Phosphatidylethanolamine-binding Protein Research Articles

Identification of Genes Associated with Lemon Floral Transition and Flower Development during Floral Inductive Water Deficits: A Hypothetical Model.

Water deficit is a key factor to induce flowering in many woody plants, but reports on the molecular mechanisms of floral induction and flowering by water deficit are scarce. Here, we analyzed the morphology, cytology, and different hormone levels of lemon buds during floral inductive water deficits. Higher levels of ABA were observed, and the initiation of floral bud differentiation was examined by paraffin sections analysis. A total of 1638 differentially expressed genes (DEGs) were identified by RNA sequencing. DEGs were related to flowering, hormone biosynthesis, or metabolism. The expression of some DEGs was associated with floral induction by real-time PCR analysis. However, some DEGs may not have anything to do with flowering induction/flower development; they may be involved in general stress/drought response. Four genes from the phosphatidylethanolamine-binding protein family were further investigated. Ectopic expression of these genes in Arabidopsis changed the flowering time of transgenic plants. Furthermore, the 5′ flanking region of these genes was also isolated and sequence analysis revealed the presence of several putative cis-regulatory elements, including basic elements and hormone regulation elements. The spatial and temporal expression patterns of these promoters were investigated under water deficit treatment. Based on these findings, we propose a model for citrus flowering under water deficit conditions, which will enable us to further understand the molecular mechanism of water deficit-regulated flowering in citrus.Highlight:Based on gene activity during floral inductive water deficits identified by RNA sequencing and genes associated with lemon floral transition, a model for citrus flowering under water deficit conditions is proposed.

A selected reaction monitoring mass spectrometric assessment of biomarker candidates diagnosing large-cell neuroendocrine lung carcinoma by the scaling method using endogenous references

Selected reaction monitoring mass spectrometry (SRM-MS) -based semi-quantitation was performed to assess the validity of 46 selected candidate proteins for specifically diagnosing large-cell neuroendocrine lung carcinoma (LCNEC) and differentiating it from other lung cancer subtypes. The scaling method was applied in this study using specific SRM peak areas (AUCs) derived from the endogenous reference protein that normalizes all SRM AUCs obtained for the candidate proteins. In a screening verification study, we found that seven out of the 46 candidate proteins were statistically significant for the LCNEC phenotype, including 4F2hc cell surface antigen heavy chain (4F2hc/CD98) (p-ANOVA ≤ 0.0012), retinal dehydrogenase 1 (p-ANOVA ≤ 0.0029), apolipoprotein A-I (p-ANOVA ≤ 0.0004), β-enolase (p-ANOVA ≤ 0.0043), creatine kinase B-type (p-ANOVA ≤ 0.0070), and galectin-3-binding protein (p-ANOVA = 0.0080), and phosphatidylethanolamine-binding protein 1 (p-ANOVA ≤ 0.0012). In addition, we also identified candidate proteins specific to the small-cell lung carcinoma (SCLC) subtype. These candidates include brain acid soluble protein 1 (p-ANOVA < 0.0001) and γ-enolase (p-ANOVA ≤ 0.0013). This new relative quantitation-based approach utilizing the scaling method can be applied to assess hundreds of protein candidates obtained from discovery proteomic studies as a first step of the verification phase in biomarker development processes.

Knockdown of PEBP4 suppresses proliferation, migration and invasion of human breast cancer cells

Phosphatidylethanolamine-binding protein 4 (PEBP4), a member of the PEBP family, plays a pivotal role in tumor progression. However, the roles of PEBP4 in breast cancer remain unclear. Therefore, in the present study, we investigated the effects of PEBP4 on breast cancer cell proliferation, migration and invasion, and the underlying mechanism was also explored. Our results showed that the expression of PEBP4 was significantly up-regulated in breast cancer cell lines. Knockdown of PEBP4 inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. Furthermore, knockdown of PEBP4 suppressed breast cancer cell migration and invasion with prevented EMT. Mechanistically, knockdown of PEBP4 inhibited breast cancer cell proliferation and migration through the inactivation of PI3K/Akt signaling pathway. In conclusion, the present study demonstrated for the first time that knockdown of PEBP4 inhibited the proliferation, invasion and tumorigenesis in breast cancer cells. Thus, PEBP4 may serve as a potential therapeutic target for the treatment of breast cancer.

