Structural Characterization of the Interaction between the αMI-Domain of the Integrin Mac-1 (αMß2) and the Cytokine Pleiotrophin.

Integrin Mac-1 (αMß2) is an adhesion receptor vital to many functions of myeloid leukocytes. It is also the most promiscuous member of the integrin family capable of recognizing a broad range of ligands. In particular, its ligand-binding αMI-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αMI-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of the αMI-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through NMR chemical shift perturbation analysis, cross saturation, NOESY, and mutagenesis studies, we found the interaction between the αMI-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5-ß5 loop of αMI-domain. The observation that increased ionic strength weakens the interaction between the proteins indicates electrostatic forces may also play a significant role in the binding. On the basis of the results from these experiments, we formulated a model of the interaction between the αMI-domain and PTN.

Arabidopsis CPK6 positively regulates ABA signaling and drought tolerance through phosphorylating ABA-responsive element-binding factors.

Abscisic acid (ABA) regulates numerous developmental processes and drought tolerance in plants. Calcium-dependent protein kinases (CPKs) are important Ca2+ sensors playing crucial roles in plant growth and development as well as responses to stresses. However, the molecular mechanisms of many CPKs in ABA signaling and drought tolerance remain largely unknown. Here we combined protein interaction studies, and biochemical and genetic approaches to identify and characterize substrates that were phosphorylated by CPK6 and elucidated the mechanism that underlines the role of CPK6 in ABA signaling and drought tolerance. The expression of CPK6 is induced by ABA and dehydration. Two cpk6 T-DNA insertion mutants are insensitive to ABA during seed germination and root elongation of seedlings; in contrast, overexpression of CPK6 showed the opposite phenotype. Moreover, CPK6-overexpressing lines showed enhanced drought tolerance. CPK6 interacts with and phosphorylates a subset of core ABA signaling-related transcription factors, ABA-responsive element-binding factors (ABFs/AREBs), and enhances their transcriptional activities. The phosphorylation sites in ABF3 and ABI5 were also identified through MS and mutational analyses. Taken together, we present evidence that CPK6 mediates ABA signaling and drought tolerance through phosphorylating ABFs/AREBs. This work thus uncovers a rather conserved mechanism of calcium-dependent Ser/Thr kinases in ABA signaling.

Advances in understanding broad-spectrum resistance to pathogens in rice.

Rice diseases caused by multiple pathogen species are a major obstacle to achieving optimal yield. Using host pathogen species-non-specific broad-spectrum resistance (BSR) for rice improvement is an efficient way to control diseases. Recent advances in rice genomics and improved understanding of the mechanisms of rice-pathogen interactions have shown that using a single gene to improve rice BSR to multiple pathogen species is technically possible and the necessary resources exist. A variety of rice genes, including major disease resistance genes and defense-responsive genes, which function in pattern-triggered immunity signaling, effector-triggered immunity signaling or quantitative resistance, can mediate BSR to two or more pathogen species independently. These genes encode diverse proteins and function differently in promoting disease resistance, thus providing a relatively broad choice for different breeding programs. This updated knowledge will facilitate rice improvement with pathogen species-non-specific BSR via gene marker-assisted selection or biotechnological approaches.

Rapeseed calcineurin B-like protein CBL4, interacting with CBL-interacting protein kinase CIPK24, modulates salt tolerance in plants.

Calcium is a ubiquitous intracellular secondary messenger in eukaryotes. Upon stress challenge, cytosolic Ca(2+) fluctuation could be sensed and bound by calcineurin B-like proteins (CBLs), which further regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs) to relay the signal and induce cellular responses. Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in rapeseed. In the present study, we characterized CBL4 gene from rapeseed. We found that CBL4 is localized at the plasma membrane and it interacted with CIPK24 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Unlike the orthologs in Arabidopsis, rapeseed CIPK24 did not interact with CBL10. Furthermore, expression of rapeseed CBL4 rescued the salt-sensitive phenotype of sos3-1 mutant and overexpression of rapeseed CBL4 in Arabidopsis showed enhanced tolerance of salt stress than wild-type. Overall, the results clarified the function of CBL4 in rapeseed.

A CCCH-type zinc finger nucleic acid-binding protein quantitatively confers resistance against rice bacterial blight disease.

