PIGN spatiotemporally regulates the spindle assembly checkpoint proteins in leukemia transformation and progression.

Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) has been linked to the suppression of chromosomal instability. The spindle assembly checkpoint complex is responsible for proper chromosome segregation during mitosis to prevent chromosomal instability. In this study, the novel role of PIGN as a regulator of the spindle assembly checkpoint was unveiled in leukemic patient cells and cell lines. Transient downregulation or ablation of PIGN resulted in impaired mitotic checkpoint activation due to the dysregulated expression of spindle assembly checkpoint-related proteins including MAD1, MAD2, BUBR1, and MPS1. Moreover, ectopic overexpression of PIGN restored the expression of MAD2. PIGN regulated the spindle assembly checkpoint by forming a complex with the spindle assembly checkpoint proteins MAD1, MAD2, and the mitotic kinase MPS1. Thus, PIGN could play a vital role in the spindle assembly checkpoint to suppress chromosomal instability associated with leukemic transformation and progression.

FERONIA restricts Pseudomonas in the rhizosphere microbiome via regulation of reactive oxygen species.

Maintaining microbiome structure is critical for the health of both plants and animals. By re-screening a collection of Arabidopsis mutants affecting root immunity and hormone crosstalk, we identified a FERONIA (FER) receptor kinase mutant (fer-8) with a rhizosphere microbiome enriched in Pseudomonas fluorescens without phylum-level dysbiosis. Using microbiome transplant experiments, we found that the fer-8 microbiome was beneficial. The effect of FER on rhizosphere pseudomonads was largely independent of its immune scaffold function, role in development and jasmonic acid autoimmunity. We found that the fer-8 mutant has reduced basal levels of reactive oxygen species (ROS) in roots and that mutants deficient in NADPH oxidase showed elevated rhizosphere pseudomonads. The addition of RALF23 peptides, a FER ligand, was sufficient to enrich P. fluorescens. This work shows that FER-mediated ROS production regulates levels of beneficial pseudomonads in the rhizosphere microbiome.

Alleles of a wall-associated kinase gene account for three of the major northern corn leaf blight resistance loci in maize.

Northern corn leaf blight, caused by the fungal pathogen Setosphaeria turcica (anamorph Exserohilum turcicum), is one of the most devastating foliar diseases of maize (Zea mays). Four genes Ht1, Ht2, Ht3 and Htn1 represent the major sources of genetic resistance against the hemibiotrophic fungus S. turcica. Differential maize lines containing these genes also form the basis to classify S. turcica races. Here, we show that Ht2 and Ht3 are identical and allelic to the previously cloned Htn1 gene. Using a map-based cloning approach and Targeting Induced Local Lesions in Genomes (TILLING), we demonstrate that Ht2/Ht3 is an allele of the wall-associated receptor-like kinase gene ZmWAK-RLK1. The ZmWAK-RLK1 variants encoded by Htn1 and Ht2/Ht3 differ by multiple amino acid polymorphisms that particularly affect the putative extracellular domain. A diversity analysis in maize revealed the presence of dozens of ZmWAK-RLK1 alleles. Ht2, Ht3 and Htn1 have been described over decades as independent resistance loci with different race spectra and resistance responses. Our work demonstrates that these three genes are allelic, which has major implications for northern corn leaf blight resistance breeding and nomenclature of S. turcica pathotypes. We hypothesize that genetic background effects have confounded the classical description of these disease resistance genes in the past.

The Interaction Network and Signaling Specificity of Two-Component System in Arabidopsis.

Two-component systems (TCS) in plants have evolved into a more complicated multi-step phosphorelay (MSP) pathway, which employs histidine kinases (HKs), histidine-containing phosphotransfer proteins (HPts), and response regulators (RRs) to regulate various aspects of plant growth and development. How plants perceive the external signals, then integrate and transduce the secondary signals specifically to the desired destination, is a fundamental characteristic of the MSP signaling network. The TCS elements involved in the MSP pathway and molecular mechanisms of signal transduction have been best understood in the model plant Arabidopsis thaliana. In this review, we focus on updated knowledge on TCS signal transduction in Arabidopsis. We first present a brief description of the TCS elements; then, the protein-protein interaction network is established. Finally, we discuss the possible molecular mechanisms involved in the specificity of the MSP signaling at the mRNA and protein levels.

