摘要
侵袭性(Invasion)和转移性(metastasis)是恶性肿瘤的主要特征,也是目前肿瘤研究和治疗中的重点和难点。肿瘤的侵袭转移是一个多步骤,多阶段的复杂过程,与肿瘤细胞的运动性、血管发生能力、自身免疫逃逸以及黏附性等都有着密切的关系。目前肿瘤的诊断和治疗以肿瘤大小、淋巴结转移数目、其它器官和组织是否转移作为标准,临床上对于某些肿瘤相关分子的免疫组化检测已经成为常规的判断肿瘤恶性程度的指标,但同时临床上的免疫组化检测并没有相对公认的金指标,无法以单一分子作为判断标准,因此新的分子检测指标的引入将对于提高临床上肿瘤恶性程度判断的准确性提供新的更为可靠的线索。
本实验室发现和正在研究的CRRF蛋白(Cancer Related RING finger)在功能上未有报道,我们对基因芯片进行初步分析发现CRRF在多种恶性肿瘤中的表达水平高于正常组织和外周血单核细胞。同时对六种侵袭能力不同的食管癌细胞进行CRRF的表达检测时,发现其在三种侵袭能力较强的KYSE180、KYSE450、E.C.9706中能够检测到,而于另三种侵袭能力较弱的KYSE2、KYSE410、KYSE450细胞中检测不到,提示着CRRF蛋白的存在与否与肿瘤细胞的侵袭水平相关。为了确定这一现象并在接近体内水平检测CRRF的表达与肿瘤恶性程度的关系,我们利用免疫组化对大样本量的胰腺癌组织和结肠癌组织的CRRF表达水平进行检测,发现CRRF主要在恶性程度较高的Ⅱ级和Ⅲ级肿瘤样本中表达比例较高,而在恶性程度较低高分化的Ⅰ级组织样本中阳性率比例较低。这一结果表明CRRF的表达状态与肿瘤恶性程度的演进有一定的联系。这一结果表明CRRF蛋白质可能在某些类型肿瘤恶化过程中起着调节的作用。
CRRF编码基因全长为1146个碱基,共编码381个氨基酸。CRRF蛋白具有N端蛋白酶相关结构域(PA domain)和C端指环结构域(RING finger domain)。N端PA结构域在多种类型的蛋白酶以及植物囊泡分选受体中有发现,但目前功能未知。PA结构域还发现能够与指环结构域位于同一蛋白质中形成一类PA-RING蛋白家族,CRRF既是家族成员之一。CRRF蛋白结构中还具有N端的信号肽和位于中间位置的跨膜区,我们通过试验证明CRRF定位于细胞浆中并呈点状分布,同时CRRF溶于表面活性剂Triton X-114而不溶于水相,推测CRRF可能是一个TypeⅠ跨膜蛋白。另外,利用衣霉素(Tunicamycin)处理细胞及PNGase F处理蛋白样品,发现CRRF的分子量减少,提示CRRF是一个N-糖基化修饰蛋白,进一步确定第88位天冬酰胺是CRRF的糖基化修饰位点。通过体外及细胞水平的泛素化检测,我们证明了CRRF具有泛素连接酶活性,并且泛素结合酶UbcH5a和UbcH5c也参与CRRF发挥其功能。对指环结构域保守氨基酸进行突变则其泛素连接酶活性即减弱甚至丧失,说明指环结构域结构的保守性及重要性。此上对CRRF蛋白性质的鉴定表明CRRF是一个位于细胞内膜系统中具有泛素连接酶活性的糖蛋白。
同时我们也发现在胰腺癌实体瘤和淋巴瘤Jurkat、Raji中,CRRF的表达与细胞对于As2O3引起的细胞凋亡的抵抗能力有关。在高表达CRRF的实体瘤和淋巴瘤细胞Raji中,细胞表现出对As2O3的敏感;而在不表达或少量表达CRRF的实体瘤和淋巴瘤细胞Jurkat中,细胞则表现出明显的抵抗,凋亡比例较少。根据已有报道,As2O3引起细胞中活性氧(Reactive Oxygen Species,ROS)的产生引起细胞分化和凋亡,因此以上现象提示着CRRF可能作用于细胞中的活性氧自由基清除酶系统而使得细胞对As2O3的敏感性存在差异。
为在分子水平上阐明CRRF的作用机理,我们对其相互作用蛋白(包括底物)进行了筛选和鉴定。筛选得到SNARE相关蛋白Snapin能够与其相互作用,并通过GST pull down、免疫沉淀、共定位试验得以验证,并且证明CRRF蛋白的C末端介导了两者的相互作用。在体外系统中,Snapin能够被CRRF泛素化。有趣的是,Snapin的表达水平在细胞转染不同剂量CRRF时的检测发现Snapin在高剂量CRRF转染的细胞中表达量反而增加,二者呈正相关,并非是经典的促进Snapin的降解。此结果提示虽然Snapin作为CRRF的泛素化底物,但CRRF可能在其它方面调节Snapin的功能,并不是介导其降解。
Invasion and metastasis of neoplasm cause lethal malignancy and poor therapy.The tumor metastasis is a multiple step and complex process that involved in such as tumor cells motility,angiogenesis,survival from the circulation.Immunohistochemisty has been widely used in the clinical diagnosis.Detection of some of cancer-related molecular markers indicates the malignance of tumor,raising evidence for clinical treatment. However,new molecular cancer-related markers should be explored and applied in order to accurately justify the malignance of patients.
