Rao P Vasantha, Peterson Yuri K, Inoue Toshihiro, Casey Patrick J
Department of Ophthalmology, Duke University School of Medicine, Durham, North Carolina 27710, USA.
Invest Ophthalmol Vis Sci. 2008 Jun;49(6):2464-71. doi: 10.1167/iovs.07-1639. Epub 2008 Mar 3.
To determine the effects of inhibition of protein geranylgeranyltransferase type I (GGTase-I), which isoprenylates so-called CaaX proteins, including the GTP-binding proteins such as Rho GTPases and the betagamma subunits of heterotrimeric G-proteins, on aqueous humor outflow and trabecular meshwork cytoskeletal integrity.
A selective small molecular inhibitor of GGTase-I, GGTI-DU40, was tested in this study to investigate its effects on actin cytoskeletal integrity, cell adhesions, cell-cell junctions, myosin II phosphosphorylation, and membrane localization of GTP-binding proteins in trabecular meshwork (TM) cells, using immunofluorescence detection and immunoblotting analysis. The effects of GGTI-DU40 on aqueous humor outflow were determined using organ-cultured, perfused anterior segments of porcine eyes.
In the TM cell lysates, GGTI-DU40 was confirmed to inhibit GGTase-I activity in a dose-dependent manner. TM cells treated with GGTI-DU40 displayed dose-dependent changes in cell morphology and reversible decreases in actin stress fibers, focal adhesions, and adherens junctions. Myosin light chain phosphorylation was decreased significantly, and membrane localization of isoprenylated small GTPases and Gbetagamma was impaired in drug-treated TM cells. Aqueous outflow facility was increased significantly in eyes perfused with GGTI-DU40.
These data demonstrate that inhibition of geranylgeranyl isoprenylation of CaaX proteins in the aqueous outflow pathway increases aqueous humor outflow, possibly through altered cell adhesive interactions and actin cytoskeletal organization in cells of the outflow pathway. This study indicates that the GGTase-I enzyme is a promising molecular target for lowering increased ocular pressure in glaucoma patients.
确定抑制蛋白质香叶基香叶基转移酶I（GGTase-I）对房水流出和小梁网细胞骨架完整性的影响。GGTase-I可使所谓的CaaX蛋白异戊二烯化，这些蛋白包括Rho GTP酶等GTP结合蛋白以及异三聚体G蛋白的βγ亚基。
在本研究中测试了一种GGTase-I的选择性小分子抑制剂GGTI-DU40，通过免疫荧光检测和免疫印迹分析，研究其对小梁网（TM）细胞中肌动蛋白细胞骨架完整性、细胞黏附、细胞间连接、肌球蛋白II磷酸化以及GTP结合蛋白膜定位的影响。使用猪眼的器官培养灌注眼前节来确定GGTI-DU40对房水流出的影响。
在TM细胞裂解物中，证实GGTI-DU40以剂量依赖性方式抑制GGTase-I活性。用GGTI-DU40处理的TM细胞表现出细胞形态的剂量依赖性变化，肌动蛋白应力纤维、粘着斑和紧密连接可逆性减少。肌球蛋白轻链磷酸化显著降低，在药物处理的TM细胞中，异戊二烯化的小GTP酶和Gβγ的膜定位受损。用GGTI-DU40灌注的眼睛房水流出率显著增加。
这些数据表明，抑制房水流出途径中CaaX蛋白的香叶基香叶基异戊二烯化可增加房水流出，可能是通过改变流出途径细胞中的细胞黏附相互作用和肌动蛋白细胞骨架组织实现的。本研究表明，GGTase-I酶是降低青光眼患者升高眼压的一个有前景的分子靶点。