ABC1K10a, an atypical kinase, functions in plant salt stress tolerance.

BACKGROUND: ABC1K (Activity of BC1 complex Kinase) is an evolutionarily primitive atypical kinase family widely distributed among prokaryotes and eukaryotes. The ABC1K protein kinases in Arabidopsis are predicted to localize either to the mitochondria or chloroplasts, in which plastid-located ABC1K proteins are involved in the response against photo-oxidative stress and cadmium-induced oxidative stress. RESULTS: Here, we report that the mitochondria-localized ABC1K10a functions in plant salt stress tolerance by regulating reactive oxygen species (ROS). Our results show that the ABC1K10a expression is induced by salt stress, and the mutations in this gene result in overaccumulation of ROS and hypersensitivity to salt stress. Exogenous application of the ROS-scavenger GSH significantly represses ROS accumulation and rescues the salt hypersensitive phenotype of abc1k10a. ROS overaccumulation in abc1k10a mutants under salt stress is likely due to the defect in mitochondria electron transport chain. Furthermore, defects of several other mitochondria-localized ABC1K genes also result in salt hypersensitivity. CONCLUSIONS: Taken together, our results reveal that the mitochondria-located ABC1K10a regulates mitochondrial ROS production and is a positive regulator of salt tolerance in Arabidopsis.

Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage.

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

Pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP1) regulates starch biosynthesis and seed development via heterotetramer formation in rice (Oryza sativa L.).

Pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP1) reversibly converts fructose 6-phosphate and pyrophosphate to fructose 1, 6-bisphosphate and orthophosphate during glycolysis, and has diverse functions in plants. However, mechanisms underlying the regulation of starch metabolism by PFP1 remain elusive. This study addressed the function of PFP1 in rice floury endosperm and defective grain filling. Compared with the wild type, pfp1-3 exhibited remarkably low grain weight and starch content, significantly increased protein and lipid content, and altered starch physicochemical properties and changes in embryo development. Map-based cloning revealed that pfp1-3 is a novel allele and encodes the regulatory ß-subunit of PFP1 (PFP1ß). Measurement of nicotinamide adenine dinucleotide (NAD+) showed that mutation of PFP1ß markedly decreased its enzyme activity. PFP1ß and three of four putative catalytic α-subunits of PFP1, PFP1α1, PFP1α2, and PFP1α4, interacted with each other to form a heterotetramer. Additionally, PFP1ß, PFP1α1 and PFP1α2 also formed homodimers. Furthermore, transcriptome analysis revealed that mutation of PFP1ß significantly altered expression of many essential enzymes in starch biosynthesis pathways. Concentrations of multiple lipid and glycolytic intermediates and trehalose metabolites were elevated in pfp1-3 endosperm, indicating that PFP1 modulates endosperm metabolism, potentially through reversible adjustments to metabolic fluxes. Taken together, these findings provide new insights into seed endosperm development and starch biosynthesis and will help in the breeding of rice cultivars with higher grain yield and quality.

The chloride anion as a signalling effector.

The specific role of the chloride anion (Cl- ) as a signalling effector or second messenger has been increasingly recognized in recent years. It could represent a key factor in the regulation of cellular homeostasis. Changes in intracellular Cl- concentration affect diverse cellular functions such as gene and protein expression and activities, post-translational modifications of proteins, cellular volume, cell cycle, cell proliferation and differentiation, membrane potential, reactive oxygen species levels, and intracellular/extracellular pH. Cl- also modulates functions in different organelles, including endosomes, phagosomes, lysosomes, endoplasmic reticulum, and mitochondria. A better knowledge of Cl- signalling could help in understanding the molecular and metabolic changes seen in pathologies with altered Cl- transport or under physiological conditions. Here we review relevant evidence supporting the role of Cl- as a signalling effector.

CoPhosK: A method for comprehensive kinase substrate annotation using co-phosphorylation analysis.

