β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent 5‐HT4 receptor signalling

G protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT₄ receptor (5‐HT₄R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the G_s pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser⁴¹²) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT₄Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling.     

Sub‐picomolar relaxin signalling by a pre‐assembled RXFP1, AKAP79, AC2, β‐arrestin 2, PDE4D3 complex

Biochemical studies suggest that G‐protein‐coupled receptors (GPCRs) achieve exquisite signalling specificity by forming selective complexes, termed signalosomes. Here, using cAMP biosensors in single cells, we uncover a pre‐assembled, constitutively active GPCR signalosome, that couples the relaxin receptor, relaxin family peptide receptor 1 (RXFP1), to cAMP following receptor stimulation with sub‐picomolar concentrations of peptide. The physiological effects of relaxin, a pleiotropic hormone with therapeutic potential in cancer metastasis and heart failure, are generally attributed to local production of the peptide, that occur in response to sub‐micromolar concentrations. The highly sensitive signalosome identified here provides a regulatory mechanism for the extremely low levels of relaxin that circulate. The signalosome includes requisite Gα_s, Gβγ and adenylyl cyclase 2 (AC2); AC2 is functionally coupled to RXFP1 through AKAP79 binding to helix 8 of the receptor; activation of AC2 is tonically opposed by protein kinase A (PKA)‐activated PDE4D3, scaffolded through a β‐arrestin 2 interaction with Ser⁷⁰⁴ of the receptor C‐terminus. This elaborate, pre‐assembled, ligand‐independent GPCR signalosome represents a new paradigm in GPCR signalling and provides a mechanism for the distal actions of low circulating levels of relaxin.     

Production and initial structural characterization of the TM4TM5 helix‐loop‐helix domain of the translocator protein

Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helix–helix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C‐terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far‐UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n‐dodecyl‐β‐d ‐maltoside, n‐octyl‐β‐d ‐glucoside, n‐dodecylphosphocholine, 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DHPC), and 1‐palmitoyl‐2‐hydroxy‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5‐DHPC complex. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Novel potato C2H2‐type zinc finger protein gene,StZFP1, which responds to biotic and abiotic stress,plays a role in salt tolerance

Many TFIIIA‐type zinc finger proteins (ZFPs) play important roles in stress responses in plants. In the present study, a novel zinc finger protein gene, StZFP1, was cloned from potato. StZFP1 is a typical TFIIIA‐type two‐finger zinc finger gene with one B‐box domain, one L‐box domain and a DLN‐box/EAR motif. The StZFP1 genes belong to a small gene family with an estimated copy number of four or five, located on chromosome I. StZFP1 is constitutively expressed in leaves, stems, roots, tubers and flowers of adult plants. Expression of StZFP1 can be induced by salt, dehydration and exogenously applied ABA. StZFP1 expression is also responsive to infection by the late blight pathogen Phytophthora infestans. Transient expression analysis of StZFP1:GFP fusion protein revealed that StZFP1 is preferentially localised in the nucleus. Ectopic expression of StZFP1, driven by the Arabidopsis rd29A promoter in transgenic tobacco, increased plant tolerance to salt stress. These results demonstrate that StZFP1 might be involved in potato responses to salt and dehydration stresses through an ABA‐dependent pathway.     

Functional Analysis of the C-Terminal Region of γ-Glutamyl Kinase of Saccharomyces cerevisiae

γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ¹-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.     

Epithelial microRNA‐9a regulates dendrite growth through Fmi‐Gq signaling in Drosophila sensory neurons

