Differential intestinal mucosal protein expression in hypercholesterolemic mice fed a phytosterol-enriched diet

The molecular mechanisms involved in the phytosterol-induced decrease in intestinal cholesterol absorption remain unclear. Further, other biological properties such as immunomodulatory activity and protection against cancer have also been ascribed to these plant compounds. To gain insight into the mechanisms underlying phytosterol actions, we conducted a proteomic study in the intestinal mucosa of phytosterol-fed apolipoprotein E-deficient hypercholesterolemic (apoE-/-) mice. With respect to control-fed apoE-/- mice, nine differentially expressed proteins were identified in whole-enterocyte homogenates using 2-D DIGE and MALDI-TOF MS. These proteins are involved in plasma membrane stabilization, cytoskeleton assembly network, and cholesterol metabolism. Four of these proteins were selected for further study since they showed the highest abundance change or had a potential functional relationship with known effects of phytosterols. Annexin A2 (ANXA2) and beta-actin decrease and annexin A4 (ANXA4) and annexin A5 (ANXA5) increase were confirmed by Western blot analysis. Intestinal gene expression of ANXA2 and A5 and beta-actin was reduced, whereas that of ANXA4 was unchanged. The main results were retested in normocholesterolemic C57BL/6J mice. ANXA4 and ANXA5 protein upregulation and ANXA2 and beta-actin downregulation were reproduced in these animals. However, no changes in gene expression were found in C57BL/6J mice in either of the four proteins selected. ANXA2, A4, and A5 and beta-actin are proteins of special interest given their pleiotropic functions that include cholesterol-ester transport from caveolae, apoptosis, and anti-inflammatory properties. Therefore, the protein expression changes identified in this study might be involved in the biological effects of phytosterols.     

Proteomics identification of annexin A2 as a key mediator in the metastasis and proangiogenesis of endometrial cells in human adenomyosis

Adenomyosis is a common estrogen-dependent disorder of females characterized by a downward extension of the endometrium into the uterine myometrium and neovascularization in ectopic lesions. It accounts for chronic pelvic pain, dysmenorrhea, menorrhagia, and infertility in 8.8-61.5% women worldwide. However, the molecular mechanisms for adenomyosis development remain poorly elucidated. Here, we utilized a two-dimensional polyacrylamide gel electrophoresis/MS-based proteomics analysis to compare and identify differentially expressed proteins in matched ectopic and eutopic endometrium of adenomyosis patients. A total of 93 significantly altered proteins were identified by tandem MS analysis. Further cluster analysis revealed a group of estrogen-responsive proteins as dysregulated in adenomyosis, among which annexin A2, a member of annexin family proteins, was found up-regulated most significantly in the ectopic endometrium of adenomyosis compared with its eutopic counterpart. Overexpression of ANXA2 was validated in ectopic lesions of human adenomyosis and was found to be tightly correlated with markers of epithelial to mesenchymal transition and dysmenorrhea severity of adenomyosis patients. Functional analysis demonstrated that estrogen could remarkably up-regulate ANXA2 and induce epithelial to mesenchymal transition in an in vitro adenomyosis model. Enforced expression of ANXA2 could mediate phenotypic mesenchymal-like cellular changes, with structural and functional alterations in a β-catenin/T-cell factor (Tcf) signaling-associated manner, which could be reversed by inhibition of ANXA2 expression. We also proved that enforced expression of ANXA2 enhanced the proangiogenic capacity of adenomyotic endometrial cells through HIF-1α/VEGF-A pathway. In vivo, we demonstrated that ANXA2 inhibition abrogated endometrial tissue growth, metastasis, and angiogenesis in an adenomyosis nude mice model and significantly alleviated hyperalgesia. Taken together, our data unraveled a dual role for ANXA2 in the pathogenesis of human adenomyosis through conferring endometrial cells both metastatic potential and proangiogenic capacity, which could serve as a potential therapeutic target for the treatment of adenomyosis patients.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.     

Tyrosine 23 phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and metastases of pancreatic cancer

The aggressiveness of pancreatic ductal adenocarcinoma (PDA) is characterized by its high metastatic potential and lack of effective therapies, which is the result of a lack of understanding of the mechanisms involved in promoting PDA metastases. We identified Annexin A2 (ANXA2), a member of the Annexin family of calcium-dependent phospholipid binding proteins, as a new molecule that promotes PDA invasion and metastases. We found ANXA2 to be a PDA-associated antigen recognized by post-treatment sera of patients who demonstrated prolonged survival following treatment with a PDA-specific vaccine. Cell surface ANXA2 increases with PDA development and progression. Knockdown of ANXA2 expression by RNA interference or blocking with anti-ANXA2 antibodies inhibits in vitro invasion of PDA cells. In addition, post-vaccination patient sera inhibits in vitro invasion of PDA cells, suggesting that therapeutic anti-ANXA2 antibodies are induced by the vaccine. Furthermore, cell-surface localization of ANXA2 is tyrosine 23 phosphorylation-dependent; and tyrosine 23 phosphorylation is required for PDA invasion. We demonstrated that tyrosine 23 phosphorylation resulting in surface expression of ANXA2 is required for TGFβ-induced, Rho-mediated epithelial-mesenchymal transition (EMT), linking the cellular function of ANXA2 which was previously shown to be associated with small GTPase-regulated cytoskeletal rearrangements, to the EMT process in PDA. Finally, using mouse PDA models, we showed that shRNA knock-down of ANXA2, a mutation at tyrosine 23, or anti-ANXA2 antibodies, inhibit PDA metastases and prolong mouse survival. Thus, ANXA2 is part of a novel molecular pathway underlying PDA metastases and a new target for development of PDA therapeutics.     