We present CoPhosK to predict kinase-substrate associations for phosphopeptide substrates detected by mass spectrometry (MS). The tool utilizes a Naïve Bayes framework with priors of known kinase-substrate associations (KSAs) to generate its predictions. Through the mining of MS data for the collective dynamic signatures of the kinases' substrates revealed by correlation analysis of phosphopeptide intensity data, the tool infers KSAs in the data for the considerable body of substrates lacking such annotations. We benchmarked the tool against existing approaches for predicting KSAs that rely on static information (e.g. sequences, structures and interactions) using publically available MS data, including breast, colon, and ovarian cancer models. The benchmarking reveals that co-phosphorylation analysis can significantly improve prediction performance when static information is available (about 35% of sites) while providing reliable predictions for the remainder, thus tripling the KSAs available from the experimental MS data providing to a comprehensive and reliable characterization of the landscape of kinase-substrate interactions well beyond current limitations.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Reassessing the role of the Escherichia coli CpxAR system in sensing surface contact.

For proper biofilm formation, bacteria must have mechanisms in place to sense adhesion to surfaces. In Escherichia coli, the CpxAR and RcsCDB systems have been reported to sense surfaces. The CpxAR system is widely considered to be responsible for sensing attachment, specifically to hydrophobic surfaces. Here, using both single-cell and population-level analyses, we confirm RcsCDB activation upon surface contact, but find that the CpxAR system is not activated, in contrast to what had earlier been reported. Thus, the role of CpxAR in surface sensing and initiation of biofilm formation should be reconsidered.

BZR1 Transcription Factor Regulates Heat Stress Tolerance Through FERONIA Receptor-Like Kinase-Mediated Reactive Oxygen Species Signaling in Tomato.

BRASSINAZOLE RESISTANT 1 (BZR1), the critical regulator of brassinosteroid (BR) response, participates in various BR-mediated developmental processes. However, the roles of BZR1 in stress tolerance are less clear. Here, we found that BZR1-like protein in tomato controls BR response and is involved in thermotolerance by regulating the FERONIA (FER) homologs. The CRISPR-bzr1 mutant showed reduced growth and was not responsive to 24-epibrassinolide (EBR) with regard to the promotion of plant growth. Mutation in BZR1 impaired the induction of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1), production of H2O2 in the apoplast and heat tolerance. Exogenous H2O2 recovered the heat tolerance of the tomato bzr1 mutant. Overexpression of BZR1 enhanced the production of apoplastic H2O2 and heat stress responses. However, silencing of RBOH1 abolished the BZR1-mediated heat tolerance. Further analysis showed that BZR1 bound to the promoters of FERONIA2 (FER2) and FER3 and induced their expression. Silencing of FER2/3 suppressed BZR1-dependent BR signaling for the induction of RBOH1 transcripts, accumulation of apoplastic H2O2 and heat tolerance. These results indicate that BZR1 regulates heat stress responses in tomato through RBOH1-dependent reactive oxygen species (ROS) signaling, which is at least partially mediated by FER2 and FER3.

Inter-relationships between the heterotrimeric Gß subunit AGB1, the receptor-like kinase FERONIA, and RALF1 in salinity response.

Plant heterotrimeric G proteins modulate numerous developmental stress responses. Recently, receptor-like kinases (RLKs) have been implicated as functioning with G proteins and may serve as plant G-protein-coupled-receptors. The RLK FERONIA (FER), in the Catharantus roseus RLK1-like subfamily, is activated by a family of polypeptides called rapid alkalinization factors (RALFs). We previously showed that the Arabidopsis G protein ß subunit, AGB1, physically interacts with FER, and that RALF1 regulation of stomatal movement through FER requires AGB1. Here, we investigated genetic interactions of AGB1 and FER in plant salinity response by comparing salt responses in the single and double mutants of agb1 and fer. We show that AGB1 and FER act additively or synergistically depending on the conditions of the NaCl treatments. We further show that the synergism likely occurs through salt-induced ROS production. In addition, we show that RALF1 enhances salt toxicity through increasing Na+ accumulation and decreasing K+ accumulation rather than by inducing ROS production, and that the RALF1 effect on salt response occurs in an AGB1-independent manner. Our results indicate that RLK epistatic relationships are not fixed, as AGB1 and FER display different genetic relationships to RALF1 in stomatal versus salinity responses.

Comparison of the effects of a kinase-dead mutation of FERONIA on ovule fertilization and root growth of Arabidopsis.

