EF-hand Domain Research Articles

Structural and functional interactions between the EF hand domain and S2–S3 loop in the type-1 ryanodine receptor ion channel

Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.

Aberrant Activity of the Calcium Sensor STIM1 Underlies Congenital Platelet Disorders and Myeloproliferative Neoplasms

MB and VA equal contributors AB and STO co-corresponding authors This study was initiated following evaluation of a 32 year-old woman who presented with a history of thrombocytopenia identified in childhood who subsequently developed features of myelofibrosis (MF). A bone marrow biopsy demonstrated hypercellularity in conjunction with megakaryocyte hyperplasia and marked reticulin fibrosis. Molecular testing for JAK2, CALR, and MPL mutations was negative. Given the unusual association between congenital thrombocytopenia and MF in this patient, exome sequencing was performed, revealing a heterozygous R304W mutation in the coiled coil (CC) domain of STIM1. Activating mutations in the CC and EF hand domains of STIM1 have been associated with Stormorken syndrome, a rare congenital platelet disorder associated with abnormal store operated calcium entry (SOCE). We subsequently identified a second patient with MF associated with a STIM1 activating mutation. This individual was found to have severe thrombocytopenia at birth, and exome sequencing revealed a heterozygous S88G mutation in the EF hand of STIM1. A bone marrow biopsy obtained at 6 months of age revealed atypical megakaryocytes and grade 1-2 MF. A repeat biopsy at age 2 showed persistence of stable MF. The unusual finding of MF in these two patients suggested the possibility of altered calcium signaling as a shared mechanism driving congenital platelet disorders such as Stormorken syndrome and myeloproliferative neoplasms (MPNs) including MF. In support of this notion, we identified elevated STIM1 expression in MF vs normal megakaryocyte progenitors, as well as in platelets from patients with essential thrombocythemia (ET) vs healthy controls. Additionally, we found that STIM1 expression was significantly elevated in CD34+ hematopoietic stem/progenitor cells (HSPCs) from both ET and MF patients vs healthy controls. Notably, STIM1 expression was increased in both JAK2 and CALR-mutant MF patients. Collectively, these findings provide evidence of aberrant STIM1 expression in MPN patient cells. To determine the functional role of STIM1 in MPN disease development, colony assays and patient-derived xenograft (PDX) experiments were performed with MF patient CD34+ cells subjected to CRISPR ablation of STIM1. Strikingly discordant results were observed with JAK2 vs CALR-mutant patient samples. Abrogation of STIM1 in CALR-mutant CD34+ cells led to decreased colony formation, and NSGS mice engrafted in parallel with STIM1-targeted cells exhibited decreased human CD45+ cell engraftment in conjunction with prolonged survival. These findings suggest an important role for STIM1 in CALR-mutant MPN disease phenotypes. In contrast, targeting of STIM1 in JAK2-mutant CD34+ cells led to increased colony formation and exacerbated disease phenotypes in vivo as manifested by enhanced human CD45+ cell engraftment, worsened splenomegaly, and early lethality. Similar results were obtained in experiments utilizing pharmacologic inhibitors of SOCE activity. Taken together, these findings indicate that the consequences of aberrant STIM1 activity may be context-dependent relating to specific MPN driver mutations. To expand these observations, we identified a separate cohort of 9 family members in Italy with Stormorken syndrome and confirmed STIM1 EF hand mutations. In ex vivo megakaryocytic differentiation assays, cells from affected individuals exhibited a defect in proplatelet formation. These observations were corroborated by initial analyses of a newly generated Stim1 R304W conditional knock-in mouse which recapitulated the characteristic thrombocytopenia found in patients with Stormorken syndrome. In summary, this study represents the first demonstration of MF development in patients with Stormorken syndrome, thereby uncovering a previously unrecognized hallmark of altered calcium signaling via aberrant STIM1 activation underlying Stormorken syndrome and MPNs. Our findings suggest distinct mechanisms relating to the interaction between JAK2 vs CALR mutation and altered STIM1 activity. Further studies of these relationships may have important ramifications for potential therapeutic approaches targeting these pathways.

Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK on the mitochondrial membrane is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding to MIRO1. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1’s EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394-431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1’s EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

EFHD1 expression is correlated with tumor-infiltrating neutrophils and predicts prognosis in gastric cancer

BackgroundGastric cancer (GC) ranks third in terms of mortality worldwide. The tumor microenvironment is critical for the progression of gastric cancer. This study investigated the association between EF-hand domain containing 1 (EFHD1) expression and its clinical significance in the tumor microenvironment (TME) of gastric cancer. MethodsWe used bioinformatic analyses to assess the relevance of EFHD1 mRNA in the TME of gastric carcinoma tissues and its relationship with clinical features. Therefore, we performed multiplex immunohistochemistry analyses to determine the potential role of the EFHD1 protein in the TME of gastric cancer. ResultsEFHD1 expression increased dramatically in gastric cancer tissues compared to levels in non-cancerous tissue samples (t = 6.246, P < 0.001). The EFHD1 protein presentation was associated with invasion depth (χ2 = 19.120, P < 0.001) and TNM stages (χ2 = 14.468, P = 0.002). Notably, EFHD1 protein expression was significantly related to CD66b + neutrophil infiltration of the intratumoral (r = 0.420, P < 0.001) and stromal (r = 0.367, P < 0.001) TME in gastric cancer. Additionally, Cox regression analysis revealed that EFHD1 was an independent prognostic predictor (hazard ratio [HR] = 2.262, P < 0.001) in patients with gastric cancer. ConclusionsOur study revealed the pattern of EFHD1 overexpression in the TME of patients with gastric cancer and demonstrated its utility as a biomarker for unfavorable clinical outcomes, thereby providing a potential immunotherapy target.

Clinical characterization of EFHD2 (swiprosin-1) in Glioma-associated macrophages and its role in regulation of immunosuppression

Glioblastoma has been extensively studied due to its high mortality and short survival. The evolution mechanism of tumor-associated macrophages (TAMs) to Glioma-associated microglia and macrophages (GAMs) in the tumor microenvironment (TME) remains to be elucidated. The tumor cell-to-cell interaction patterns have not been well defined yet. The EF-Hand Domain Family Member D2 (EFHD2) has been reported to be differentially expressed as an immunomodulatory molecule in a variety of cancers. But large-scale clinical data from multiple ethnic communities have not been used to investigate the role of EFHD2 in glioma. RNA-seq data from 313 or 657 glioma patients from the Chinese Glioma Genome Atlas (CGGA) database and 603 glioma patients from the Cancer Genome Atlas (TCGA) database were analyzed retrospectively. Cell localization was performed using single-cell sequencing data from the CGGA database and the GSE131928 dataset. Mouse glioma cell lines and primary macrophages isolated from Efhd2 knockout mice were co-cultured to validate the immunomodulatory effects of EFHD2 on macrophages and the remodeling of TME of glioblastoma. EFHD2 is enriched in high-grade gliomas, isocitrate dehydrogenase wild-type, and 1p/19q non-co-deficient gliomas. It is a potential biomarker of glioma-proneuronal subtypes and an independent prognostic factor for overall survival in patients with malignant glioblastoma. EFHD2 regulates the monocyte-macrophage system function and positively correlates with immunosuppressive checkpoints. Further experimental data demonstrates that Efhd2 influences the polarization state of GAMs and inhibits the secretion of TGF-β1. In vitro experiments have revealed that macrophages lacking Efhd2 suppress the vitality of two glioma cell lines and decelerate the growth of glioma xenografts. In conclusion, EFHD2 promises to be a key target for TME-related immunotherapy.

Evolution and diversification of CaM/CML gene family in green plants

Calmodulin (CaM) and calmodulin-like (CML) proteins are crucial Ca2+ sensors, which are widely involved in different biological processes of plants, including their growth and development, and stress responses. However, the origin and evolution of the CaM/CML gene family in plants remain elusive. In this study, 2133 CaM and 23094 CML genes were identified from the 1000 plants project (1 KP) species and the sequenced plants, covering algae, mosses, monilophytes, lycophytes, flowering plants, and all other green plant branches. Analysis showed that the size of the CML subfamily was correlated with the genome size of corresponding plant species, as well as the total gene number in the genome. Moreover, with the evolution from algae to angiosperms, the number of CML genes in plants increased gradually which could have been driven mainly by genome-wide segmental duplication events, while the number of CaMs remained basically stable at 2–3. Phylogenetic analysis demonstrated that CaM first appeared in green algae, while CML appeared earlier and has already been presented in dinoflagellates. Further analysis showed that the number and sequence of EF-hand domain in CaMs are highly conserved, while those of CMLs are diverse among different plant taxa. Expression analysis revealed that the expression level of CaMs was generally higher than that of CMLs, indicating that the high-expression genes have essential functions, while the low-expression genes are the main reasons for the functional diversity of the CaM/CML gene family in plants. The results might contribute to understanding the evolution of CaM/CML genes as well as their molecular functions.

