Kim Kwangsoo, Mitra Sharmistha, Wu Gang, Berka Vladimir, Song Jinmei, Yu Ye, Poget Sebastien, Wang Da-Neng, Tsai Ah-Lim, Zhou Ming
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine , Houston, Texas 77030, United States.
Division of Hematology, Department of Internal Medicine, University of Texas-McGovern Medical School , Houston, Texas 77030, United States.
Biochemistry. 2016 Dec 6;55(48):6673-6684. doi: 10.1021/acs.biochem.6b00610. Epub 2016 Nov 17.
STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe and Cu. Ferrous STEAP1 also reacts readily with O through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with ∼80 and ∼20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, K, was ∼30 μM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.
前列腺六跨膜上皮抗原1（STEAP1）在多种癌细胞中强烈表达，尤其是在前列腺癌中，抑制其表达可降低肿瘤细胞增殖速率。然而，STEAP1的生理功能仍不清楚。在此，我们首次以毫克水平纯化了一种哺乳动物（兔）的STEAP1，从而能够对其进行高质量的生化和生物物理表征。我们发现，STEAP1可能组装成同源三聚体，与STEAP2共表达时形成异源三聚体。每个STEAP1原体结合一个血红素辅基，该辅基主要为低自旋，有一对组氨酸轴向配体，还有一小部分高自旋和P450型血红素。处于亚铁状态时，STEAP1能够还原铁和铜的过渡金属离子络合物。亚铁STEAP1还能通过外层球氧化还原机制与氧迅速反应。与所有三种底物反应的动力学都是双相的，快速和慢速阶段分别约为80%和20%，这与其血红素的异质性一致。纯化过程中，STEAP1保留了低水平的结合黄素腺嘌呤二核苷酸（FAD），结合平衡常数K约为30μM。这些结果突出了STEAP作为一种新型金属还原酶和超氧化物合酶的特性，并为进一步研究STEAP1活性如何影响癌症进展奠定了坚实基础。
