FgGyp8 as a putative FgRab1 GAP is required for growth and pathogenesis by regulating FgSnc1-mediated secretory vesicles fusion in Fusarium graminearum

Fusarium graminearum is an important plant pathogenic fungus that causes disease and yield reduction in many cereal crops, such as wheat and barley. Gyp8 stimulates GTP hydrolysis on Ypt1 in yeast. However, the functions of Gyp8 in plant pathogenic fungi are still unknown. In this study, we investigated the roles of Fg Gyp8 in F. graminearum by genetic and pathological analyses. Through gene knockout and phenotypic analyses, we found that Fg Gyp8 is required for vegetative growth in F. graminearum. The conidiation, conidial size and number of septa per conidium of ΔFggyp8 mutant are significantly reduced when compared to the wild type PH-1. Furthermore, Fg Gyp8 is crucial for pathogenicity on wheat coleoptiles and wheat heads. Fg Gyp8 contains a conserved TBC domain. Domain deletion analysis showed that the TBC domain, C-and N-terminal regions of Fg Gyp8 are all important for its biological functions in F. graminearum. Moreover, we showed that Fg Gyp8 catalyzes the hydrolysis of the GTP on Fg Rab1 to GDP in vitro, indicating that Fg Gyp8 is a GTPase-activating protein(GAP) for Fg Rab1. In addition, we demonstrated that Fg Gyp8 is required for Fg Snc1-mediated fusion of secretory vesicles with the plasma membrane in F. graminearum. Finally, we showed that Fg Gyp8 has functional redundancy with another Fg Rab1 GAP, Fg Gyp1, in F. graminearum. Taken together, we conclude that Fg Gyp8 is required for vegetative growth, conidiogenesis, pathogenicity and acts as a GAP for Fg Rab1 in F. graminearum.

Inhibitory Effects of Rap1GAP Overexpression on Proliferation and Migration of Endothelial Cells via ERK and Akt Pathways

Rapl is expressed in human umbilical vein endothelial cells （HUVECs）. Rapl-GTPase activating protein （RaplGAP）, with its specific target, Rapl, has been shown to be important in the regulation of many physiological and certain pathological processes. In this study, we investigated the effect of RaplGAP expression on endothelial cell function, or, more specifically, proliferation and migration of endothelial cells. HUVECs were transfected with pcDNA3.1 （empty vector）, pcDNA3.1 containing Flag-tagged-RaplGAP or Myc-tagged-RaplN17. The proliferation, migration and tube formation were examined and compared among the 3 groups. Expression of Rapl, RaplGAP, extracellular signal-regulated kinase （ERK）, phospho-ERK, Akt, phosphor-Akt was detected by Western blotting. The results showed that the proliferation, migration and tube formation were significantly reduced in RaplGAP- and RaplN17-transfected HUVECs as compared with empty vector-transfected control. These changes were coincident with increased expression of Rap 1GAP and decreased expression of activated Rap l, phospho-ERK and -Akt. After treatment of Rap l GAP-transfected HUVECs with a stimulator of Rapl guanine-nucleotide-exchange factor （RaplGEF） 8CPT-2＇OMe-cAMP, it was found that Rapl activity was decreased as compared with empty vector-transfected control. Pretreatment of HU- VECs with an ERK inhibitor PD98059 or a PI3K inhibitor LY294002 prior to stimulation not only blocked 8CPT-2＇OMe-cAMP-induced phosphorylation of ERK and Akt, but also significantly reduced cell proliferation and migration. Finally, we examined the effect of vascular endothelial growth factor （VEGF） on HUVECs overexpressing RaplGAP. VEGF-stimulated Rapl activity, phosphorylation of ERK and Akt, cyclin D1 expression and cell proliferation were repressed in HUVECs overexpressing RaplGAP as compared to empty vector-transfected Control. Taken together, our findings demonstrate that RaplGAP/Rapl and their downstream effectors regulate proliferation and migration of HUVECs via ERK and Akt pathways.

Rap1 GTP酶激活蛋白1、基质金属蛋白酶2与基质金属蛋白酶9在结直肠癌中的表达及其意义

目的 探讨Rap1 GTP酶激活蛋白1（Rap1GAP）、基质金属蛋白酶2（MMP-2）与基质金属蛋白酶9（MMP-9）在结直肠癌中的表达及其与临床病理特征的关系。方法 采用免疫组织化学法检测Rap1GAP、MMP-2与MMP-9在结直肠癌、绒毛状腺瘤、管状腺瘤及正常结直肠组织中的表达，分析各组间表达的差异及与临床病理参数的关系。结果 Rap1GAP在结直肠癌、绒毛状腺瘤、管状腺瘤和正常结直肠组织中的阳性率分别为30.4 %（14／46）、77.8 %（14／18）、69.6 %（16／23）、95.2 %（20／21），各组间表达差异有统计学意义（χ^2＝30.659，P＝0.000）；MMP-2的阳性率分别为71.7 %（33／46）、55.6 %（10／18）、52.2 %（12／16）、9.5 %（2／21），各组间表达差异有统计学意义（χ^2＝22.459，P＝0.000）；MMP-9的阳性率分别为76.1 %（35／46）、61.1 %（11／18）、56.5 %（13／23）、14.3 %（3／21），各组间表达差异有统计学意义（χ^2＝22.643，P＝0.000）。在结直肠癌中，Rap1GAP阳性率与肿瘤分化程度有关（χ^2＝5.275，P＝0.022），与患者性别、年龄及淋巴结转移无关（均P＞0.05）；MMP-2、MMP-9阳性率与淋巴结转移有关（χ^2＝6.661，P＝0.010；χ^2＝8.475，P＝0.040），与患者性别、年龄、分化程度均无关（均P＞0.05）。在结直肠癌中，Rap1GAP的表达与MMP-2及MMP-9呈负相关（r＝-0.424，P＝0.003；r＝-0.294，P＝0.048）；MMP-2和MMP-9的表达无相关性（r＝0.101，P＝0.505）。结论 Rap1GAP、MMP-2与MMP-9在结直肠癌的恶性生物学行为中起重要作用。在结直肠癌中，Rap1GAP与MMP-2和MMP-9的表达呈负相关，三者相互作用影响着结直肠癌的发生、发展。

Crystal Structure of C-terminal of ACAP1 in Endocytic Recycling

ACAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF) 6, is part of a novel clathrin coat complex that is regulated by ARF6 for endocytic recycling in