Three FT and multiple CEN and BFT genes regulate maturity, flowering, and vegetative phenology in kiwifruit.

Kiwifruit is a woody perennial horticultural crop, characterized by excessive vegetative vigor, prolonged juvenility, and low productivity. To understand the molecular factors controlling flowering and winter dormancy, here we identify and characterize the kiwifruit PEBP (phosphatidylethanolamine-binding protein) gene family. Five CEN-like and three BFT-like genes are differentially expressed and act as functionally conserved floral repressors, while two MFT-like genes have no impact on flowering time. FT-like genes are differentially expressed, with AcFT1 confined to shoot tip and AcFT2 to mature leaves. Both act as potent activators of flowering, but expression of AcFT2 in Arabidopsis resulted in a greater impact on plant morphology than that of AcFT1. Constitutive expression of either construct in kiwifruit promoted in vitro flowering, but AcFT2 displayed a greater flowering activation efficiency than AcFT1, leading to immediate floral transition and restriction of leaf development. Both leaf and flower differentiation were observed in AcFT1 kiwifruit lines. Sequential activation of specific PEBP genes in axillary shoot buds during growth and dormancy cycles indicated specific roles in regulation of kiwifruit vegetative and reproductive phenologies. AcCEN and AcCEN4 marked active growth, AcBFT2 was associated with suppression of latent bud growth during winter, and only AcFT was activated after cold accumulation and dormancy release.

Role of the PEBP4 protein in the development and metastasis of gastric cancer.

Phosphatidylethanolamine-binding protein 4 (PEBP4) has previously been reported to be upregulated in various cancers. However, the physiological functions of PEBP4 in gastric cancer are still unknown. Aiming to clarify the properties and role of PEBP4 in the development and invasion of gastric cancer, we performed several biological assays and a knockdown assay. The expression level of PEBP4 was shown to be significantly upregulated in gastric cancer tissue samples, and knockdown of the expression of PEBP4 induced significant inhibitory effects on cell proliferation, migration and invasiveness. In addition, it was demonstrated that PEBP4 was associated with the development and invasion of gastric cancer cells through activation of the PI3K/Akt signaling pathway. Our findings supported the hypothesis that PEBP4 might be a novel potential drug target for the treatment of gastric cancer.

Cryopreservation of bull semen is associated with carbonylation of sperm proteins

Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level and motility of spermatozoa. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein M1, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation-involved proteins or could indicate cryopreservation-induced premature capacitation.

Differential gene expression in porcine oviduct during the oestrous cycle.

The oviduct undergoes changes under the influence of steroid hormones during the oestrous cycle. However, the molecular mechanisms underlying oviductal regulation are not fully understood. The aim of the present study was to identify the gene expression profile of the porcine oviduct in different stages of the cycle using microarray technology. A systematic study was performed on animals at four different stage: prepubertal gilts, and sows in the preovulatory, postovulatory and luteal phase of the oestrous cycle. The porcine oviduct expressed a total of 4929 genes. Moreover, significant differences in the expression of several genes were detected as the oestrous cycle progressed. Analysis of the differentially expressed genes indicated that a total of 86, 89 and 15 genes were upregulated in prepubertal gilts, preovulatory and luteal sows respectively compared with levels observed in postovulatory sows. Moreover, 80, 51 and 64 genes were downregulated in prepubertal, preovulatory and luteal animals respectively compared with the postovulatory sows. The concentrations of 10 selected transcripts were quantified by real-time reverse transcription-polymerase chain reaction to validate the cDNA array hybridisation data. Conversely, for some genes, localisation of corresponding protein expression in the oviduct was analysed by immunohistochemistry (i.e. cholecystokinin, glutathione peroxidase 2, mucin 1, phosphatidylethanolamine binding protein 4 and tachykinin 3) and mass spectrometry analysis of oviductal fluid allowed identification of peptides from all five proteins. The results of the present study demonstrate that gene expression in the porcine oviduct is clearly regulated during the oestrous cycle, with some oviductal proteins that could be related to several reproductive processes described here for the first time.

Expansion of the phosphatidylethanolamine binding protein family in legumes: a case study of Lupinus angustifolius L. FLOWERING LOCUS T homologs, LanFTc1 and LanFTc2.