STIM1: The lord of the rings?

STIM1 and Orai1 are the central core of the Store Operated Calcium Entry (SOCE). This calcium influx mechanism is triggered after the activation of Gq protein-coupled receptors at the plasma membrane (PM) that activate phospholipase C. The phospholipase C produces Inositol triphosphate (IP3) which rapidly diffuses throughout the cytosol, resulting in the binding and activation of IP3 receptors (IP3R) and the rapid efflux of calcium from the endoplasmic reticulum (ER) to the cytosol. The calcium depletion in the ER is sensed by the stromal interaction molecule 1 (STIM1) a single-pass transmembrane protein at the ER that binds intraluminal calcium through an EF-hand domain in its amino terminal region (Fig. 1A). The cytosolic portion of STIM1 contains multiple domains. The region that interacts and activates Orai channels is known as SOAR (the STIM1 Orai activating region) [1]. For SOAR be accessible to Orai1, STIM1 must get an extended conformation that unlocks SOAR from its coiled-coil 1 (CC1) region [2]. The extended conformation is triggered by calcium depletion at the ER that oligomerizes STIM1. The oligomers of STIM1 then translocate to a close distance between two opposing membranes, forming what is known as ER-PM junctions. STIM1 accumulates at ER-PM junctions conforming the denominated STIM1 puncta.

An X-Domain Phosphoinositide Phospholipase C (PI-PLC-like) of Trypanosoma brucei Has a Surface Localization and Is Essential for Proliferation

Trypanosoma brucei is the causative agent of African trypanosomiasis, a deadly disease that affects humans and cattle. There are very few drugs to treat it, and there is evidence of mounting resistance, raising the need for new drug development. Here, we report the presence of a phosphoinositide phospholipase C (TbPI-PLC-like), containing an X and a PDZ domain, that is similar to the previously characterized TbPI-PLC1. TbPI-PLC-like only possesses the X catalytic domain and does not have the EF-hand, Y, and C2 domains, having instead a PDZ domain. Recombinant TbPI-PLC-like does not hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and does not modulate TbPI-PLC1 activity in vitro. TbPI-PLC-like shows a plasma membrane and intracellular localization in permeabilized cells and a surface localization in non-permeabilized cells. Surprisingly, knockdown of TbPI-PLC-like expression by RNAi significantly affected proliferation of both procyclic and bloodstream trypomastigotes. This is in contrast with the lack of effect of downregulation of expression of TbPI-PLC1.

Kinetics of Ca2+ Dissociation from Cod Parvalbumin Studied by Fluorescent Stopped-flow Method.

Small Ca2+-binding protein parvalbumin possesses two strong Ca2+/Mg2+- binding sites located within two EF-hand domains. Most parvalbumins have no tryptophan residues, while cod protein contains a single tryptophan residue, which fluorescence (spectrum maximum position and fluorescence quantum yield) is highly sensitive to the Ca2+ association/dissociation. Intrinsic protein fluorescence of cod parvalbumin can be used for elucidating the mechanism of Ca2+ binding to this protein. Fluorescence of the single tryptophan residue of cod parvalbumin has been used to monitor Ca2+-induced changes in the protein, both in steady-state and kinetic mode. Steady-state fluorescence spectra of cod parvalbumin were measured using Cary Eclipse spectrofluorimeter. Stopped-flow accessories in combination with a novel high-speed spectrofluorimeter were used for measurements of kinetics of Ca2+ dissociation from cod parvalbumin after fast mixing of Ca2+-loaded protein with a chelator of divalent metal cations ethylenediaminetetraacetic acid (EDTA). The fluorescent phase plots (fluorescence intensity at a fixed wavelength plotted against a fluorescence intensity at another fixed wavelength), constructed from steady state and kinetical data, shows a break at [Ca2+]/[parvalbumin] ratio close to 1. This means that the transition passes through an intermediate state, which is a protein with one bound calcium ion. These observations indicate that the binding of Ca2+ to cod parvalbumin is sequential. The results of the present spectral study showed that the binding of Ca2+ to cod parvalbumin is a sequential process. Calcium dissociation rate constants for the two binding sites of cod parvalbumin evaluated from the kinetic data are koff1 = 1.0 s-1 and koff2 = 1.5 s-1.