Bacterial blight is a devastating disease of rice (Oryza sativa) caused by Xanthomonas oryzae pv oryzae (Xoo). Zinc finger proteins harboring the motif with three conserved cysteine residues and one histidine residue (CCCH) belong to a large family. Although at least 67 CCCH-type zinc finger protein genes have been identified in the rice genome, their functions are poorly understood. Here, we report that one of the rice CCCH-type zinc finger proteins, C3H12, containing five typical CX(8)-CX(5)-CX(3)-H zinc finger motifs, is involved in the rice-Xoo interaction. Activation of C3H12 partially enhanced resistance to Xoo, accompanied by the accumulation of jasmonic acid (JA) and induced expression of JA signaling genes in rice. In contrast, knockout or suppression of C3H12 resulted in partially increased susceptibility to Xoo, accompanied by decreased levels of JA and expression of JA signaling genes in rice. C3H12 colocalized with a minor disease resistance quantitative trait locus to Xoo, and the enhanced resistance of randomly chosen plants in the quantitative trait locus mapping population correlated with an increased expression level of C3H12. The C3H12 protein localized in the nucleus and possessed nucleic acid-binding activity in vitro. These results suggest that C3H12, as a nucleic acid-binding protein, positively and quantitatively regulates rice resistance to Xoo and that its function is likely associated with the JA-dependent pathway.

A network pharmacological-based study of the mechanism of Liuwei Dihuang pill in the treatment of chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) is a progressive disease that poses a huge economic burden to society. Liuwei Dihuanng pill is an effective treatment for chronic kidney disease, but its treatment mechanism is unclear. The rapid development of network pharmacology has provided new strategies for studying Chinese medicine. METHOD: The traditional Chinese medicine systems pharmacology database and analysis platform was used to obtain the bioactive components and targets of Liuwei Dihuanng pill. The sources for the CKD-related targets were then obtained from the Genecards, OMIM, TTD, and DisGeNET databases. R was used to identify the intersecting genes for Liuwei Dihuang pill and CKD-related targets. Analysis of protein-protein interactions (PPI) was performed using STRING, and PPI networks and drug-component-target networks were constructed using Cytoscape software. Kyoto encyclopedia of genes and genomes pathway and gene ontology enrichment analyses were performed using R. Finally, molecular docking was performed to determine the binding activity between bioactive components and the targets. RESULT: After screening and data de-duplication of 74 active components, 209 drug targets, and 14,794 disease targets, a total of 204 drug-disease targets were acquired. Subsequently, a drug-component-target network and PPI network were established. The primary components of Liuwei Dihuang pill included quercetin, stigmasterol, kaempferol, beta-sitosterol, tetrahydroalstonine, kadsurenone, hederagenin, hancinone C, diosgenin, and sitosterol. In addition, JUN, AKT1, TP53, RELA, MAPK1, FOS, TNF, IL6, ESR1, and RXRA were identified as the main targets. Gene ontology function enrichment analysis revealed that these targets were involved in reactive oxygen species metabolic processes, responses to metal ions and to chemical stimuli, G protein-coupled amine receptor activity, and nuclear factor receptor activity. Kyoto encyclopedia of genes and genomes enrichment analysis showed that these targets were involved in the AGE-RAGE signaling pathway, IL-17 signaling pathway, TNF signaling pathway, and so on. Molecular docking results indicated good binding activity between the core targets and core components. CONCLUSION: The potential mechanism of Liuwei Dihuanng pill in the treatment of CKD was preliminarily discussed in this study, providing a theoretical basis and evidence for further experimental research.

Engineering an Enzyme for Direct Electrical Monitoring of Activity.

Proteins have been shown to be electrically conductive if tethered to an electrode by means of a specific binding agent, allowing single molecules to be wired into an electrical sensing circuit. Such circuits allow enzymes to be used as sensors, detectors, and sequencing devices. We have engineered contact points into a Φ29 polymerase by introducing biotinylatable peptide sequences. The modified enzyme was bound to electrodes functionalized with streptavidin. Φ29 connected by one biotinylated contact, and a second nonspecific contact showed rapid small fluctuations in current when activated. Signals were greatly enhanced with two specific contacts. Features in the distributions of DC conductance increased by a factor 2 or more over the open to closed conformational transition of the polymerase. Polymerase activity is manifested by a rapid (millisecond) large (25% of background) current fluctuations imposed on the DC conductance.

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice.

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 microg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.