BackgroundThe Arabidopsis FLOWERING LOCUS T (FT) gene, a member of the phosphatidylethanolamine binding protein (PEBP) family, is a major controller of flowering in response to photoperiod, vernalization and light quality. In legumes, FT evolved into three, functionally diversified clades, FTa, FTb and FTc. A milestone achievement in narrow-leafed lupin (Lupinus angustifolius L.) domestication was the loss of vernalization responsiveness at the Ku locus. Recently, one of two existing L. angustifolius homologs of FTc, LanFTc1, was revealed to be the gene underlying Ku. It is the first recorded involvement of an FTc homologue in vernalization. The evolutionary basis of this phenomenon in lupin has not yet been deciphered.ResultsBacterial artificial chromosome (BAC) clones carrying LanFTc1 and LanFTc2 genes were localized in different mitotic chromosomes and constituted sequence-specific landmarks for linkage groups NLL-10 and NLL-17. BAC-derived superscaffolds containing LanFTc genes revealed clear microsyntenic patterns to genome sequences of nine legume species. Superscaffold-1 carrying LanFTc1 aligned to regions encoding one or more FT-like genes whereas superscaffold-2 mapped to a region lacking such a homolog. Comparative mapping of the L. angustifolius genome assembly anchored to linkage map localized superscaffold-1 in the middle of a 15 cM conserved, collinear region. In contrast, superscaffold-2 was found at the edge of a 20 cM syntenic block containing highly disrupted collinearity at the LanFTc2 locus. 118 PEBP-family full-length homologs were identified in 10 legume genomes. Bayesian phylogenetic inference provided novel evidence supporting the hypothesis that whole-genome and tandem duplications contributed to expansion of PEBP-family genes in legumes. Duplicated genes were subjected to strong purifying selection. Promoter analysis of FT genes revealed no statistically significant sequence similarity between duplicated copies; only RE-alpha and CCAAT-box motifs were found at conserved positions and orientations.ConclusionsNumerous lineage-specific duplications occurred during the evolution of legume PEBP-family genes. Whole-genome duplications resulted in the origin of subclades FTa, FTb and FTc and in the multiplication of FTa and FTb copy number. LanFTc1 is located in the region conserved among all main lineages of Papilionoideae. LanFTc1 is a direct descendant of ancestral FTc, whereas LanFTc2 appeared by subsequent duplication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3150-z) contains supplementary material, which is available to authorized users.

Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein

Mouse Apolipoprotein L9 (ApoL9) is an understudied cytoplasmic, interferon-inducible protein. The details of its intracellular localization and normal cellular functions are unclear. We report here that ApoL9 localizes to small puncta diffusely distributed in the cytoplasm, as well as to larger granules of varying size and number that are similar to aggresome-like induced structures (ALIS) and contain the autophagy receptor Sqstm1/p62, the autophagosome marker Lc3, and ubiquitin. Transfection of B16F10 mouse melanoma cells stably expressing ApoL9 (B16F10L9) with certain liposome-based transfection reagents causes dramatic disturbances in its subcellular distribution. We reasoned that these disturbances may be due to the interaction of ApoL9 with dioleoylphosphatidylethanolamine (DOPE), the helper lipid component of several transfection reagents. Recombinant ApoL9 produced in E. coli, as well as ApoL9 expressed in HEK293T cells, specifically bind phosphatidylethanolamine (PE) in vitro. ApoL9 is expressed at high levels in liver and brain, organs enriched in PE. Since PE is known to facilitate replication of positive strand RNA viruses, we examined the role of ApoL9 during replication of Japanese encephalitis virus (JEV), a positive strand virus of the family Flaviviridae. JEV titres in B16F10L9 cells are higher than those in B16F10 cells. We propose that ApoL9 is a PE-binding protein that may have important roles in several cellular processes that involve this phospholipid.

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3

ABSTRACTAutophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Characterization and Functional Analysis of PEBP Family Genes in Upland Cotton (Gossypium hirsutum L.).