The leucine zipper EF-hand containing transmembrane protein-1 EF-handis an atypical tripartite calcium, temperature, and pH sensor.

Mitochondrial calcium (mtCa2+) is integral to ATP production; however, excessive Ca2+ leads to cell death. Both these processes are crucial to cellular function, and thus mtCa2+ homeostasis is tightly regulated. Leucine zipper EF-hand containing transmembrane protein-1 (LETM1) is an essential inner-mitochondrial membrane protein that regulates mtCa2+ through exchange of Ca2+ for protons (H+). LETM1 haploinsufficiency is associated with Wolf-Hirschhorn syndrome, characterized by microcephaly, growth retardation, and epileptic seizures, indicating a fundamental function for LETM1 in humans. LETM1 contains a conserved Ca2+ binding EF-hand motif (EF1). Deletion of EF1 abrogates LETM1 mediated mtCa2+ flux, but the precise mechanism by which EF1 regulates exchanger function remains unknown. To begin understanding the role of EF1 in LETM1 function, we biophysical and structurally characterized isolated EF1. Our data show that EF1 exhibits augmented α-helical secondary structure, solvent exposed hydrophobicity and oligomerization in the presence of Ca2+ due to a weak but specific, Ca2+-binding affinity. The low affinity is, in part, due to a terminal Asp within the binding loop, where mutation to Glu increases the affinity ∼4-fold. EF1 is structurally sensitive to pH changes within the physiological range for mitochondria, and remarkably, unfolds at high and low temperatures. Using solution NMR spectroscopy, we reveal that EF1 adopts an atypical EF-hand arrangement by pairing with an upstream loop-helix motif. Collectively, our data suggest that EF1 functions as a divergent multipartite environmental sensor within the EF-hand protein family by adopting an inimitable three-helix EF-hand domain arrangement in LETM1.

Genome-wide identification and expression analysis of calmodulin-like proteins in cucumber.

The calmodulin-like (CML) protein is a crucial Ca2+-binding protein that can sense and conduct the Ca2+ signal in response to extracellular stimuli. The CML protein families have been identified and characterized in many species. Nevertheless, scarce information on cucumber CML is retrievable. In this study, bioinformatic analyses, including gene structure, conserved domain, phylogenetic relationship, chromosome distribution, and gene synteny, were comprehensively performed to identify and characterize CsCML gene members. Spatiotemporal expression analysis in different organs and environment conditions were assayed with real-time quantitative polymerase chain reaction (qRT-PCR). Forty-four CsCMLs family members were well characterized, and the results showed that the 44 CsCML proteins contained one to four EF-hand domains without other functional domains. Most of the CsCML proteins were intron-less and unevenly distributed on seven chromosomes; two tandemly duplicated gene pairs and three segmentally duplicated gene pairs were identified in the cucumber genome. Cis-acting element analysis showed that the hormone, stress, and plant growth and development-related elements were in the promotor regions. In addition, spatiotemporal expression analysis revealed distinctive expression patterns for CsCML genes in different tissues and environmental conditions, and a putative protein interaction network also confirmed their potential role in responding to various stimuli. These results provide a foundation for understanding CsCMLs and provide a theoretical basis for further study of the physiological functions of CsCMLs.

Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy.

