Torrent Gerard, Benito Antoni, Castro Jessica, Ribó Marc, Vilanova Maria
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n, E-17071 Girona, Spain.
Biol Chem. 2008 Aug;389(8):1127-36. doi: 10.1515/BC.2008.114.
Onconase, a member of the pancreatic type ribonuclease family, is currently used as a chemotherapeutic agent for the treatment of different types of cancer. It is widely accepted that one of the properties that renders this enzyme cytotoxic is its ability to evade the cytosolic ribonuclease inhibitor (RI). In the present work, we produced and characterized an onconase variant that lacks the disulfide bond C30/C75. This variant mimics the stable unfolding intermediate des(30-75) produced in the reductive unfolding pathway of onconase. We found that the reduction of the C30/C75 disulfide bond does not significantly alter the cytotoxic properties of onconase, although the variant possesses a notably reduced conformational stability. Interestingly, both its catalytic activity and its ability to evade RI are comparable to wild-type onconase under mild reductive conditions in which the three disulfide containing intermediate des(30-75) is present. These results suggest that the C30/C75 disulfide bond could easily be reduced under physiological redox conditions.
癌抑素是胰腺型核糖核酸酶家族的一员，目前用作治疗不同类型癌症的化疗药物。人们普遍认为，使这种酶具有细胞毒性的特性之一是其逃避胞质核糖核酸酶抑制剂（RI）的能力。在本研究中，我们制备并表征了一种缺乏二硫键C30/C75的癌抑素变体。该变体模拟了癌抑素还原解折叠途径中产生的稳定解折叠中间体des(30-75)。我们发现，尽管该变体的构象稳定性显著降低，但C30/C75二硫键的还原并未显著改变癌抑素的细胞毒性特性。有趣的是，在存在含三个二硫键的中间体des(30-75)的温和还原条件下，其催化活性和逃避RI的能力与野生型癌抑素相当。这些结果表明，在生理氧化还原条件下，C30/C75二硫键很容易被还原。
