BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) over-activation is highly involved in cardiovascular diseases (CVDs), with the Gαq-PLCβ3 axis acting as a core node of RAAS. PLCβ3 is a potential target of CVDs, and the lack of inhibitors has limited its drug development.
OBJECTIVE: Sinapine (SP) is a potential leading compound for treating CVDs. Thus, we aimed to elucidate the regulation of SP towards the Gαq-PLCβ3 axis and its molecular mechanism.
METHODS: Aldosteronism and hypertension animal models were employed to investigate SP\'s inhibitory effect on the abnormal activation of the RAAS through the Gαq-PLCβ3 axis. We used chemical biology methods to identify potential targets and elucidate the underlying molecular mechanisms.
METHODS: The effects of SP on aldosteronism and hypertension were evaluated using an established animal model in our laboratory. Target identification and underlying molecular mechanism research were performed using activity-based protein profiling with a bio-orthogonal click chemistry reaction and other biochemical methods.
RESULTS: SP alleviated aldosteronism and hypertension in animal models by targeting PLCβ3. The underlying mechanism for blocking the Gαq-PLCβ3 interaction involves targeting the EF hands through the Asn-260 amino acid residue. SP regulated the Gαq-PLCβ3 axis more precisely than the Gαq-GEFT or Gαq-PKCζ axis in the cardiovascular system.
CONCLUSIONS: SP alleviated RAAS over-activation via Gαq-PLCβ3 interaction blockade by targeting the PLCβ3 EF hands domain, which provided a novel PLC inhibitor for treating CVDs. Unlike selective Gαq inhibitors, SP reduced the risk of side effects compared to Gαq inhibitors in treating CVDs.

Rv1211 is a conserved hypothetical protein in Mycobacterium tuberculosis and is required for the growth and pathogenesis of the bacteria. The protein has been suggested as a calmodulin-like calcium-binding protein with an EF-hand motif and as a target of trifluoperazine, a calmodulin antagonist in eukaryotes that inhibits mycobacterial growth. Here, we expressed the recombinant protein of Rv1211 and performed structural and biochemical studies of Rv1211 and its interaction with Ca2+ or trifluoperazine. Surprisingly, Rv1211 exhibited an elution property typical of a natively unfolded protein. Subsequent circular dichroism experiments with temperature elevation and trifluoroethanol treatment showed that Rv1211 has unfolded structure. Additional NMR experiment confirmed the unfolded state of the protein and further showed that it does not bind to Ca2+. Still, Rv1211 did bind to trifluoperazine, as evidenced by the two-dimensional NMR spectra of 15N-labeled Rv1211. However, there were no peak shifts upon binding, showing that Rv1211 retained its unfolded state even after the trifluoperazine binding. The residues involved in the binding were clustered in the C-terminal region, as identified by the sequence assignment. Isothermal titration calorimetry showed that the Kd of trifluoperazine-Rv1211 binding is 41 μM and that the stoichiometry is 1 : 2 (Rv1211: trifluoperazine). Our results argue against the suggestion of Rv1211 as a Ca2+-binding calmodulin-like protein, and show that Rv1211 is a natively unfolded protein that binds to trifluoperazine. In addition, our results suggest the evidence of the \"Fuzziness\" in the Rv1211-trifluoperazine interaction that differs from the conventional binding-induced folding of natively unfolded proteins.