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

Calcineurin B inhibits calcium oxalate crystallization, growth and aggregation via its high calcium-affinity property

Calcineurin inhibitors (CNIs) are widely used in organ transplantation to suppress immunity and prevent allograft rejection. However, some transplant patients receiving CNIs have hypocitraturia, hyperoxaluria and kidney stone with unclear mechanism. We hypothesized that CNIs suppress activities of urinary calcineurin, which may serve as the stone inhibitor. This study aimed to investigate effects of calcineurin B (CNB) on calcium oxalate monohydrate (COM) stone formation. Sequence and structural analyses revealed that CNB contained four EF-hand (Ca2+-binding) domains, which are known to regulate Ca2+ homeostasis and likely to affect COM crystals. Various crystal assays revealed that CNB dramatically inhibited COM crystallization, crystal growth and crystal aggregation. At an equal amount, degrees of its inhibition against crystallization and crystal growth were slightly inferior to total urinary proteins (TUPs) from healthy subjects that are known to strongly inhibit COM stone formation. Surprisingly, its inhibitory effect against crystal aggregation was slightly superior to TUPs. While TUPs dramatically inhibited crystal-cell adhesion, CNB had no effect on this process. Ca2+-affinity assay revealed that CNB strongly bound Ca2+ at a comparable degree as of TUPs. These findings indicate that CNB serves as a novel inhibitor of COM crystallization, growth and aggregation via its high Ca2+-affinity property.

Genome Wide Characterization of CBL-CIPK Family Genes and Their Responsive Expression in Rosa chinensis

Calcium (Ca2+) plays a pivotal role in various signal transduction pathways. Calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ signals by activating the plant specific protein kinase known as the CBL-interacting protein kinase (CIPK). In plants, the CBL-CIPK signaling network regulates multiple signals in response to different extracellular cues including abiotic stress. However, the genome wide annotation and expression patterns of CBLs and CIPKs in woody cutting flower plants are still unclear. In this study, a total number of 7 CBLs (RcCBLs) and 17 CIPKs (RcCIPKs) genes, divided into four and five subfamilies, respectively, were identified from the rose genome. All RcCBLs possess a classic elongation factor-hand (EF-hand) domain, while all RcCIPKs possess both the classic kinase and NAF domains. Most RcCBLs were predicted to be plasma membrane localized, whereas most RcCIPKs were predicted to be cytoplasmic localized. Synteny analysis showed that one RcCBL gene pair and five RcCIPK gene pairs have gone through whole genome duplication events. Promoter cis-element prediction assays indicated that RcCBLs and RcCIPKs could function in different abiotic stress responses in rose plants. Further quantitative real-time PCR analysis demonstrated that RcCBLs and RcCIPKs were expressed in different organs with overlapped but distinct patterns in response to various abiotic stresses. The findings in this work will provide fundamental information and gene resources for further functional research on RcCBLs and RcCIPKs.

Abstract 1775: Ca2+ Induces Dimeric Dual Oxidase's EF-hand Domain to Monomer: an implication for its activation Mechanism

Abstract 1775: Ca2+ Induces Dimeric Dual Oxidase's EF-hand Domain to Monomer: an implication for its activation Mechanism

TPC1-Type Channels in Physcomitrium patens: Interaction between EF-Hands and Ca2.

Two-pore channels (TPCs) are members of the superfamily of ligand-gated and voltage-sensitive ion channels in the membranes of intracellular organelles of eukaryotic cells. The evolution of ordinary plant TPC1 essentially followed a very conservative pattern, with no changes in the characteristic structural footprints of these channels, such as the cytosolic and luminal regions involved in Ca2+ sensing. In contrast, the genomes of mosses and liverworts encode also TPC1-like channels with larger variations at these sites (TPC1b channels). In the genome of the model plant Physcomitrium patens we identified nine non-redundant sequences belonging to the TPC1 channel family, two ordinary TPC1-type, and seven TPC1b-type channels. The latter show variations in critical amino acids in their EF-hands essential for Ca2+ sensing. To investigate the impact of these differences between TPC1 and TPC1b channels, we generated structural models of the EF-hands of PpTPC1 and PpTPC1b channels. These models were used in molecular dynamics simulations to determine the frequency with which calcium ions were present in a coordination site and also to estimate the average distance of the ions from the center of this site. Our analyses indicate that the EF-hand domains of PpTPC1b-type channels have a lower capacity to coordinate calcium ions compared with those of common TPC1-like channels.

Comparative time-course transcriptome analysis of two contrasting alfalfa (Medicago sativa L.) genotypes reveals tolerance mechanisms to salt stress.