microRNA‐9 (miR‐9) is highly expressed in the nervous system across species and plays essential roles in neurogenesis and axon growth; however, little is known about the mechanisms that link miR‐9 with dendrite growth. Using an in vivo model of Drosophila class I dendrite arborization (da) neurons, we show that miR‐9a, a Drosophila homolog of mammalian miR‐9, downregulates the cadherin protein Flamingo (Fmi) thereby attenuating dendrite development in a non‐cell autonomous manner. In miR‐9a knockout mutants, the dendrite length of a sensory neuron ddaE was significantly increased. Intriguingly, miR‐9a is specifically expressed in epithelial cells but not in neurons, thus the expression of epithelial but not neuronal Fmi is greatly elevated in miR‐9a mutants. In contrast, overexpression of Fmi in the neuron resulted in a reduction in dendrite growth, suggesting that neuronal Fmi plays a suppressive role in dendrite growth, and that increased epithelial Fmi might promote dendrite growth by competitively binding to neuronal Fmi. Fmi has been proposed as a G protein‐coupled receptor (GPCR), we find that neuronal G protein Gαq (Gq), but not Go, may function downstream of Fmi to negatively regulate dendrite growth. Taken together, our results reveal a novel function of miR‐9a in dendrite morphogenesis. Moreover, we suggest that Gq might mediate the intercellular signal of Fmi in neurons to suppress dendrite growth. Our findings provide novel insights into the complex regulatory mechanisms of microRNAs in dendrite development, and further reveal the interplay between the different components of Fmi, functioning in cadherin adhesion and GPCR signalling. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 225–237, 2016     

Sequence variation of intergenic mitochondrial DNA spacers (mtDNA‐IGS) of Phytophthora infestans (Oomycetes) and related species

The potato late‐blight disease is caused by the pseudofungus Phytophthora infestans (Oomycetes). This pathogen was of historical importance as it caused the Irish Potato Famine. There is currently a worldwide resurgence of the disease. Following worldwide migrations as well as being able to discriminate P. infestans from related species are key issues. We present sequence variation of five inter‐genic mitochondrial DNA spacers (mtDNA‐IGS) for P. infestans and four related taxa. Intra and inter‐taxon variation was observed showing potential for both molecular ecology and molecular systematic.     

Sl‐lncRNA15492 interacts with Sl‐miR482a and affects Solanum lycopersicum immunity against Phytophthora infestans

Long non‐coding RNAs (lncRNAs) are involved in the resistance of plants to infection by pathogens via interactions with microRNAs (miRNAs). Long non‐coding RNAs are cleaved by miRNAs to produce phased small interfering RNAs (phasiRNAs), which, as competing endogenous RNAs (ceRNAs), function as decoys for mature miRNAs, thus inhibiting their expression, and contain pre‐miRNA sequences to produce mature miRNAs. However, whether lncRNAs and miRNAs mediate other molecular mechanisms during plant resistance to pathogens is unknown. In this study, as a positive regulator, Sl‐lncRNA15492 from tomato (Solanum lycopersicum Zaofen No. 2) plants affected tomato resistance to Phytophthora infestans. Gain‐ and loss‐of‐function experiments and RNA ligase‐mediated 5′‐amplification of cDNA ends (RLM‐5′ RACE) also revealed that Sl‐miR482a was negatively involved in tomato resistance by targeting Sl‐NBS‐LRR genes and that silencing of Sl‐NBS‐LRR1 decreased tomato resistance. Sl‐lncRNA15492 inhibited the expression of mature Sl‐miR482a, whose precursor was located within the antisense sequence of Sl‐lncRNA15492. Further degradome analysis and additional RLM‐5′ RACE experiments verified that mature Sl‐miR482a could also cleave Sl‐lncRNA15492. These results provide a mechanism by which lncRNAs might inhibit precursor miRNA expression through antisense strands of lncRNAs, and demonstrate that Sl‐lncRNA15492 and Sl‐miR482a mutually inhibit the maintenance of Sl‐NBS‐LRR1 homeostasis during tomato resistance to P. infestans.     

Potato StMPK7 is a downstream component of StMKK1 and promotes resistance to the oomycete pathogen Phytophthora infestans