In \'Tsuda\' turnip, the swollen root peel accumulates anthocyanin pigments in a light-dependent manner, but the mechanism is unclear. Here, mutant g120w which accumulated extremely low levels of anthocyanin after light exposure was identified. Segregation analysis showed that the anthocyanin-deficient phenotype was controlled by a single recessive gene. By using bulked-segregant analysis sequencing and CAPS marker-based genetic mapping analyses, a 21.6-kb region on chromosome A07 was mapped, in which a calcium-binding EF hand family protein named BrLETM2 was identified as the causal gene. RNA sequencing analysis showed that differentially expressed genes (DEGs) between wild type and g120w in light-exposed swollen root peels were enriched in anthocyanin biosynthetic process and reactive oxygen species (ROS) biosynthetic process GO term. Furthermore, nitroblue tetrazolium (NBT) staining showed that the ROS level decreased in g120w mutant. Anthocyanins induced by UV-A were abolished by the pre-treatment of seedlings with DPI (an inhibitor of nicotinamide adenine nucleoside phosphorylase (NADPH) oxidase) and decreased in g120w mutant. These results indicate that BrLETM2 modulates ROS signaling to promote anthocyanin accumulation in turnip under UV-A and provides new insight into the mechanism of how ROS and light regulate anthocyanin production.

Calcium is an important second messenger in mediating adaptation responses of plants to abiotic and biotic stresses. Calmodulin-like (CML) protein is an important calcium-signaling protein that can sense and decode Ca2+ signal in plants. Medicago truncatula is a model legume plant; however, investigations of MtCML proteins are limited. Using genome analysis and BLAST database searches, fifty MtCML proteins that possess EF-hand motifs were identified. Phylogenetic analysis showed that CML homologs between M. truncatula, Arabidopsis thaliana and Oryza sativa shared close relationships. Gene structure analysis revealed that these MtCML genes contained one to four conserved EF-hand motifs. All MtCMLs are localized to eight chromosomes and underwent gene duplication. In addition, MtCML genes were differentially expressed in different tissues of M. truncatula. Cis-acting elements in promoter region and expression analysis revealed the potential response of MtCML protein to abiotic stress and hormones. The results provide a basis of further functional research on the MtCML gene family and facilitate their potential use for applications in the genetic improvement on M. truncatula in drought, cold and salt stress environments.

The marine diatom Phaeodactylum tricornutum originated from a series of secondary symbiotic events and has been used as a model organism for studying diatom biology. A novel type II homodimeric isocitrate dehydrogenase from P. tricornutum (PtIDH1) was expressed, purified, and identified in detail through enzymatic characterization. Kinetic analysis showed that PtIDH1 is NAD+-dependent and has no detectable activity with NADP+. The catalytic efficiency of PtIDH1 for NAD+ is 0.16 μM-1·s-1 and 0.09 μM-1·s-1 in the presence of Mn2+ and Mg2+, respectively. Unlike other bacterial homodimeric NAD-IDHs, PtIDH1 activity was allosterically regulated by the isocitrate. Furthermore, the dimeric structure of PtIDH1 was determined at 2.8 Å resolution, and each subunit was resolved into four domains, similar to the eukaryotic homodimeric NADP-IDH in the type II subfamily. Interestingly, a unique and novel C-terminal EF-hand domain was first defined in PtIDH1. Deletion of this domain disrupted the intact dimeric structure and activity. Mutation of the four Ca2+-binding sites in the EF-hand significantly reduced the calcium tolerance of PtIDH1. Thus, we suggest that the EF-hand domain could be involved in the dimerization and Ca2+-coordination of PtIDH1. The current report, on the first structure of type II eukaryotic NAD-IDH, provides new information for further investigation of the evolution of the IDH family.

The homologous leucine zipper/EF-hand-containing transmembranes (LETMs) are highly conserved across a broad range of eukaryotic organisms. The LETM functional characteristics involved in biological process have been identified primarily in animals, but little is known about the LETM biological function mode in plants. Based on the results of the current investigation, the GhLETM1 gene crucially affects filament elongation and anther dehiscence of the stamen in cotton. Both excessive and lower expression of the GhLETM1 gene lead to defective stamen development, resulting in shortened filaments and indehiscent anthers with pollen abortion. The results also showed that the phenotype of the shortened filaments was negatively correlated with anther defects in the seesaw model under the ectopic expression of GhLETM1. Moreover, our results notably indicated that the gene requires accurate expression and exhibits a sensitive dose effect for its proper function. This report has important fundamental and practical significance in crop science, and has crucial prospects for genetic engineering of new cytoplasmic male sterility lines and breeding of crop hybrid varieties.