Upland cotton (Gossypium hirsutum L.) is a naturally occurring photoperiod-sensitive perennial plant species. However, sensitivity to the day length was lost during domestication. The phosphatidylethanolamine-binding protein (PEBP) gene family, of which three subclades have been identified in angiosperms, functions to promote and suppress flowering in photoperiod pathway. Recent evidence indicates that PEBP family genes play an important role in generating mobile flowering signals. We isolated homologues of the PEBP gene family in upland cotton and examined their regulation and function. Nine PEBP-like genes were cloned and phylogenetic analysis indicated the genes belonged to four subclades (FT, MFT, TFL1 and PEBP). Cotton PEBP-like genes showed distinct expression patterns in relation to different cotton genotypes, photoperiod responsive and cultivar maturity. The GhFT gene expression of a semi-wild race of upland cotton were strongly induced under short day condition, whereas the GhPEBP2 gene expression was induced under long days. We also elucidated that GhFT but not GhPEBP2 interacted with FD-like bZIP transcription factor GhFD and promote flowering under both long- and short-day conditions. The present result indicated that GhPEBP-like genes may perform different functions. This work corroborates the involvement of PEBP-like genes in photoperiod response and regulation of flowering time in different cotton genotypes, and contributes to an improved understanding of the function of PEBP-like genes in cotton.

Effect of a chemical inhibitor of human phosphatidylethanolamine-binding protein 4 on radiosensitivity of rectal cancer cells.

BackgroundHuman phosphatidylethanolamine-binding protein 4 (hPEBP4) is a well-established antiapoptosis molecule in recent years. It has also been demonstrated to be involved in the radioresistance of rectal cancer. The objective of this study was to determine whether IOI-42, a chemical inhibitor of hPEBP4, could sensitize rectal cancer cells.MethodsRectal cancer cells were treated with IOI-42 alone or in combination with irradiation. Clonogenic survival assays and tumor volume growth analysis were used, respectively, to study the effect of IOI-42 in vitro and in vivo. Western blot was adopted to measure the activation of signal pathway.ResultsClonogenic survival assays showed that IOI-42, combined with irradiation, caused a significant decrease in colony formation compared with radiation alone, which was associated with the downregulation of Akt activation. And we also confirmed the effect of IOI-42 in nude mice transplanted with human rectal cancer subcutaneously.ConclusionsThese data suggest that IOI-42 has a potential to enhance the radiosensitivity of rectal cancer cells, providing a rationale to further investigate the feasibility of combining of IOI-42 with radiation, keeping in mind that this may result in unexpected toxicities.

Understanding perspectives of signalling mechanisms regulating PEBP1 function.

Phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein, belongs to PEBP family of proteins. It is known to interact with many proteins that are mainly involved in pathways that monitor cell proliferation and differentiation. PEBP1 in many cells interacts with several pathways, namely MAPK, GRK2, NF-кB, etc. that keeps the cell proliferation and differentiation in check. This protein is expressed by many cells in humans, including neurons where it is predominantly involved in production of choline acetyltransferase. Deregulated PEBP1 is known to cause cancer, diabetic nephropathy and neurodegenerative diseases like Alzheimer's and dementia. Recent research led to the discovery of many drugs that mainly target the interaction of PEBP1 with its partners. These compounds are known to bind PEBP1 in its conserved domain which abrogate its association with interacting partners in several different pathways. We outline here the latest developments in understanding of PEBP1 function in maintaining cell integrity. Copyright © 2016 John Wiley & Sons, Ltd. Phosphatidylethanolamine-binding protein is crucial in regulation of MAPK and PKC pathways. Its diverse roles, including regulating these pathways keep cell differentiation and proliferation in check. This review outlines some latest findings which greatly add to our current knowledge of phosphatidylethanolamine-binding protein.

Revisiting the phosphatidylethanolamine‐binding protein (PEBP) gene family reveals cryptic FLOWERING LOCUS T gene homologs in gymnosperms and sheds new light on functional evolution

Angiosperms and gymnosperms are two major groups of extant seed plants. It has been suggested that gymnosperms lack FLOWERING LOCUS T (FT), a key integrator at the core of flowering pathways in angiosperms. Taking advantage of newly released gymnosperm genomes, we revisited the evolutionary history of the plant phosphatidylethanolamine-binding protein (PEBP) gene family through phylogenetic reconstruction. Expression patterns in three gymnosperm taxa and heterologous expression in Arabidopsis were studied to investigate the functions of gymnosperm FT-like and TERMINAL FLOWER 1 (TFL1)-like genes. Phylogenetic reconstruction suggests that an ancient gene duplication predating the divergence of seed plants gave rise to the FT and TFL1 genes. Expression patterns indicate that gymnosperm TFL1-like genes play a role in the reproductive development process, while GymFT1 and GymFT2, the FT-like genes resulting from a duplication event in the common ancestor of gymnosperms, function in both growth rhythm and sexual development pathways. When expressed in Arabidopsis, both spruce FT-like and TFL1-like genes repressed flowering. Our study demonstrates that gymnosperms do have FT-like and TFL1-like genes. Frequent gene and genome duplications contributed significantly to the expansion of the plant PEBP gene family. The expression patterns of gymnosperm PEBP genes provide novel insight into the functional evolution of this gene family.

