GATA binding protein 2 mediated ankyrin repeat domain containing 26 high expression in myeloid-derived cell lines

BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.

Targeting neuronal PAS domain protein 2 and KN motif/ankyrin repeat domains 1:Advances in type 2 diabetes therapy

This editorial summarizes the latest literature on the roles of neuronal PAS domain protein 2 and KN motif/ankyrin repeat domain 1 in type 2 diabetes(T2D).We highlight their involvement inβ-cell dysfunction,explore their potential as therapeutic targets,and discuss the implications for new treatment strategies.We offer valuable insights into relevant gene regulation and cellular mechanisms relevant for the targeted management of T2D.

结直肠癌组织中RREB1和ANKRD1的表达与临床病理特征和预后的相关性研究

目的探讨结直肠癌(colorectal cancer,CRC)组织ras反应元件结合蛋白1(ras-responsive element binding protein 1,RREB1),ankyrin重复结构域1(ankyrin repeat domain 1,ANKRD1)的表达及与临床病理特征和预后不良的关系。方法选取2013年1月~2016年12月复旦大学附属中山医院厦门医院收治的105例CRC患者,应用免疫组织化学SP法检测CRC患者癌组织和癌旁组织RREB1和ANKRD1蛋白表达,分析RREB1和ANKRD1蛋白表达与患者临床病理特征的关系,患者均随访5年,比较不同RREB1和ANKRD1表达患者的预后情况,并分析CRC患者预后不良的影响因素。采用Spearman相关系数分析RREB1与ANKRD1表达的相关性。结果CRC癌组织RREB1蛋白阳性率(59.05%)显著高于癌旁组织(27.61%),差异有统计学意义(χ^(2)=21.120,P=0.000)。CRC癌组织ANKRD1蛋白阳性率(31.43%)显著低于癌旁组织(60.95%),差异有统计学意义(χ^(2)=18.410,P=0.000)。TNM分期Ⅲ期、淋巴结转移N1~N2患者RREB1蛋白阳性率高于TNM分期Ⅰ~Ⅱ期、淋巴结转移N0患者,差异有统计学意义(χ^(2)=4.263,8.199,均P<0.05)。TNM分期Ⅲ期、淋巴结转移N1~N2患者ANKRD1蛋白阳性率低于TNM分期Ⅰ~Ⅱ期、淋巴结转移N0患者,差异有统计学意义(χ^(2)=5.515,7.411,均P<0.05)。RREB1高表达组5年生存率(54.84%)低于RREB1低表达组(74.42%),差异有统计学意义(χ^(2)=5.459,P=0.020);ANKRD1高表达组的5年生存率(78.79%)高于ANKRD1低表达组(55.56%),差异有统计学意义(χ^(2)=5.130,P=0.024)。RREB1高表达(HR=2.437,95%CI:1.113~4.684)、ANKRD1低表达(HR=0.573,95%CI:0.185~1.952)、TNM分期Ⅲ期(HR=2.202,95%CI:1.357~4.215)和淋巴结转移N1~N2(HR=1.247,95%CI:1.532~4.368)是CRC患者预后不良的危险因素(均P<0.05)。Spearman秩相关分析结果,CRC癌组织中RREB1与ANKRD1表达呈负相关(r=-0.389,P=0.036)。结论CRC癌组织中RREB1表达升高,而ANKRD1表达降低,二者共同参与CRC的发生发展,有望成为评估CRC患者预后的组织肿瘤标志物。

Daikenchuto (Da-Jian-Zhong-Tang) ameliorates intestinal fibrosis by activating myofibroblast transient receptor potential ankyrin 1 channel

AIM To investigate the anti-fibrotic effects of the traditional oriental herbal medicine Daikenchuto(DKT) associated with transient receptor potential ankyrin 1(TRPA1) channels in intestinal myofibroblasts. METHODS Inflammatory and fibrotic changes were detected in a2,4,6-trinitrobenzenesulfonic acid(TNBS) chronic colitis model of wild-type and TRPA1-knockout(TRPA1-KO) mice via pathological staining and immunoblotting analysis.Ca^(2+) imaging experiments examined the effects of DKT and its components/ingredients on intestinal myofibroblast(In Myo Fib) cell TRPA1 channel function.Profibrotic factors and transforming growth factor (TGF) -β1-associated signaling were tested in an In Myo Fib cell line by q PCR and immunoblotting experiments.Samples from non-stenotic and stenotic regions of the intestines of patients with Crohn’s disease (CD) were used for pathological analysis. RESULTS Chronic treatment with TNBS caused more severe inflammation and fibrotic changes in TRPA1-KO than in wild-type mice.A one-week enema administration of DKT reduced fibrotic lesions in wild-type but not in TRPA1-KO mice.The active ingredients of DKT,i.e.,hydroxyα-sanshool and 6-shogaol,induced Ca^(2+) influxes in In Myo Fib,and this was antagonized by co-treatment with a selective TRPA1 channel blocker,HC-030031.DKT counteracted TGF-β1-induced expression of TypeⅠcollagen andα-smooth muscle actin (α-SMA) ,which were accompanied by a reduction in the phosphorylation of Smad-2 and p38-mitogen-activated protein kinase (p38-MAPK) and the expression of myocardin.Importantly,24-h incubation with a DKT active component Japanese Pepper increased the m RNA and protein expression levels of TRPA1 in In Myo Fibs,which in turn negatively regulated collagen synthesis.In the stenotic regions of the intestines of CD patients,TRPA1 expression was significantly enhanced.CONCLUSION The effects of DKT on the expression and activation of the TRPA1 channel could be advantageous for suppressing intestinal fibrosis,and benefit inflammatory bowel disease treatment.

