Effects of covalent conjugation with quercetin and its glycosides on the structure and allergenicity of Bra c p from bee pollen.

Profilin family members are potential pan-allergens in foods, presenting public health hazards. However, studies on the allergenicity modification of profilin allergens are limited. Herein, quercetin and its glycosides (isoquercitrin and rutin) were applied to modify the allergenicity of a profilin allergen (Bra c p) from Brassica campestris bee pollen. Results showed that only quercetin can be closely covalently bound to Bra c p among the three, and the binding site was located at the Cys98 residue. After covalently conjunction, the relative content of α-helix structure in Bra c p was reduced by 40.05%, while random coil was increased by 42.89%; moreover, the Tyr and Phe residues in Bra c p were masked. These structural changes could alter the conformational antigenic epitopes of Bra c p, resulting in its allergenicity reduction. Our findings might provide a technical foundation for reducing the allergenicity of bee pollen and foods containing profilin family allergens.

Profilin promotes formin-mediated actin filament assembly and vesicle transport during polarity formation in pollen.

Pollen germination is critical for the reproduction of flowering plants. Formin-dependent actin polymerization plays vital roles in vesicle trafficking and polarity establishment during this process. However, how formin-mediated actin assembly is regulated in vivo remains poorly understood. Here, we investigated the function of reproductive profilin 4 and 5 (PRF4 and PRF5) in polarity establishment during pollen germination in Arabidopsis thaliana. Our data showed that the actin filament content was reduced in the prf4 prf5 double mutant and substantially increased in both PRF4- and PRF5-overexpressing pollen grains. By contrast, the positive effect of profilin in promoting actin polymerization was abolished in a formin mutant, atfh5. In addition, the interaction between Arabidopsis formin homology 5 (AtFH5) and actin filaments was attenuated and the trafficking of AtFH5-labeled vesicles was slowed in prf4 prf5 pollen grains. Formation of the collar-like structure at the germination pore was also defective in prf4 prf5 pollen grains as the fast assembly of actin filaments was impaired. Together, our results suggest that PRF4 and PRF5 regulate vesicle trafficking and polarity establishment during pollen germination by promoting AtFH5-mediated actin polymerization and enhancing the interaction between AtFH5 and actin filaments.

Identification of profilin 1 as the primary target for the anti-cancer activities of Furowanin A in colorectal cancer.

BACKGROUND: Furowanin A (Fur A) is a flavonoid compound isolated from medicinal plant Millettia pachycarpa Benth. This study aims to explore the effect of Fur A on Colorectal cancer (CRC) and its molecular mechanisms. METHODS: Cell proliferative capacity of CRC cells was assessed by CCK-8 assay. Cell apoptosis and cell cycle distribution were detected by flow cytometry. Cell migration and invasion were detected by wound healing and Transwell assay, respectively. EMT markers, apoptosis and profilin 1(Pfn1) expression were detected by immunohistochemistry (IHC). The protein expression levels were examined by western blotting. i-TRAQ analyses were conducted to identify the differentially expressed genes in CRC cells. CRC xenograft model was also used to validate the in vivo anti-cancer activity of Fur A. RESULTS: Fur A exhibited anti-prolifertive, blocked cell cycle progression and promoted apoptotic cell death in CRC cells. Fur A suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) in vitro, and tumor growth and pulmonary metastasis in vivo, without causing obvious toxicity. iTRAQ analysis identified Pfn1 as a gene up-regulated by Fur A. In xenograft tumor tissue, the expression of Pfn1 was also elevated by Fur A treatment. In clinical CRC samples, high expression of Pfn1 was correlated with lower stage and longer survival. Knockdown of Pfn1 significantly dampened the pro-apoptotic and anti-metastatic activities of Fur A in CRC cells. Ectopic Pfn1 expression augmented the anti-neoplastic activities of Fur A. CONCLUSION: Fur A exhibited anti-cancer activities in vitro and in vivo in CRC by up-regulating Pfn1.

Mechanism of CAP1-mediated apical actin polymerization in pollen tubes.

Srv2p/CAP1 is an essential regulator of actin turnover, but its exact function in regulating actin polymerization, particularly the contribution of its actin nucleotide exchange activity, remains incompletely understood. We found that, although Arabidopsis CAP1 is distributed uniformly in the cytoplasm, its loss of function has differential effects on the actin cytoskeleton within different regions of the pollen tube. Specifically, the F-actin level increases in the shank but decreases in the apical region of cap1 pollen tubes. The reduction in apical F-actin results mainly from impaired polymerization of membrane-originated actin within cap1 pollen tubes. The actin nucleotide exchange activity of CAP1 is involved in apical actin polymerization. CAP1 acts synergistically with pollen ADF and profilin to promote actin turnover in vitro, and it can overcome the inhibitory effects of ADF and synergize with profilin to promote actin nucleotide exchange. Consistent with its role as a shuttle molecule between ADF and profilin, the cytosolic concentration of CAP1 is much lower than that of ADF and profilin in pollen. Thus, CAP1 synergizes with ADF and profilin to drive actin turnover in pollen and promote apical actin polymerization in pollen tubes in a manner that involves its actin nucleotide exchange activity.