The Evolution of Photoperiod-Insensitive Flowering in Sorghum, A Genomic Model for Panicoid Grasses.

Of central importance in adapting plants of tropical origin to temperate cultivation has been selection of daylength-neutral genotypes that flower early in the temperate summer and take full advantage of its long days. A cross between tropical and temperate sorghums [Sorghum propinquum (Kunth) Hitchc.×S. bicolor (L.) Moench], revealed a quantitative trait locus (QTL), FlrAvgD1, accounting for 85.7% of variation in flowering time under long days. Fine-scale genetic mapping placed FlrAvgD1 on chromosome 6 within the physically largest centiMorgan in the genome. Forward genetic data from “converted” sorghums validated the QTL. Association genetic evidence from a diversity panel delineated the QTL to a 10-kb interval containing only one annotated gene, Sb06g012260, that was shown by reverse genetics to complement a recessive allele. Sb06g012260 (SbFT12) contains a phosphatidylethanolamine-binding (PEBP) protein domain characteristic of members of the “FT” family of flowering genes acting as a floral suppressor. Sb06g012260 appears to have evolved ∼40 Ma in a panicoid ancestor after divergence from oryzoid and pooid lineages. A species-specific Sb06g012260 mutation may have contributed to spread to temperate regions by S. halepense (“Johnsongrass”), one of the world’s most widespread invasives. Alternative alleles for another family member, Sb02g029725 (SbFT6), mapping near another flowering QTL, also showed highly significant association with photoperiod response index (P = 1.53×10 − 6). The evolution of Sb06g012260 adds to evidence that single gene duplicates play large roles in important environmental adaptations. Increased knowledge of Sb06g012260 opens new doors to improvement of sorghum and other grain and cellulosic biomass crops.

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site.

Protein modifications, whether chemically induced or post-translational (PTMs), play an essential role for the biological activity of proteins. Understanding biological processes and alterations thereof will rely on the quantification of these modifications on individual residues. Here we present SSPaQ, a subtractive method for the parallel quantification of the extent of modification at each possible site of a protein. The method combines uniform isotopic labeling and proteolysis with MS, followed by a segmentation approach, a powerful tool to refine the quantification of the degree of modification of a peptide to a segment containing a single modifiable amino acid. The strength of this strategy resides in: (1) quantification of all modifiable sites in a protein without prior knowledge of the type(s) of modified residues; (2) insensitivity to changes in the solubility and ionization efficiency of peptides upon modification; and (3) detection of missed cleavages caused by the modification for mitigation. The SSPaQ method was applied to quantify modifications resulting from the interaction of human phosphatidyl ethanolamine binding protein 1 (hPEBP1), a metastasis suppressor gene product, with locostatin, a covalent ligand and antimigratory compound with demonstrated activity towards hPEBP1. Locostatin is shown to react with several residues of the protein. SSPaQ can more generally be applied to induced modification in the context of drugs that covalently bind their target protein. With an alternate front-end protocol, it could also be applied to the quantification of protein PTMs, provided a removal tool is available for that PTM. Graphical Abstract ᅟ.

Strawberry MOTHER OF FT AND TFL1 regulates seed germination and post-germination growth through integrating GA and ABA signaling in Arabidopsis

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) are known to play antagonistic roles in the control of seed germination and seedling establishment. Here, the regulatory roles of woodland strawberry MOTHER OF FT AND TFL1 (FvMFT) involved in GA and ABA signaling were investigated. FvMFT, which encodes a phosphatidylethanolamine-binding protein, was predominantly expressed in young strawberry fruits. Transient over-expression of FvMFT repressed the expression of several DELLA genes in strawberry fruits. Ectopic over-expression of FvMFT in Arabidopsis thaliana markedly altered the expression levels of ABA-INSENSITIVE (ABI) and DELLA genes, which is accompanied by partially restoring the impaired post-germination growth when the seeds were germinated on sugar-free medium. The same transgenic Arabidopsis plants also exhibited hypersensitivity to GA3 and ABA treatments during seed germination and post-germination growth. In addition, the fluorescence-tagged FvMFT proteins exhibited intriguing subcellular localizations in integument cells and endosperm cells of transgenic Arabidopsis. Taken together, our results support the notion that FvMFT could act as a dual regulator of seed germination and post-germination growth in response to GA and ABA signaling in plants.

