A Novel Ubiquitin-Specific Protease, UBP43, Cloned from Leukemia Fusion Protein AML1-ETO-Expressing Mice, Functions in Hematopoietic Cell Differentiation

Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation.

本研究采用PCR偶联消减筛选-代表性差异分析（PCR-coupled subtractive screening-representational difference analysis）技术，从AML1-ETO敲入小鼠（AML1-ETO knockin mice）中克隆获得一个全新基因。该基因在敲入小鼠的卵黄囊与胎肝中呈高表达状态。核苷酸序列分析结果显示，其cDNA（complementary DNA）包含一段长1107 bp的开放阅读框，可编码一条含368个氨基酸残基的多肽链。进一步的蛋白质序列与蛋白翻译分析表明，该基因属于泛素特异性蛋白酶（ubiquitin-specific proteases, UBP）家族，分子质量为43 kDa，因此我们将该基因命名为UBP43。与其他泛素蛋白酶类似，UBP43蛋白具备去泛素化酶活性。蛋白质泛素化修饰参与诸多重要的细胞生命活动。在野生型成年小鼠中，UBP43在胸腺与腹腔巨噬细胞中呈高表达水平。在本次分析的9种不同小鼠造血细胞系中，UBP43仅在单核细胞谱系相关的细胞系中可被检测到表达。此外，其表达会在细胞因子诱导的单核细胞分化过程中受到调控。本研究针对UBP43在造血髓系细胞系M1中的功能展开探究：通过逆转录病毒基因转染技术将UBP43导入M1细胞，成功获得数个UBP43高表达克隆用于后续研究。对UBP43/M1细胞进行形态学与细胞表面标志物检测后发现，UBP43过表达会阻断细胞因子诱导的单核细胞终末分化过程。上述实验数据表明，UBP43在造血过程中发挥重要调控作用，其机制可能是在髓系细胞分化阶段，通过调控泛素依赖的蛋白水解通路，或是改变其他调控因子的泛素化修饰状态来实现功能。