The loss-of-function mutations and down-regulated expression of ASB3 gene promote the growth and metastasis of colorectal cancer cells

Background: Ankyrin repeat and SOCS box protein 3(ASB3) is a member of ASB family and contains ankyrin repeat sequence and SOCS box domain. Previous studies indicated that it mediates the ubiquitination and degradation of tumor necrosis factor receptor 2 and is likely involved in inflammatory responses. However, its effects on oncogenesis are unclear. This study aimed to investigate the effects of ASB3 on the growth and metastasis of colorectal cancer(CRC).Methods: We used next?generation sequencing or Sanger sequencing to detect ASB3 mutations in CRC specimens or cell lines, and used real?time quantitative polymerase chain reaction, Western blotting, and immunohistochemical or immunofluorescence assay to determine gene expression. We evaluated cell proliferation by MTT and colony for?mation assays, tested cell cycle distribution by flow cytometry, and assessed cell migration and invasion by transwell and wound healing assays. We also performed nude mouse experiments to evaluate tumorigenicity and hepatic metastasis potential of tumor cells.Results: We found that ASB3 gene was frequently mutated(5.3%) and down?regulated(70.4%) in CRC cases. Knock?down of endogenous ASB3 expression promoted CRC cell proliferation, migration, and invasion in vitro and facilitated tumorigenicity and hepatic metastasis in vivo. Conversely, the ectopic overexpression of wild?type ASB3, but not that of ASB3 mutants that occurred in clinical CRC tissues, inhibited tumor growth and metastasis. Further analysis showed that ASB3 inhibited CRC metastasis likely by retarding epithelial?mesenchymal transition, which was characterized by the up?regulation of β?catenin and E?cadherin and the down?regulation of transcription factor 8, N?cadherin, and vimentin.Conclusion: ASB3 dysfunction resulted from gene mutations or down?regulated expression frequently exists in CRC and likely plays a key role in the pathogenesis and progression of CRC.

Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites

The homozygous inv （inversion of embryonic turning） mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs （Xinv-1 and Xinv-2） of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites （IQ motifs） are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs ofXenopus inv and mouse inv proteins may regulate their function in different ways.

Murine Cytomegalovirus IE3 Protein Interacts with Ankrd17

Murine cytomegalovirus（MCMV） IE3 protein is a multifunctional viral protein that inter-acts with several target proteins of both viral and host cellular origin.To investigate the biological func-tion of IE3 in the pathogenesis of the brain disorders caused by CMV,a screening for host cellular pro-teins that could interact with IE3 was performed.By yeast two-hybrid screening,ankyrin repeats do-main 17（Ankrd17,also known as Gtar） was identified as a host factor that could interact with IE3.This interaction was verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.Map-ping analysis suggested that the 1-148 residues of IE3 were responsible for the interaction.These results suggested that the interaction between Ankrd17 and IE3 may play a key role in the pathogenesis of MCMV-associated disease.

Severe hyperbilirubinemia in a neonate with hereditary spherocytosis due to a de novo ankyrin mutation: A case report

BACKGROUND Hereditary spherocytosis(HS)is a common type of hemolytic anemia caused by a red cell membrane disorder.HS type 1(HS1)is mostly caused by mutations in ankyrin(ANK1).Newborns with HS1 usually only exhibit anemia and mild jaundice.We herein report a case of HS1 and discuss its clinical characteristics.CASE SUMMARY A 2-d-old male full-term newborn was admitted to our hospital with severe,intractable neonatal jaundice.Laboratory investigations showed hemolytic anemia and hyperbilirubinemia and excluded immune-mediated hemolysis.The patient underwent two exchange transfusions and one plasmapheresis resulting in significantly reduced serum bilirubin.Hematologic analyses and genomic DNA sequencing studies were performed.The trio clinical exome sequencing revealed a de novo null heterozygous mutation in the patient's ANK1 gene:c.841C>T(p.Arg281Ter).This mutation results in the premature termination of the ANK1 protein.CONCLUSION Our case demonstrates that genetic analysis can be an essential method for diagnosing HS when a newborn has severe hyperbilirubinemia.