Detection of profilin in SPT extracts that are supposed to contain it.

INTRODUCTION AND OBJECTIVES: Profilin is a panallergen contained in pollen, plant foods and latex. Although cross-reactivity is expected while performing skin prick tests (SPT) with allergens that contain profilin, this is not always noticed. The purpose of this study was to detect if profilin is contained in the commercial SPT extracts of pollen and plant foods which, in their fresh form, contain determined epitopes of profilin. MATERIAL AND METHODS: Commercial SPT extracts of different pharmaceuticals were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The study included purified palm date profilin, peach (whole, pulp and peel extracts), hazelnut, Olea europea, Parietaria judaica and Phleum pratense. RESULTS: Profilin was detected in all, but peach extracts; it was neither contained in the whole peach extract nor in the ones of peel or pulp. CONCLUSION: The only accurate way to detect sensitization to profilin, while performing SPT, is the use of purified profilin extract. Even if a plant food or pollen contain an identified molecule of profilin, the relevant SPT commercial extract may not.

Structure-based virtual screening identifies a small-molecule inhibitor of the profilin 1-actin interaction.

Profilin 1 (Pfn1) is an important regulator of the actin cytoskeleton and plays a vital role in many actin-based cellular processes. Therefore, identification of a small-molecule intervention strategy targeted against the Pfn1-actin interaction could have broad utility in cytoskeletal research and further our understanding of the role of Pfn1 in actin-mediated biological processes. Based on an already resolved Pfn1-actin complex crystal structure, we performed structure-based virtual screening of small-molecule libraries to seek inhibitors of the Pfn1-actin interaction. We identified compounds that match the pharmacophore of the key actin residues of Pfn1-actin interaction and therefore have the potential to act as competitive inhibitors of this interaction. Subsequent biochemical assays identified two candidate compounds with nearly identical structures that can mitigate the effect of Pfn1 on actin polymerization in vitro As a further proof-of-concept test for cellular effects of these compounds, we performed proximity ligation assays in endothelial cells (ECs) to demonstrate compound-induced inhibition of Pfn1-actin interaction. Consistent with the important role of Pfn1 in regulating actin polymerization and various fundamental actin-based cellular activities (migration and proliferation), treatment of these compounds reduced the overall level of cellular filamentous (F) actin, slowed EC migration and proliferation, and inhibited the angiogenic ability of ECs both in vitro and ex vivo In summary, this study provides the first proof of principle of small-molecule-mediated interference with the Pfn1-actin interaction. Our findings may have potential general utility for perturbing actin-mediated cellular activities and biological processes.

Guttiferone K suppresses cell motility and metastasis of hepatocellular carcinoma by restoring aberrantly reduced profilin 1.

Hepatocellular carcinoma (HCC) is an aggressive malignancy and the 5-year survival rate of advanced HCC is < 10%. Guttiferone K (GUTK) isolated from the Garcinia genus inhibited HCC cells migration and invasion in vitro and metastasis in vivo without apparent toxicity. Proteomic analysis revealed that actin-binding protein profilin 1 (PFN1) was markedly increased in the presence of GUTK. Over-expression of PFN1 mimicked the effect of GUTK on HCC cell motility and metastasis. The effect of GUTK on cell motility was diminished when PFN1 was over-expressed or silenced. Over-expression of PFN1 or incubation with GUTK decreased F-actin levels and the expression of proteins involved in actin nucleation, branching and polymerization. Moreover, a reduction of PFN1 protein levels was common in advanced human HCC and associated with poor survival rate. In conclusion, GUTK effectively suppresses the motility and metastasis of HCC cells mainly by restoration of aberrantly reduced PFN1 protein expression.

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation.

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASP(pS157), but no changes in cofilin1/VASP(pS239) in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASP(pS157) or cofilin1/VASP(pS239) suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220-31. ©2016 AACR.

Pro j 2 is mesquite profilin: molecular characteristics and specific IgE binding activity.