A plasma membrane receptor protein kinase, FERONIA (FER), regulates various aspects of plant reproductive and vegetative growth. In roots, binding of a peptide ligand to FER causes rapid suppression of cell elongation whereas in ovules, FER is involved in gametophyte interactions. Here, we examined the effect of a mutation that eliminates kinase activity, on both ovule fertilization and root growth, using the same batch of seeds containing a kinase-dead mutation. The kinase-dead mutation of FER reduced the ability to complement fer-4 knockout phenotypes, compared with wild-type sequence in root, but not in ovules. Our results support a model in which cell type-specific regulatory mechanisms, such as different interacting partners and/or downstream signaling events, lead to cell type-specific functions of FER.

Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation.

Selenophosphate synthetase (SEPHS) synthesizes selenophosphate, the active selenium donor, using ATP and selenide as substrates. SEPHS was initially identified and isolated from bacteria and has been characterized in many eukaryotes and archaea. Two SEPHS paralogues, SEPHS1 and SEPHS2, occur in various eukaryotes, while prokaryotes and archaea have only one form of SEPHS. Between the two isoforms in eukaryotes, only SEPHS2 shows catalytic activity during selenophosphate synthesis. Although SEPHS1 does not contain any significant selenophosphate synthesis activity, it has been reported to play an essential role in regulating cellular physiology. Prokaryotic SEPHS contains a cysteine or selenocysteine (Sec) at the catalytic domain. However, in eukaryotes, SEPHS1 contains other amino acids such as Thr, Arg, Gly, or Leu at the catalytic domain, and SEPHS2 contains only a Sec. Sequence comparisons, crystal structure analyses, and ATP hydrolysis assays suggest that selenophosphate synthesis occurs in two steps. In the first step, ATP is hydrolyzed to produce ADP and gamma-phosphate. In the second step, ADP is further hydrolyzed and selenophosphate is produced using gamma-phosphate and selenide. Both SEPHS1 and SEPHS2 have ATP hydrolyzing activities, but Cys or Sec is required in the catalytic domain for the second step of reaction. The gene encoding SEPHS1 is divided by introns, and five different splice variants are produced by alternative splicing in humans. SEPHS1 mRNA is abundant in rapidly proliferating cells such as embryonic and cancer cells and its expression is induced by various stresses including oxidative stress and salinity stress. The disruption of the SEPHS1 gene in mice or Drosophila leads to the inhibition of cell proliferation, embryonic lethality, and morphological changes in the embryos. Targeted removal of SEPHS1 mRNA in insect, mouse, and human cells also leads to common phenotypic changes similar to those observed by in vivo gene knockout: the inhibition of cell growth/proliferation, the accumulation of hydrogen peroxide in mammals and an unidentified reactive oxygen species (ROS) in Drosophila, and the activation of a defense system. Hydrogen peroxide accumulation in SEPHS1-deficient cells is mainly caused by the down-regulation of genes involved in ROS scavenging, and leads to the inhibition of cell proliferation and survival. However, the mechanisms underlying SEPHS1 regulation of redox homeostasis are still not understood.

Enzyme sequestration by the substrate: An analysis in the deterministic and stochastic domains.

This paper is concerned with the potential multistability of protein concentrations in the cell. That is, situations where one, or a family of, proteins may sit at one of two or more different steady state concentrations in otherwise identical cells, and in spite of them being in the same environment. For models of multisite protein phosphorylation for example, in the presence of excess substrate, it has been shown that the achievable number of stable steady states can increase linearly with the number of phosphosites available. In this paper, we analyse the consequences of adding enzyme docking to these and similar models, with the resultant sequestration of phosphatase and kinase by the fully unphosphorylated and by the fully phosphorylated substrates respectively. In the large molecule numbers limit, where deterministic analysis is applicable, we prove that there are always values for these rates of sequestration which, when exceeded, limit the extent of multistability. For the models considered here, these numbers are much smaller than the affinity of the enzymes to the substrate when it is in a modifiable state. As substrate enzyme-sequestration is increased, we further prove that the number of steady states will inevitably be reduced to one. For smaller molecule numbers a stochastic analysis is more appropriate, where multistability in the large molecule numbers limit can manifest itself as multimodality of the probability distribution; the system spending periods of time in the vicinity of one mode before jumping to another. Here, we find that substrate enzyme sequestration can induce bimodality even in systems where only a single steady state can exist at large numbers. To facilitate this analysis, we develop a weakly chained diagonally dominant M-matrix formulation of the Chemical Master Equation, allowing greater insights in the way particular mechanisms, like enzyme sequestration, can shape probability distributions and therefore exhibit different behaviour across different regimes.