Transient Receptor Potential Ankyrin 1 Ion Channel Facilitates Acute Inflammation Induced by Surgical Incision in Mice

Background: Transient receptor potential ankyrin (TRPA) 1 is known as a peripheral initiator of acute inflammation and hyperalgesia. However, its role in the facilitation of innate immune responses followed by resolution of the inflammation triggered by a surgical incision has not been fully investigated. Therefore, we evaluated the mechanism by which TRPA1 regulates the inflammatory responses mainly facilitated by neutrophils and macrophages in the early course of wound repair after an incision. Methods: Plantar incision was performed in wild-type and TRPA1-/- mice. The infiltration of polymorphonuclear neutrophils, macrophage phenotype, and induction of inflammatory mediators were assessed for 7 days postoperatively. Results: TRPA1-/-?mice exhibited decreased infiltration of polymorphonuclear neutrophilscompared with wild-type mice on day 1. Consistently, the influx of F4/80+ iNOS+ proinflammatory M1 macrophages to incised sites was markedly decreased on day 2. Similarly, F4/80+ CD206+M2 macrophages, which regulate the resolution of inflammation and promote wound healing in the later phase of acute inflammation, were significantly decreased in TRPA1-/- compared with those in wild-type mice on day 7. In addition, the induction of heme oxygenase-1, which promotes wound healing by switching phenotype of macrophages, was decreased in the early phase of acute inflammation, whereas the expression of proinflammatory mediators such as tumor necrosis factor and cyclooxygenase-2, and 15-lipoxygenase, which are involved in the resolution of inflammation was increased in the late phase in TRPA1-/- mice. Conclusions: Early innate immune responses including neutrophil infiltration and macrophage polarization at incised sites were inhibited in TRPA1-/- mice, associated with increased pro-inflammatory mediators in later phase. Peripheral TRPA1 may facilitate the acute inflammatory process, leading to the promotion of macrophage-mediated resolution of inflammation and wound repair after a surgical incision.

Zhenxin Anshen formula(镇心安神方)ameliorates atopic der-matitis-like skin dysfunction in mice and in vitro via regulation of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 in Neural pathways

OBJECTIVE:To investigate the efficacy of Zhenxin Anshen formula(镇心安神方,ZXAS)on atopic dermatitis(AD)by transient receptor potential vanilloid 1(TRPV1)and transient receptor potential ankyrin 1(TRPA1)signalling pathway in mice and in vitro.METHODS:AD-like lesions were induced by 1-chloro-2,4-dinitrobenzene(DNCB)to the shaved dorsal skin of BALB/c mice.BALB/c mice were divided into five groups:normal control,model control,cetirizine,low-,medium-,and high-dose of ZXAS.After ZXAS in-tervention,the skin lesions and blood samples were collected for hematoxylin and eosin-stained and measuring the concentrations of inflammatory cytokines.Immunoglobulin E(IgE),interleukin(IL)-4,IL-5,IL-13,and thymic stromal lymphopoietin(TSLP)were de-tected by Enzyme-linked immunosorbent assay(ELISA).The spinal cords were collected for measuring the expression of gastrin-releasing peptide receptor(GRPR),TRPV1,and TRPA1 by using immunohistochemistry,western blotting,and quantitative real-time polymerase chain reaction(qR T-PCR)analyses.In addition,3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT)assay,flow cytometry,ELISA,and Western blotting were conducted for analysis of primary dorsal root ganglia(DRG)neurons in vitro.RESULTS:ZXAS treatment improved DNCB-induced AD-like lesions through reducing dermatitis score,number of scratching and epidermal thickness,accompanied by the de-creased IgE and Th2 inflammatory cytokines.ZXAS also supressed the mRNA and protein expression of GRPR,TRPV1,and TRPA1 in the spinal cord.The medicated sera of ZXAS decreased capsaicin-induced Ca^(2+)influx and downregulated the expression of TRPV1,TRPA1,and phospholipase C in DRG neurons.CONCLUSIONS:The therapeutic effect of ZXAS on AD may be related to the regulation of TRPV1 and TRPA1 and inhibition of Ca^(2+)signals in neurons.

