruses is manifested by curling and puckering of leaves and thickening of veins, resembling developmental defects. This is probably due to the long-term altered regulation of expression of development related gene(s). Our results show that in the infected leaves the transcript level of TORNADO1 (SlTRN1), a gene important for cell expansion and vein formation, increased significantly. SlTRN1 is transcribed from two start sites. The preferential usage of one start site governs its expression in viral-stressed plants. To investigate the role of specific promoter elements in mediating differential expression of SlTRN1, we performed SlTRN1 promoter analysis. The promoter-regulatory sequences harbor multiple W-boxes. The SlWRKY16 transcription factor actively interacts with one of the W-boxes. WRKY proteins are commonly induced by salicylic acid (SA), and consequently SA treatment increased transcript level of SlWRKY16 and SlTRN1. Further mutational analyses confirmed the role of W-boxes in mediating SlTRN1 induction during ToLCNDV infection or SA treatment. We postulate that the activation of SA pathway during stress-response in tomato induces WRKY16, which in turn modulates transcription of SlTRN1 gene. This study unravels the mechanism of regulation of a developmental gene during stress-response, which may affect the severity of symptoms.

Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control.

The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.

Regulation of transcriptional activation function of rat estrogen receptor α (ERα) by novel C-terminal splice inserts.

Estrogen receptor α (ERα) mediates estrogen diverse actions on tissues. ERα gene has eight constitutively expressing exons and is known to have multiple isoforms generated by alternative initiation of transcription and splicing events including exon skipping. We have discovered two novel exons inserted between exon 5 and 6 of rat ERα that can add independently or in tandem 18 and 14 amino acids to the estrogen binding/activator function 2 domain of the receptor. Their transcript expression is three to six fold higher in heart compared to brain, aorta, liver, ovary and uterus. In heart, the new variants increased ~2 fold with animal growth from prenatal to adulthood, and had a minor increment in aged animals (28 months). Inclusion of these exons yields a receptor with practically no binding capacity for estrogen and reduced dimerization. The new variants show nuclear localization but are less efficient in binding to estrogen responsive elements (EREs) and failed to transcriptionally activate promoters containing EREs (mSlo, KCNE2). Thus, the new variants can regulate the wild-type receptor function and may contribute to the regulatory action of estrogen, especially in the maturing heart where they are more abundant.

The first transmembrane domain (TM1) of ß2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels.

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary ß-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in ß-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the ß2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the ß2-subunit physically binds to the transmembrane S1 domain of the α-subunit.

Distinct transcriptional regulation of human large conductance voltage- and calcium-activated K+ channel gene (hSlo1) by activated estrogen receptor alpha and c-Src tyrosine kinase.

Estrogen receptor α (ERα) regulates gene transcription via "genomic" (binding directly or indirectly, typically via Sp1 or AP-1 sites, to target genes) and/or "nongenomic" (signaling) mechanisms. ERα activation by estrogen up-regulates the murine Ca(2+)-activated K(+) channel α subunit gene (mSlo1) via genomic mechanisms. Here, we investigated whether ERα also drives transcription of the human (hSlo1) gene. Consistent with this view, estrogen increased hSlo1 transcript levels in primary human smooth muscle cells. Promoter studies revealed that estrogen/hERα-mediated hSlo1 transcription was nearly 6-fold more efficient than for mSlo1 (EC(50), 0.07 versus 0.4 nM). Unlike the genomic transcriptional mechanism employed by mSlo1, hSlo1 exhibits a nongenomic hERα-mediated regulatory mechanism. This is supported by the following: 1) efficient hSlo1 transcription after disruption of the DNA-binding domain of hERα or knockdown of Sp1, and 2) lack of AP-1 sites in the hSlo1 promoter. Three nongenomic signaling pathways were explored: Src, Rho, and PI3K. Inhibition of Src with 10 µM PP2, and reported downstream ERK with 25 µM PD98059 did not prevent estrogen action but caused an increase in hSlo1 basal transcription; conversely, constitutively active c-Src (Y527F) decreased hSlo1 basal transcription even preventing its estrogen/hERα-mediated transcriptional activation. Rho inhibition by coexpressed Clostridium botulinum C3 transferase did not alter estrogen action. In contrast, inhibition of PI3K activity with 10 µM LY294002 decreased estrogen-stimulated hSlo1 transcription by ∼40%. These results indicate that the nongenomic PI3K signaling pathway plays a role in estrogen/hERα-stimulated hSlo1 gene expression; whereas c-Src activity leads to hSlo1 gene tonic repression independently of estrogen, likely through ERK activation.