The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens.

Nonsense-mediated mRNA decay (NMD) is important for RNA quality control and gene regulation in eukaryotes. NMD targets aberrant transcripts for decay and also directly influences the abundance of non-aberrant transcripts. In animals, the SMG1 kinase plays an essential role in NMD by phosphorylating the core NMD factor UPF1. Despite SMG1 being ubiquitous throughout the plant kingdom, little is known about its function, probably because SMG1 is atypically absent from the genome of the model plant, Arabidopsis thaliana. By combining our previously established SMG1 knockout in moss with transcriptome-wide analysis, we reveal the range of processes involving SMG1 in plants. Machine learning assisted analysis suggests that 32% of multi-isoform genes produce NMD-targeted transcripts and that splice junctions downstream of a stop codon act as the major determinant of NMD targeting. Furthermore, we suggest that SMG1 is involved in other quality control pathways, affecting DNA repair and the unfolded protein response, in addition to its role in mRNA quality control. Consistent with this, smg1 plants have increased susceptibility to DNA damage, but increased tolerance to unfolded protein inducing agents. The potential involvement of SMG1 in RNA, DNA and protein quality control has major implications for the study of these processes in plants.

Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter.

Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.

Structure-Based Virtual Screening of Protein Tyrosine Phosphatase Inhibitors: Significance, Challenges, and Solutions.

Phosphorylation, a critical mechanism in biological systems, is estimated to be indispensable for about 30% of key biological activities, such as cell cycle progression, migration, and division. It is synergistically balanced by kinases and phosphatases, and any deviation from this balance leads to disease conditions. Pathway or biological activity-based abnormalities in phosphorylation and the type of involved phosphatase influence the outcome, and cause diverse diseases ranging from diabetes, rheumatoid arthritis, and numerous cancers. Protein tyrosine phosphatases (PTPs) are of prime importance in the process of dephosphorylation and catalyze several biological functions. Abnormal PTP activities are reported to result in several human diseases. Consequently, there is an increased demand for potential PTP inhibitory small molecules. Several strategies in structure-based drug designing techniques for potential inhibitory small molecules of PTPs have been explored along with traditional drug designing methods in order to overcome the hurdles in PTP inhibitor discovery. In this review, we discuss druggable PTPs and structure-based virtual screening efforts for successful PTP inhibitor design.

Unphosphorylated EIIANtr induces ClpAP-mediated degradation of RpoS in Azotobacter vinelandii.

The nitrogen-related phosphotransferase system (PTSNtr ) is composed of the EINtr , NPr and EIIANtr proteins that form a phosphorylation cascade from phosphoenolpyruvate. PTSNtr is a global regulatory system present in most Gram-negative bacteria that controls some pivotal processes such as potassium and phosphate homeostasis, virulence, nitrogen fixation and ABC transport activation. In the soil bacterium Azotobacter vinelandii, unphosphorylated EIIANtr negatively regulates the expression of genes related to the synthesis of the bioplastic polyester poly-ß-hydroxybutyrate (PHB) and cyst-specific lipids alkylresorcinols (ARs). The mechanism by which EIIANtr controls gene expression in A. vinelandii is not known. Here, we show that, in presence of unphosphorylated EIIANtr , the stability of the stationary phase sigma factor RpoS, which is necessary for transcriptional activation of PHB and ARs synthesis related genes, is reduced, and that the inactivation of genes coding for ClpAP protease complex in strains that carry unphosphorylated EIIANtr , restored the levels and in vivo stability of RpoS, as well as the synthesis of PHB and ARs. Taken together, our results reveal a novel mechanism, by which EIIANtr globally controls gene expression in A. vinelandii, where the unphosphorylated EIIANtr induces the degradation of RpoS by the proteolytic complex ClpAP.