hASB-8相互作用蛋白的初步研究

hASB 8基因是对肿瘤细胞生长具有明显抑制作用的人类新基因 .其编码蛋白属于人ASB蛋白家族中的一个成员 ,与小鼠中的ASB 8蛋白同源性达 96 % .保守结构域分析显示hASB 8在N端包含 4个Ankyrinre peats ,在C端包含了一个SOCSbox .利用酵母双杂交技术 ,筛选了人的胎盘 (Placenta)cDNA文库 ,获得了与hASB 8相互作用的 2个蛋白 ,ElonginC和CDK4bindingprotein ;并在二倍体酵母体内进行了验证 .这些试验提示hASB 8蛋白可能介导肿瘤细胞中靶蛋白和泛素复合体之间的相互作用 。

Multifunctional protein: cardiac ankyrin repeat protein

Cardiac ankyrin repeat protein （CARP） not only serves as an important component of muscle sarcomere in the cytoplasm, but also acts as a transcription co-factor in the nucleus. Previous studies have demonstrated that CARP is up-regulated in some cardiovascular disorders and muscle diseases; however, its role in these diseases remains controversial now. In this review, we will discuss the continued progress in the research related to CARP, including its discovery, structure, and the role it plays in cardiac development and heart diseases.

Downregulation of Notch-regulated Ankyrin Repeat Protein Exerts Antitumor Activities against Growth of Thyroid Cancer

Background： The Notch-regulated ankyrin repeat protein （NRARP） is recently found to promote proliferation of breast cancer cells. The role of NRARP in carcinogenesis deserves extensive investigations. This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation, apoptosis, cell cycle, and invasion in thyroid cancer. Methods： Thirty-four cases with thyroid cancer were collected from the Department of General Surgery, Xinhua Hospital, Shanghai ]iao Tong University School of Medicine between 2011 and 2012. lmmunohistochemistry was used to detect the level of N RARP in cancer tissues. Lentivirus carrying NRARP-shRNA （Lenti-NRARP-shRNA） was applied to down-regulate NRARP expression. Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. Apoptosis and cell cycle distribution were determined by flow cytometry. Cell invasion was tested using Transwell invasion assay. In addition, expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection. Student＇s t-test, one-way analysis of variance （ANOVA）, or Kaplan Meier were used to analyze the differences between two group or three groups. Results： N RARP was highly expressed in thyroid cancer tissues. Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of l 0 or higher （P 〈 0.05）. Lenti-NRARP-shRNA-induced G 1 arrest （BHTl 01 ： 72.57% ± 5.32%; g305C： 75.45% ± 5.26%） by promoting p21 expression, induced apoptosis by promoting bax expression and suppressing bcl-2 expression, and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression. Conclusion： Downregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer, that suggests a potential role of NRARP in thyroid cancer targeted therapy.

Multifaceted roles of ASB proteins and its pathological significance

BACKGROUND： Post-translational （PT） modification in cells regulates many intracellular events like signal transduction, transcription, cell cycle, protein quality control, apoptosis and cellular development. Ubiquitination is one of the PT modifications which ftmctions as a marker for degradation of target proteins by the proteasome and as a regulatory mechanism for several signalling pathways. The ubiquitination mechanism requires multiple enzymes, including El, E2, and E3 ligases. Among them, E3 ligases play a major role in recognizing target proteins and an essential feature of protein homeostatic mechanisms within the cell. Most of the ASB （ankyrin repeat SOCS box） proteins fimction as RING family of E3 ubiquitin ligases characterized by the presence of two conserved domains N-terminal ankyrin repeat and C-terminal SOCS box domain METHODS and RESULTS： Current studies have shown that some ASBs function as important regulators of several signalling pathways. This review gives an overview of ASB proteins on numerous cellular processes such as insulin signalling, spermatogenesis, myogenesis and in cellular development. Including various pathological situations, such as cancer, primary open-angle glaucoma, and inflammation, indicating that ASBs has important functions in both normal and pathological development CONCLUSIONS： This article provides a precise comprehensive focus on ASBs protein structure, its biological functions, and their pathological significance.

Neurons with Multiple Axons Have Functional Axon Initial Segments

Neurons grow multiple axons after treatment with glycogen synthase kinase-3（GSK-3） inhibitors. However,whether they are electrically active is not known. Here, we examined the role of multiple axons as electrophysiological components during neuronal firing. Combining pharmacological, immunofluorescence, and electrophysiological methods, we found that more neurons had multiple axon initial segments（AISs） after inhibition of GSK-3 activity with SB415286. The multiple AISs induced by GSK-3 inhibition were enriched with voltage-gated sodium channels. The depolarization rate of the multiple-AIS neurons was increased, but their action potential threshold and halfwidth were normal. By calculating derivatives of the actionpotential rising phase, an extra d2 V/dt2 peak from the extra AIS was distinguished; this indicated that the extra AIS fired ahead of the soma and increased the rate of depolarization.Our study demonstrates that the multiple axons induced by GSK-3 inhibition have AIS structures that are electrically active, and provides insight for axon and AIS studies.