Thromboxane A2 receptor and MaxiK-channel intimate interaction supports channel trans-inhibition independent of G-protein activation.

Large conductance voltage- and calcium-activated potassium channels (MaxiK, BK(Ca)) are well known for sustaining cerebral and coronary arterial tone and for their linkage to vasodilator ß-adrenergic receptors. However, how MaxiK channels are linked to counterbalancing vasoconstrictor receptors is unknown. Here, we show that vasopressive thromboxane A2 receptors (TP) can intimately couple with and inhibit MaxiK channels. Activation of the receptor with its agonist trans-inhibits MaxiK independently of G-protein activation. This unconventional mechanism is supported by independent lines of evidence: (i) inhibition of MaxiK current by thromboxane A2 mimetic, U46619, occurs even when G-protein activity is suppressed; (ii) MaxiK and TP physically associate and display a high degree of proximity; and (iii) Förster resonance energy transfer occurs between fluorescently labeled MaxiK and TP, supporting a direct interaction. The molecular mechanism of MaxiK-TP intimate interaction involves the receptor's first intracellular loop and C terminus, and it entails the voltage-sensing conduction cassette of MaxiK channel. Further, physiological evidence of MaxiK-TP physical interaction is given in human coronaries and rat aorta, and by confirming TP role (with antagonist SQ29,548) in the U46619-induced MaxiK inhibition in human coronaries. We propose that vasoconstrictor TP receptor and MaxiK-channel direct interaction facilitates G-protein-independent TP to MaxiK trans-inhibition, which would promote vasoconstriction.

Hormonal regulation of cardiac KCNE2 gene expression.

The KCNE2 gene encodes a single transmembrane domain protein that modulates a variety of K+ channel functions in various tissues. Here we show that cardiac KCNE2 transcript levels are approximately 10-fold upregulated at the end of pregnancy. This upregulation was mimicked by 17-beta estradiol but not by 5alpha-dihydrotestosterone treatments in ovariectomized mice. To investigate the mechanism of KCNE2 transcriptional regulation by estrogen, we experimentally identified KCNE2 transcription start sites, delineated its gene structure and characterized its promoter region. Estrogen treatment stimulated KCNE2 promoter activity in a dose-dependent manner and ICI 182,780 blocked estrogen stimulation. A direct genomic mechanism was demonstrated by (i) the loss of estrogen responsiveness in the presence of a DNA-binding domain mutant estrogen receptor alpha or mutant KCNE2 ERE and (ii) binding of ERalpha to the KCNE2 ERE. These findings show that a genomic mechanism of estrogen action alters KCNE2 expression, which may have important physiological implications.

c-Src tyrosine kinase, a critical component for 5-HT2A receptor-mediated contraction in rat aorta.

Serotonin (5-hydroxytryptamine, 5-HT) receptors (5-HTRs) play critical roles in brain and cardiovascular functions. In the vasculature, 5-HT induces potent vasoconstrictions, which in aorta are mainly mediated by activation of the 5-HT(2A)R subtype. We previously proposed that one signalling mechanism of 5-HT-induced vasoconstriction could be c-Src, a member of the Src tyrosine kinase family. We now provide evidence for a central role of c-Src in 5-HT(2A)R-mediated contraction. Inhibition of Src kinase activity with 10 mum 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) prior to contraction resulted in approximately 90-99% inhibition of contractions induced by 5-HT or by alpha-methyl-5-HT (5-HT(2)R agonist). In contrast, PP2 pretreatment only partly inhibited contractions induced by angiotensin II and the thromboxane A(2) mimetic, U46619, and had no significant action on phenylephrine-induced contractions. 5-Hydroxytryptamine increased Src kinase activity and PP2-sensitive tyrosine-phosphorylated proteins. As expected for c-Src identity, PP2 pretreatment inhibited 5-HT-induced contraction with an IC(50) of approximately 1 mum. Ketanserin (10 nm), a 5-HT(2A) antagonist, but not antagonists of 5-HT(2B)R (100 nm SB204741) or 5-HT(2C)R (20 nm RS102221), prevented 5-HT-induced contractions, mimicking PP2 and implicating 5-HT(2A)R as the major receptor subtype coupled to c-Src. In HEK 293T cells, c-Src and 5-HT(2A)R were reciprocally co-immunoprecipitated and co-localized at the cell periphery. Finally, 5-HT-induced Src activity was unaffected by inhibition of Rho kinase, supporting a role of c-Src upstream of Rho kinase. Together, the results highlight c-Src activation as one of the early and pivotal mechanisms in 5-HT(2A)R contractile signalling in aorta.