Evolution of the PEBP gene family and selective signature on FT‐like clade

AbstractThe PEBP family of proteins, which encode a phosphatidyl ethanolamine‐binding protein (PEBP) domain, serve as a hub in the network of integrating environmental and developmental signals to regulate flowering in all angiosperms. To understand how PEBP duplication genes arose and evolved during plant evolution, we identified 259 genes involved in the PEBP domain from 25 species, including angiosperms, gymnosperms, and embryophytes. We found that plant PEBP genes could be divided into three monophyletic groups, including FT‐like, TFL‐like and MFT‐like subgroups. Based on the phylogeny, FT‐like and TFL‐like subgroups split before the angiosperm divergence. The likelihood ratio test indicates that the FT‐like clade is significantly different in Ka/Ks with TFL‐like and MFT‐like clades. We found this shift in mutation might be attributed to the positive selection imposed on the fourth exon of recent FT duplications. Divergent expression patterns of FT paralogs indicated that they had undergone subfunctionalization after duplication. In addition, some of the FT‐like genes have been targets of selection during domestication in rice and maize, for which the flowering time is an important agronomic trait. These results imply potential recent positive selection on FT‐like genes with potential to increase the prevalence of novel functional alleles leading to extensive plant flowering time diversity and adaptation to environmental changes.

PEBP4 silencing inhibits hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer cells.

Hypoxia induced epithelial-to-mesenchymal transition (EMT) to facilitate the tumor biology. Phosphatidylethanolamine-binding protein 4 (PEBP4) is a member of the PEBP family and has been reported to be upregulated in various cancer types. The definite function of PEBP4 in regulating the EMT of prostate cancer, however, is still unclear. Here, we examined the functional role of PEBP4 and the underlying molecular mechanisms in hypoxia-induced EMT in prostate cancer cells. Our results showed that PEBP4 mRNA and protein expression was markedly increased in the human prostate cancer tissues and cell lines. Knockdown of PEBP4 significantly inhibited hypoxia-induced migration/invasion and EMT program. Furthermore, knockdown of PEBP4 prevented hypoxia-induced the expression of p-Akt and p-mTOR in prostate cancer cells. Taken together, this study reported here provided evidence that knockdown of PEBP4 inhibited hypoxia-induced EMT in prostate cancer cells. Our study uncovered a novel role for PEBP4 in prostate cancer progression, which might support the potential for PEBP4 targeting in prostate cancer therapy.

Phosphatidylethanolamine binding protein 4 (PEBP4) is a secreted protein and has multiple functions

Phosphatidylethanolamine binding proteins (PEBP) represent a superfamily of proteins that are conserved from bacteria to humans. In mammals, four members have been identified, PEBP1–4. To determine the functional differences among PEBP1–4 and the underlying mechanism for their actions, we performed a sequence alignment and found that PEBP4 contains a signal peptide and potential glycosylation sites, whereas PEBP1–3 are intracellular proteins. To test if PEBP4 is secreted, we made constructs with Myc epitope at the amino (N) terminus or carboxyl (C) terminus to mask the signal sequence or keep it free, respectively. Our data revealed that both mouse and human PEBP4 were secreted when the epitope was tagged at their C-terminus. To our surprise, secretion was dependent upon the C-terminal conserved domain in addition to the N-terminal signal sequence. When the epitope was placed to the N-terminus, the recombinant protein failed to secrete and instead, was retained in the cytoplasm. Mass spectrometry detected asparagine (N)-glycosylation on the secreted PEBP4. Although overexpression of N-terminal tagged PEBP4 resulted in an inhibition of ERK activation by EGF, that with a C-terminal epitope tag did not have such an effect. Likewise, transfection of PEBP4 shRNA did not appear to affect ERK activation, suggesting that PEBP4 does not participate in the regulation of this pathway. In contrast, PEBP4 siRNA suppressed phosphorylation of Act at S473. Therefore, our results suggest that PEBP4 is a multifunctional protein and can be secreted. It will be important to investigate the mechanism by which PEBP4 is secreted and regulates cellular events.