A cascade formed by phosphorylation events of mitogen-activated protein kinases (MAPKs) takes part in plant stress responses. However, the roles of these MAPKs in resistance of potato (Solanum tuberosum) against Phytophthora pathogens is not well studied. Our previous work showed that a Phytophthora infestans RXLR effector targets and stabilizes the negative regulator of MAPK kinase 1 of potato (StMKK1). Because in Arabidopsis thaliana the AtMPK4 is the downstream phosphorylation target of AtMKK1, we performed a phylogenetic analysis and found that potato StMPK4/6/7 are closely related and are orthologs of AtMPK4/5/11/12. Overexpression of StMPK4/7 enhances plant resistance to P. infestans and P. parasitica. Yeast two-hybrid analysis revealed that StMPK7 interacts with StMKK1, and StMPK7 is phosphorylated on flg22 treatment and by expressing constitutively active StMKK1 (CA-StMKK1), indicating that StMPK7 is a direct downstream signalling partner of StMKK1. Overexpression of StMPK7 in potato enhances potato resistance to P. infestans. Constitutively active StMPK7 (CA-StMPK7; StMPK7^{D198G, E202A}) was found to promote immunity to Phytophthora pathogens and to trigger host cell death when overexpressed in Nicotiana benthamiana leaves. Cell death triggered by CA-StMPK7 is SGT1/RAR1-dependent. Furthermore, cell death triggered by CA-StMPK7 is suppressed on coexpression with the salicylate hydroxylase NahG, and StMPK7 activation promotes salicylic acid (SA)-responsive gene expression. We conclude that potato StMPK7 is a downstream signalling component of the phosphorelay cascade involving StMKK1 and StMPK7 plays a role in immunity to Phytophthora pathogens via an SA-dependent signalling pathway.     

DnaQ exonuclease‐like domain of Cas2 promotes spacer integration in a type I‐E CRISPR‐Cas system

CRISPR‐Cas systems constitute an adaptive immune system that provides acquired resistance against phages and plasmids in prokaryotes. Upon invasion of foreign nucleic acids, some cells integrate short fragments of foreign DNA as spacers into the CRISPR locus to memorize the invaders and acquire resistance in the subsequent round of infection. This immunization step called adaptation is the least understood part of the CRISPR‐Cas immunity. We have focused here on the adaptation stage of Streptococcus thermophilus DGCC7710 type I‐E CRISPR4‐Cas (St4) system. Cas1 and Cas2 proteins conserved in nearly all CRISPR‐Cas systems are required for spacer acquisition. The St4 CRISPR‐Cas system is unique because the Cas2 protein is fused to an additional DnaQ exonuclease domain. Here, we demonstrate that St4 Cas1 and Cas2‐DnaQ form a multimeric complex, which is capable of integrating DNA duplexes with 3′‐overhangs (protospacers) in vitro. We further show that the DnaQ domain of Cas2 functions as a 3′–5′‐exonuclease that processes 3′‐overhangs of the protospacer to promote integration.     

A rapid fluorogenic GPCR–β‐arrestin interaction assay

Detection of protein–protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein‐coupled receptor (GPCR)–β‐arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal‐to‐noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease‐activated receptor‐1 activation developed in minutes and required specific serine–threonine residues in the receptor carboxyl tail, consistent with a classical G protein‐coupled receptor kinase dependent β‐arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.     

A novel NOD1‐ and CagA‐independent pathway of interleukin‐8 induction mediated by the Helicobacter pylori type IV secretion system

The type IV secretion system (T4SS) of Helicobacter pylori triggers massive inflammatory responses during gastric infection by mechanisms that are poorly understood. Here we provide evidence for a novel pathway by which the T4SS structural component, CagL, induces secretion of interleukin‐8 (IL‐8) independently of CagA translocation and peptidoglycan‐sensing nucleotide‐binding oligomerization domain 1 (NOD1) signalling. Recombinant CagL was sufficient to trigger IL‐8 secretion, requiring activation of α₅β₁ integrin and the arginine–glycine–aspartate (RGD) motif in CagL. Mutation of the encoded RGD motif to arginine‐glycine‐alanine (RGA) in the cagL gene of H. pylori abrogated its ability to induce IL‐8. Comparison of IL‐8 induction between H. pylori ΔvirD4 strains bearing wild‐type or mutant cagL indicates that CagL‐dependent IL‐8 induction can occur independently of CagA translocation. In line with this notion, exogenous CagL complemented H. pylori ΔcagL mutant in activating NF‐κB and inducing IL‐8 without restoring CagA translocation. The CagA translocation‐independent, CagL‐dependent IL‐8induction involved host signalling via integrin α₅β₁, Src kinase, the mitogen‐activated protein kinase (MAPK) pathway and NF‐κB but was independent of NOD1. Our findings reveal a novel pathway whereby CagL, via interaction with host integrins, can trigger pro‐inflammatory responses independently of CagA translocation or NOD1 signalling.