Oscillations in cytosolic free calcium determine the polarity of tip-growing root hairs. The Ca2+ channel cyclic nucleotide gated channel 14 (CNGC14) contributes to the dynamic changes in Ca2+ concentration gradient at the root hair tip. However, the mechanisms that regulate CNGC14 are unknown. In this study, we detected a direct interaction between calmodulin 7 (CaM7) and CNGC14 through yeast two-hybrid and bimolecular fluorescence complementation assays. We demonstrated that the third EF-hand domain of CaM7 specifically interacts with the cytosolic C-terminal domain of CNGC14. A two-electrode voltage clamp assay showed that CaM7 completely inhibits CNGC14-mediated Ca2+ influx, suggesting that CaM7 negatively regulates CNGC14-mediated calcium signaling. Furthermore, CaM7 overexpressing lines phenocopy the short root hair phenotype of a cngc14 mutant and this phenotype is insensitive to changes in external Ca2+ concentrations. We, thus, identified CaM7-CNGC14 as a novel interacting module that regulates polar growth in root hairs by controlling the tip-focused Ca2+ signal.

Tic20 is an important translocon protein that plays a role in protein transport in the chloroplast. The sequence of Tic20 was determined in the lower brown alga Saccharina japonica. Structural analysis of SjTic20 revealed a noncanonical structure consisting of an N-terminal non-cyanobacterium-originated EF-hand domain (a helix-loop-helix structural domain) and a C-terminal cyanobacterium-originated Tic20 domain. Subcellular localization and transmembrane analysis indicated that SjTic20 featured an \"M\"-type Nin-Cin-terminal orientation, with four transmembrane domains in the innermost membrane of the chloroplast in the microalga Phaeodactylum tricornutum, and the EF-hand domain was entirely extruded into the chloroplast stroma. Our study provides information on the structure, localization, and topological features of SjTic20, and further functional analysis of SjTic20 in S. japonica is needed.

Centrin is an evolutionarily conserved EF-hand-containing protein, which is present in eukaryotic organisms as diverse as algae, yeast, and humans. Centrins are associated with the microtubule-organizing center and with centrosome-related structures, such as basal bodies in flagellar and ciliated cells, and the spindle pole body in yeast. Five centrin genes have been identified in Trypanosoma brucei (T. brucei), a protozoan parasite that causes sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. In the present study, we identified that centrin5 of T. brucei (TbCentrin5) is localized throughout the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds Ca2+ ions with a high affinity and constructed a model of TbCentrin5 bound by Ca2+ ions. Meanwhile, we observed that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 and that the interactions are Ca2+ -dependent, suggesting that TbCentrin5 is able to form different complexes with other TbCentrins to participate in relevant cellular processes. Our study provides a foundation for better understanding of the biological roles of TbCentrin5.

The transcription factor IRF3 is phosphorylated in response to viral infection, and it subsequently forms a homodimer and translocates into the nucleus to induce the transcription of genes important for antiviral immunity, such as type I interferons (IFNs). This multistep process is essential for host defense against viral infection, but its regulation remains elusive. Here, we report that the EF-hand protein calmodulin-like 6 (CALML6) directly bound to the phosphorylated serine-rich (SR) region of IRF3 and impaired its dimerization and nuclear translocation. Enforced CALML6 expression suppressed viral infection-induced production of IFN-β and expression of IFN-stimulated genes (ISGs), whereas CALML6 deficiency had the opposite effect. In addition, impaired IFN-β and ISG expression in bone-marrow-derived macrophages and tissues of CALML6 transgenic mice promoted viral replication. These findings identify a phosphorylation-dependent negative feedback loop that maintains the homeostasis of antiviral innate immunity.