Gallery Melissa, Blank Jonathan L, Lin Yinghui, Gutierrez Juan A, Pulido Jacqueline C, Rappoli David, Badola Sunita, Rolfe Mark, Macbeth Kyle J
Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, USA.
Mol Cancer Ther. 2007 Jan;6(1):262-8. doi: 10.1158/1535-7163.MCT-06-0542.
Poh1 deubiquitinase activity is required for proteolytic processing of polyubiquitinated substrates by the 26S proteasome, linking deubiquitination to complete substrate degradation. Poh1 RNA interference (RNAi) in HeLa cells resulted in a reduction in cell viability and an increase in polyubiquitinated protein levels, supporting the link between Poh1 and the ubiquitin proteasome pathway. To more specifically test for any requirement of the zinc metalloproteinase motif of Poh1 to support cell viability and proteasome function, we developed a RNAi complementation strategy. Effects on cell viability and proteasome activity were assessed in cells with RNAi of endogenous Poh1 and induced expression of wild-type Poh1 or a mutant form of Poh1, in which two conserved histidines of the proposed catalytic site were replaced with alanines. We show that an intact zinc metalloproteinase motif is essential for cell viability and 26S proteasome function. As a required enzymatic component of the proteasome, Poh1 is an intriguing therapeutic drug target for cancer.
Poh1去泛素化酶活性是26S蛋白酶体对多泛素化底物进行蛋白水解加工所必需的，它将去泛素化与底物的完全降解联系起来。在HeLa细胞中进行Poh1 RNA干扰（RNAi）导致细胞活力降低和多泛素化蛋白水平升高，这支持了Poh1与泛素蛋白酶体途径之间的联系。为了更具体地测试Poh1的锌金属蛋白酶基序对维持细胞活力和蛋白酶体功能的任何需求，我们开发了一种RNAi互补策略。在内源性Poh1进行RNAi并诱导表达野生型Poh1或Poh1的突变形式（其中假定催化位点的两个保守组氨酸被丙氨酸取代）的细胞中评估对细胞活力和蛋白酶体活性的影响。我们表明，完整的锌金属蛋白酶基序对于细胞活力和26S蛋白酶体功能至关重要。作为蛋白酶体必需的酶成分，Poh1是一种引人关注的癌症治疗药物靶点。