CRRF is a novel gene and its expression level is higher in malignant cancers than that of normal tissues according to analysis of genes expression by microarray.At the same time,CRRF protein could be detected in three higher invasive esophageal cancer cells KYSE180,KYSE450 and E.C.9706,but in another three esophageal cancer cells KYSE2,KYSE410 and KYSE510 the expression of CRRF could not be detected.It suggested that CRRF is relative to invasive ability of cancer cells.In order to identify the relationship of CRRF expression and the malignance in cancer patients,CRRF expression was detected in pancreatic cancer and colon cancer tissue arrays.The CRRF expression was significantly positively related with moderate and poor differentiated cancer tissues and not to well differentiated cancer tissues,that indicated that CRRF expression is involved in cancer malignance.
The coding region of CRRF gene contains 1146 bases,it encodes 381 amino acids. According to domains analysis,two main domains,N-terminal protease-associated domain(PA domain) and C-terminal RING finger domain were predicted.CRRF protein also contains signal peptide and transmembrane domain.CRRF showed punctuated localization in cytosol and dissolved in Triton X-114,suggested a possible TypeⅠtransmembrane protein.In addition,molecular weight of CRRF decreased after cells treated with Tunicamycin and protein samples treated with PNGase F.Using N-glycosylated putative point mutations,Asn88 was identified as the N-glycosylated site of CRRF.In vitro and in vivo ubiquitination assay suggested that CRRF demonstrates E3 ligase activity and UbcH5a and UbcH5c mediates this activity.Mutations in RING finger domain of CRRF abolished the E3 ligase activity,suggested the conservation and importance of RING finger domain for the function of CRRF.
CRRF expression is also found to be related with solid tumor and lymphoma cancer cells sensitivity to As2O3.Pancreatic cancer cells and Raji cells,which express higher CRRF,showed sensitive to As2O3.However,pancreatic caner cells and Jurkat cells, which are hard to detect CRRF expression,showed insensitive to As2O3.
Snapin was screened and identified as CRRF interacting protein.Proximal C terminal of CRRF mediated the interaction between CRRF and Snapin.Using in vitro ubiquitination assay CRRF ubiquitinates Snapin.Finally,Snapin and CRRF were co-transfected into COS-7 cells to detect the expression relationship between these two proteins,higher Snapin protein level is detected in higher CRRF protein expressed cells. This data suggested CRRF mediating Snapin ubiquitination maybe is involved in Snapin other function and not for proteasome degradation.
引文
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