BACKGROUND: Pollens from mesquite (Prosopis juliflora) are potent allergen responsible in causing immediate hypersensitivity reactions in susceptible people in tropical countries. OBJECTIVE: This study aimed to clone, express and purify the mesquite pollen profilin (Pro j 2) as well as evaluating its nucleotide sequence homology in order to predict allergenic cross-reactivity with profilins of common allergenic plants. METHODS: Immunoblotting assay and specific ELISA were applied to determine the immunoreactivity of sera from 35 patients who were allergic to mesquite pollen. The mesquite profilin-coding sequence was cloned into PTZ57R/T vector and amplified. The cDNA of mesquite pollen profilin was then expressed in Escherichia coli using pET-21b (+) vector and puri?ed by one-step Ni2+ a?nity chromatography. IgE binding capacity of the recombinant mesquite profiling (rPro j 2) was analyzed by specific ELISA, immunoblotting, and inhibition assays. RESULTS: cDNA nucleotide sequencing revealed an open reading frame of 399bp encoding for 133 amino acids which belongs to the profilin family. Seventeen patients (17/35, 48.57%) had significant specific IgE level for rPro j 2. Immunodetection and inhibition assays indicated that puri?ed rPro j 2 might be similar as that in the crude extract. CONCLUSION: Pro j 2, as a new allergen from mesquite pollen, was produced in E. coli with an IgE-reactivity similar to that of its natural counterpart. The amino acid sequences homology analysis of mesquite profilin and several profilin molecules from other plants showed high degree of cross-reactivity among plant-derived profilins from unrelated families.

Variability of Corylus avellana, L. CorA and profilin pollen allergens expression.

Corylus avellana is the source of inhalant allergies induced by hazel pollen as well as food allergies induced after ingestion of hazelnuts. In this study, real-time PCR approach was used to analyse expression of hazel pollen allergens on the molecular level. Relative quantity of hazelnut allergens Corylus avellana, L. CorA and Corylus avellana, L. pollen profiling in samples from different Ukraine areas were determining and comparing. Differences among the levels of both analysed allergen transcripts were found for hazel CorA and profillin. In both cases, the expression within the urbanized growth conditions was higher when compared to the sample from village area. The average expression for CorA was 0.84 times higher than for profilin and the results are very variable depending on the place of growth. Expression levels here were within the range of 2.957 up to the 52.936. Profilin expression was the highest in the sample from the polluted place of growth-cement plant area with the value of 52 times higher when compared to the sample from the village area. In this study, comparison of expression levels of hazel CorA and profiling pollen allergens was performed for the first time. Real-time PCR assay developed in this study proved the sensitivity for detection of the changes of the hazel pollen allergens expression levels and could benefit labs by fast and reproducible detection method of these allergens.

[Therapeutic effect and mechanism of Phoenix roebelenii pollen vaccine for treatment of mice with allergic rhinitis].

OBJECTIVE: To apply Phoenix roebelenii pollen vaccine to murine models of allergic rhinitis and observe the pathological changes of allergic rhinitis in mice, and to study the efficacy and mechanism of the vaccine for the treatment of allergic rhinitis. METHODS: BALB/c mice models of allergic rhinitis were established by intraperitoneal injection, and then treated with immunotherapy of allergen vaccine by subcutaneous injection. The mice were examined for the levels of airway hyperresponsiveness by a noninvasive lung function detector, for the specific antibodies IgE and IgG2a in serum and cytokines by indirect ELISA, and for the pathological changes of ultrastructure of nasal mucosa of the mice by transmission electron microscopy before and after the treatment. RESULTS: After the immunotherapy, nasal symptoms and airway hyperresponsiveness of the mice were relieved. The level of specificity antibody IgG2a in serum was elevated, and IgE dropped significantly. In the culture supernatant of spleen cells, INF-γ and IL-10 levels increased and the production of IL-4 decreased. CONCLUSION: The recombinant profilin of the Phoenix roebelenii pollen as vaccine has a certain therapeutic effect for the pollen allergic rhinitis, and it works maybe through promoting the transition of Th2 to Th1 and regulating the balance of helper T cells.

Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize.

Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription-PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein-protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize.

[Molecular aspects of allergy to plant products. Part III. Panallergens and breeding of hypoallergenic cultivars]. / Molekularne aspekty alergii na produkty pochodzenia roslinnego. Cz. III. Panalergeny oraz hodowla odmian hypoalergogennych.

In addition to major allergens, also minor allergens, i.e. panallergens have been shown to be responsible for many IgE cross-reactions even between unrelated pollen and plant food allergen sources. It can be explained also by cross-allergenicity underlying the T cell response to conserved regions of panallergens. In this article, we focus on known panallergens which presently comprise a few protein families, including non-specific lipid transfer proteins (nsLTP) (PR-14), thaumatin like proteins (TLP) (PR-5), profilins, and polcalcins. Food allergy has an impact on the quality of life of an allergic patient. The way of developing novel plant cultivars with decreased allergenicity and possibility of down-regulating the expression of an allergen by genetic modification are discussed.

Effects of dietary supplementation with phytonutrients on vaccine-stimulated immunity against infection with Eimeria tenella.

