TRIM50 Protein Regulates Vesicular Trafficking for Acid Secretion in Gastric Parietal Cells

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.     

Structure and function of the SPRY/B30.2 domain proteins involved in innate immunity

The SPRY domain is a protein interaction module found in 77 murine and ～100 human proteins, and is implicated in important biological pathways, including those that regulate innate and adaptive immunity. The current definition of the SPRY domain is based on a sequence repeat discovered in the sp lA kinase and ry anodine receptors. The greater SPRY family is divided into the B30.2 (which contains a PRY extension at the N‐terminus) and “SPRY‐only” sub‐families. In this brief review, we examine the current structural and biochemical literature on SPRY/B30.2 domain involvement in key immune processes and highlight a PRY‐like 60 amino acid region in the N‐terminus of “SPRY‐only” proteins. Phylogenetic, structural, and functional analyses suggest that this N‐terminal region is related to the PRY region of B30.2 and should be characterized as part of an extended SPRY domain. Greater understanding of the functional importance of the N‐terminal region in “SPRY only” proteins will enhance our ability to interrogate SPRY interactions with their respective binding partners.     

miRNA-27a靶向SPRY2对退变椎间盘血管长入生成作用的影响

摘要 目的：探讨miRNA-27a靶向调控 Sprouty 同源物2（Sprouty Homolog 2, SPRY2）对人髓核细胞系（nucleus pulposus cells, NPCs）诱导人微血管内皮细胞（Human microvascular vascular endothelial cells, HMEC-1）血管生成作用的影响。方法：收集脊柱侧弯和椎间盘退变（Intervertebral disc degeneration, IDD）患者的椎间盘组织分别作为对照组和IDD组,通过miRNA芯片筛选差异表达的miRNA。RT-qPCR和荧光原位杂交实验验证组织中miR-27a的表达水平。慢病毒转染细胞,细胞分为Control组（未转染）;NC组（转染慢病毒空载）;sh-miR-27a组（转染 miR-27a抑制慢病毒）;miR-27a组（转染 miR-27a过表达慢病毒）;SPRY2组（转染 SPRY2 过表达慢病毒）及miR-27a +SPRY2组（转染miR-27a和SPRY2 过表达慢病毒）。RT-qPCR检测髓核细胞中miR-27a和SPRY2的表达。双荧光素酶报告基因实验验证miR-27a和SPRY2的靶向关系。将经过不同处理的髓核细胞条件培养基与完全培养基混合培养HMEC-1细胞,Transwell和管腔形成实验检测HMEC-1细胞的侵袭和血管生成能力。免疫荧光和ELISA检测髓核细胞和混合培养基中转化生长因子-β1(Transforming growth factor-β1, TGF-β1)含量。结果：与对照组椎间盘组织相比,IDD组miR-27a表达明显增加。与NC组相比,SPRY2在sh-miR-27a组中表达升高（P<0.05）,miR-27a组中表达降低（P<0.05）。与NC组相比,miR-27a组HMEC-1细胞侵袭和血管生成能力增强（P<0.05）,TGF-β1表达上升（P<0.05）;SPRY2组HMEC-1细胞侵袭数减少（P<0.05）,管腔样结构未形成。与miR-27a组相比,miR-27a+SPRY2组HMEC-1细胞侵袭和血管生成能力下降（P<0.05）,TGF-β1表达下降（P<0.05）。结论：miRNA-27a通过靶向抑制SPRY2的表达促进髓核细胞诱导HMEC-1细胞成血管能力。     

长链非编码RNA SPRY4-IT1对髓母细胞瘤增殖及侵袭转移的影响

目的：探讨长链非编码RNA SPRY4-IT1（LncRNA）对髓母细胞瘤增殖及侵袭转移的影响。方法：髓母细胞瘤Daoy细胞分为对照组和si-SPRY4-IT1组,分别利用脂质体Lipofectamine 2000将阴性对照荧光序列和SPRY4-IT1-siRNA转入细胞中,采用Real-time PCR检测转染后各组细胞中SPRY4-IT1的表达情况,CCK-8实验及平板克隆形成实验检测细胞增殖能力的变化,以细胞体外侵袭、迁移实验分别检测细胞侵袭、迁移能力的变化,Western blot检测SPRY4-IT1对基质金属蛋白酶（MMP）-2和MMP-9蛋白的影响。结果：si-SPRY4-IT1组SPRY4-IT1 mRNA表达水平、细胞体外增殖能力、细胞侵袭、迁移能力、细胞MMP-2蛋白表达均较对照组明显降低（P<0.05）,而MMP-9蛋白表达未见明显变化。结论：干扰长链非编码RNA SPRY4-IT1在髓母细胞瘤Daoy细胞中的表达能显著抑制细胞的增殖、侵袭和迁移能力。