大鼠不同发育时期胰腺相关蛋白的差异表达

探讨大鼠胰腺不同发育时期相关蛋白的差异表达,应用显微技术分离了大鼠孕15.5天,孕18.5天胚胎胰腺和新生鼠及成年鼠的胰腺,提取其蛋白质后,用固相pH梯度双向聚丙烯酰胺凝胶电泳和质谱分析等蛋白质组学方法,得到了4个不同发育时期的蛋白质表达谱.对其中的6个在孕18.5天胚胎胰腺中有高丰度表达,而在成年鼠胰腺中缺失的蛋白质点,4个在成年胰腺中特异表达的蛋白质点, 8个在成年胰腺中表达明显下调的蛋白质点和1个在成年中表达上调的点,进行了肽质量指纹分析和蛋白质鉴定,共获得18个点的肽质量指纹图.经BIOWORK等软件搜索大鼠非冗余蛋白质数据库来鉴定其身份,发现其中7个点为大鼠甲胎蛋白（AFP）、5个点为胰脂酶相关蛋白1前体、1个点为微管蛋白β、2个点为蛋白二硫异构酶、1个为FLN29基因产物的类似物、1个为胰蛋白酶V-A前体、1个为过氧化物氧化还原酶4.其中AFP为特异表达于大鼠胚胎期及新生期胰腺的蛋白质,在孕18.5天的胰腺中表达量最高,在成年胰腺中极低表达.对它们的功能和与胚胎胰腺代谢调节功能完善过程的可能关系进行了初步探讨.     

Characterization of the nuclear matrix proteins in a transgenic mouse model for prostate cancer

The nuclear matrix (NM) contains a number of proteins that have been found to be associated with transformation. We have previously identified changes in the NM associated with prostate cancer. In this study, we examine the molecular changes that are associated with prostate cancer development in transgenic adenocarcinoma of mouse prostate (TRAMP) model by studying the differences in the NM proteins (NMPs). We collected prostates from the TRAMP males at six critical time points: 6 weeks (puberty), 11 and 19 weeks (development of mild hyperplasia), 25 weeks (development of severe hyperplasia), 31 and 37 weeks (development of neoplasia). The nuclear matrices from the prostates collected at these time points were then isolated and the NMPs were characterized by high-resolution two-dimensional gel electrophoresis. We found three NMPs (E1A, E1B, and E1C) that were present in the 6-week-old prostate and two NMPs (E2A and E2B) that were present in the 11-week-old prostate. These NMPs were absent in the 31- and 37-week-old prostate. We also found five NMPs (E3A-E3E) that were present in the 31-week-old prostate, but absent in the earlier time points. In addition, three NMPs (Le1, Le2, Le3) were present at higher expression in the 6-, 11-, 19-, and 25-weeks old TRAMP prostates, but they were expressed lower during the development of neoplasia at 31- and 37-weeks old. Identification of these NMPs permits the development of novel markers that can characterize various stages of prostate cancer development as well as potentially therapeutic targets.     

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The kcat of the E. coli FBPase was 14.6 s(-1) as compared to 21 s(-1) for the pig kidney enzyme, while the K(m) of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg2+ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg2+, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg2+ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.     

Inflammation and Cancer: Role of Annexin A1 and FPR2/ALX in Proliferation and Metastasis in Human Laryngeal Squamous Cell Carcinoma

The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1_2–26 (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1_2–26 treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1_2–26 treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.     

Interaction of human neutrophils with endothelial cells regulates the expression of endogenous proteins annexin 1, galectin-1 and galectin-3

Annexin 1 (ANXA1), galectin-1 (Gal-1) and galectin-3 (Gal-3) proteins have been identified as important mediators that promote or inhibit leukocyte migration. The expression of these proteins was studied in human neutrophils and endothelial cells (ECs) during a transmigration process induced by IL-8. Upon neutrophil adhesion to EC, a significant increase in the cleaved ANXA1 (LCS3, raised against all ANXA1 isoforms) expression was detected in the plasma membrane of adhered neutrophils and ECs compared to intact ANXA1 isoform (LCPS1, against N-terminus of protein). Adherent neutrophils had elevated Gal-3 levels in the nucleus and cytoplasm, and ECs in their plasma membranes. In contrast, a decrease in the total amounts of Gal-1 was detected in migrated compared to non-migrated neutrophils. Therefore, ANXA1 and Gal-3 changed in their content and localization when neutrophils adhere to endothelia, suggesting a process of sensitive-balance between two endogenous anti- and pro-inflammatory mediators.