Two phytonutrient mixtures, VAC (carvacrol, cinnamaldehyde, and Capsicum oleoresin), and MC (Capsicum oleoresin and turmeric oleoresin), were evaluated for their effects on chicken immune responses following immunization with an Eimeria profilin protein. Chickens were fed with a non-supplemented diet, or with VAC- or MC-supplemented diets, immunized with profilin, and orally challenged with virulent oocysts of Eimeria tenella. Immunity against infection was evaluated by body weight, fecal oocyst shedding, profilin antibody levels, lymphocyte recall responses, cytokine expression, and lymphocyte subpopulations. Following immunization and infection, chickens fed the VAC- or MC-supplemented diets showed increased body weights, greater profilin antibody levels, and/or greater lymphocyte proliferation compared with non-supplemented controls. Prior to Eimeria infection, immunized chickens on the MC-supplemented diet showed reduced IFN-γ and IL-6 levels, but increased expression of TNFSF15, compared with non-supplemented controls. Post-infection levels of IFN-γ and IL-6 were increased, while IL-17F transcripts were decreased, with MC-supplementation. For VAC-supplemented diets, decreased IL-17F and TNFSF15 levels were observed only in infected chickens. Finally, immunized chickens fed the MC-supplemented diet exhibited increased MHC class II(+), CD4(+), CD8(+), TCR1+, or TCR2(+) T cells compared with nonsupplemented controls. Animals on the VAC-containing diet only displayed an increase in K1(+) macrophages. In conclusion, dietary supplementation with VAC or MC alters immune parameters following recombinant protein vaccination against avian coccidiosis.

Identification of a new allergen from Amaranthus retroflexus pollen, Ama r 2.

BACKGROUND: Pollinosis from Amaranthus retroflexus pollen is a common cause of respiratory allergy in Iran with a high positive rate (68.8%) among Iranian allergic patients. The aim of the present study was to evaluate the allergenicity of the A. retroflexus pollen profilin. METHODS: Using sera from twelve patients allergic to A. retroflexus pollen, IgE-binding proteins from the A. retroflexus pollen extract was identified by immunoblotting. The cDNA of A. retroflexus pollen profilin was amplified, then cloned into the pET-21b (+) vector, expressed in Escherichia coli, and finally purified by metal affinity chromatography. The IgE-binding capacity of the recombinant protein was then analyzed by the ELISA, immunoblotting, and inhibition assays, as well as by the skin prick test (SPT). RESULTS: Immunoblotting results indicated a 14.6kDa protein with IgE-reactivity to 33% (4/12) among A. retroflexus pollen-allergic patients. Nucleotide sequencing of the cDNA revealed an open reading frame of 399 bp encoding for 133 amino acid residues which was belonged to the profilin family and designated as Ama r 2. A recombinant Ama r 2 (rAma r 2) was then produced in E. coli as a soluble protein which showed a strong IgE-reactivity via ELISA confirmed by the SPT. Inhibition experiments revealed high IgE cross-reactivities with the profilins from other plants. CONCLUSIONS: The profilin from the A. retroflexus pollen, Ama r 2, was firstly identified as an allergen. Moreover, rAma r 2 was produced in E. coli as a soluble immunoreactive protein with an IgE-reactivity similar to that of its natural counterpart.

Molecular cloning and characterization of a profilin gene BnPFN from Brassica nigra that expressing in a pollen-specific manner.

Brassica nigra is a newly found invasive species in Zhejiang Province, China. It distributes alongside the roads, in vegetable fields and on riversides. When it blooms, some natives there will suffer from allergic rhinitis. We designed gene-specific primer pairs according to reported profilin genes and successfully isolated their homolog from flower bud cDNA of B. nigra. The gene, designated BnPFN, was submitted to GenBank under accession number EU004073. BnPFN was 405 bp in length encoding 134 amino acids. Expression analysis of BnPFN gene was carried out by means of RT-PCR. The results showed that BnPFN express only in anthers and pollens, and there was no detection in roots, leaves, stems, sepals, petals and pistils. We suggest that BnPFN is a pollen-specific gene and may be responsible for pollen anaphylactic reactions in those invading areas when B. nigra blooms.

Pollen food syndrome: update on the allergens.

Pollen food syndrome results from cross-reactivity between pollen-specific IgE and homologous proteins found in fruits and vegetables. These proteins can be grouped into several categories based on structure and include profilins, pathogenesis-related proteins, and cross-reactive carbohydrate determinants. Although cooking the reactive fruits and vegetables has been shown to destroy IgE-binding epitopes, evidence suggests that the remaining linear epitopes can bind cross-reactive T cells and enhance T-cell activation in vitro. Several methods of diagnosing food allergies exist, including skin prick tests and double-blind food challenges; however, diagnosing pollen food syndrome depends almost exclusively on clinical history. Immunotherapy has been studied as a treatment for pollen food syndrome, with highly variable results.