Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Consejo Nacional de Investigaciones Científicas y Técnicas CONICET-Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas IQUIFIB, Buenos Aires, Argentina.
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Consejo Nacional de Investigaciones Científicas y Técnicas CONICET-Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas IQUIFIB, Buenos Aires, Argentina.
Arch Biochem Biophys. 2022 Dec 15;732:109450. doi: 10.1016/j.abb.2022.109450. Epub 2022 Oct 31.
The Spf1p protein from Saccharomyces cerevisiae belongs to the family of P5A-ATPases that have recently been shown to protect the endoplasmic reticulum by dislocating misinserted membrane proteins. The loss of function of P5A-ATPases leads to endoplasmic reticulum stress with a pleiotropic phenotype including protein, sterol and metal ion dyshomeostasis. Like other P-ATPases, Spf1p requires Mg. We found that free Mg stimulated the Spf1p ATPase activity along a double hyperbolic curve with two components of K = 14 and 800 μM Ca, Mn and Co lowered about 50% of the Spf1p ATPase with relatively low affinity (K ∼75 μM) and the activity was fully recovered after metal ion chelation with EGTA. In contrast, low concentrations of Zn and Cddecreased the activity to less than 20% and lead to slow irreversible inactivation of the enzyme. After the treatment with Zn, Spf1p exhibited a reduced apparent affinity for ATP and formed lower levels of the catalytic phosphoenzyme. The inactivation by Zn occurred preferentially at a pH > 6 and could be prevented by adding either ATP or ADP to the inactivation media. These results suggest that Zn inactivated Spf1p by binding to amino acid residues from the nucleotide binding-phosphorylation domains that are protonated at lower pH. Alternatively the binding of nucleotides may indirectly compete with a conformational change leading to the Zn-inactive form of the enzyme. Exposure of yeast cells to high concentrations of Zn led to changes similar to the phenotype characteristic of the Spf1Δ cells. Altogether, our data, point out a possible mechanism by which the inhibition of P5A-ATPases could potentiate metal ion-induced ER stress and proteotoxicity.
酿酒酵母的 Spf1p 蛋白属于 P5A-ATP 酶家族，最近的研究表明，该酶家族通过将错误插入的膜蛋白移位来保护内质网。P5A-ATP 酶功能丧失会导致内质网应激，表现出多种表型，包括蛋白质、固醇和金属离子的稳态失衡。与其他 P-ATP 酶一样，Spf1p 需要 Mg。我们发现游离 Mg 以双双曲线的形式刺激 Spf1p ATP 酶活性，有两个组成部分，K = 14 和 800 μM Ca、Mn 和 Co 以相对较低的亲和力（K∼75 μM）降低 Spf1p ATP 酶活性约 50%，金属离子螯合后用 EGTA 可完全恢复活性。相比之下，低浓度的 Zn 和 Cd 使活性降低到小于 20%，并导致酶的缓慢不可逆失活。用 Zn 处理后，Spf1p 对 ATP 的表观亲和力降低，形成的催化磷酸酶水平降低。Zn 的失活优先发生在 pH > 6，可通过在失活介质中添加 ATP 或 ADP 来阻止。这些结果表明，Zn 通过结合在较低 pH 下质子化的核苷酸结合-磷酸化结构域中的氨基酸残基来失活 Spf1p。或者，核苷酸的结合可能会间接与导致酶的 Zn 失活形式的构象变化竞争。酵母细胞暴露于高浓度的 Zn 会导致类似于 Spf1Δ 细胞特征的表型变化。总之，我们的数据指出了一种可能的机制，即 P5A-ATP 酶的抑制可能会增强金属离子诱导的内质网应激和蛋白毒性。