Salt stress is a major abiotic stress affecting plant growth and crop yield. For the successful cultivation of alfalfa (Medicago sativa L.), a key legume forage, in saline-affected areas, it's essential to explore genetic modifications to improve salt-tolerance.Transcriptome assay of two comparative alfalfa genotypes, Adina and Zhaodong, following a 4 h and 8 h's 300 mM NaCl treatment was conducted in this study in order to investigate the molecular mechanism in alfalfa under salt stress conditions. Results showed that we obtained 875,023,571 transcripts and 662,765,594 unigenes were abtained from the sequenced libraries, and 520,091 assembled unigenes were annotated in at least one database. Among them, we identified 1,636 differentially expression genes (DEGs) in Adina, of which 1,426 were up-regulated and 210 down-regulated, and 1,295 DEGs in Zhaodong, of which 565 were up-regulated and 730 down-regulated. GO annotations and KEGG pathway enrichments of the DEGs based on RNA-seq data indicated that DEGs were involved in (1) ion and membrane homeostasis, including ABC transporter, CLC, NCX, and NHX; (2) Ca2+ sensing and transduction, including BK channel, EF-hand domain, and calmodulin binding protein; (3) phytohormone signaling and regulation, including TPR, FBP, LRR, and PP2C; (4) transcription factors, including zinc finger proteins, YABBY, and SBP-box; (5) antioxidation process, including GST, PYROX, and ALDH; (6) post-translational modification, including UCH, ubiquitin family, GT, MT and SOT. The functional roles of DEGs could explain the variations in salt tolerance performance observed between the two alfalfa genotypes Adina and Zhaodong. Our study widens the understanding of the sophisticated molecular response and tolerance mechanism to salt stress, providing novel insights on candidate genes and pathways for genetic modification involved in salt stress adaptation in alfalfa.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

Calmodulin regulates the calcium homeostasis in mantle of Crassostrea gigas under ocean acidification

The biosynthesis of shell is a complicated calcification process in the marine bivalve, which can be severely impacted by ocean acidification (OA). Calmodulin (CaM) is a pivotal calcium regulator and thought to be crucial for calcification. In the present study, a CaM (designated CgCaM) with calcium-binding activity was identified from the Pacific oyster Crassostrea gigas with the objective to understand its possible role in the regulation of calcium homeostasis under acidification treatment. The open reading frame (ORF) of CgCaM was of 474 bp encoding a 17.5 kDa protein with four continuous EF-hand domains. CgCaM shared high similarity with CaMs from other invertebrates and vertebrates. The mRNA transcript of CgCaM was constitutively expressed in all detected tissues with the higher expression level in mantle, especially highest in the middle fold of the three folds of mantle. CgCaM was found to be mainly distributed in the mantle epithelium. When the oysters were exposed to acidified seawater, the expression level of CgCaM in the middle fold of mantle and the content of Ca2+ in this fold both decreased significantly. These results collectively suggested that CgCaM was involved in the regulation of calcium homeostasis in the middle fold of mantle under acidification treatment.

Genomic collation revealed the significance of Piriformospora indica (Serendipita indica) PiEF-hand protein in vesicle trafficking and fungal hyphal growth

EF-hand domain proteins are Ca2+ binding proteins with helix-loop-helix structural pattern and classified as CaMs, CMLs, CBLs, CDPKs, Rbohs, Myosin light chains (Mlcs) and others. Mlcs are important proteins that function through binding with IQ motifs of Myosins and respond to changes in Ca2+ ion concentration. Present study reports in silico analysis of Piriformospora indica EF-hand (PiEF-hand) protein annotated as probable myosin regulatory light chain Cdc4. The genomic analysis revealed sequence & structure attributes, orthologs identification, evolutionary perspective, protein-protein interactions and functionality. PiEF-hand protein is 141 aa long acidic protein lacking cysteine. Structural analysis highlighted four EF-hand motifs and conservation of Myosin heavy chain IQ motif interacting sites. Further, evolutionary relationships across 79 eukaryotic organisms were drawn classifying orthologs in two groups- CaMs and Mlcs. The PiEF-hand protein is grouped along with Mlcs underlining its possible role as myosin light chain. Separate sequence logos of each EF-hand motifs for CaMs and Mlcs revealed poor sequence conservation among Mlcs, whereas, CaMs EF-hand motifs were well conserved. The protein-protein interaction network analysis glimpsed involvement of PiEF-hand protein in vesicular trafficking and polarized fungal hyphal growth.