Stocki Pawel, Chapman Daniel C, Beach Lori A, Williams David B
Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Canada.
Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Canada.
J Biol Chem. 2014 Aug 15;289(33):23086-23096. doi: 10.1074/jbc.M114.570911. Epub 2014 Jul 2.
Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This "hyperoxidation" phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.
内质网中的蛋白质折叠由分子伴侣和折叠催化剂协助，这些分子伴侣和折叠催化剂包括蛋白质二硫键异构酶和肽基脯氨酰异构酶家族的成员。在本报告中，我们研究了肽基脯氨酰异构酶的亲环蛋白亚群对蛋白质折叠的贡献，并确定除了亲环蛋白B之外，亲环蛋白C也是一种内质网（ER）亲环蛋白。使用白蛋白和转铁蛋白作为人肝癌细胞中含顺式脯氨酸蛋白质的模型，我们发现亲环蛋白B和C的联合敲低延迟了转铁蛋白的分泌，但令人惊讶的是，却导致了白蛋白更有效的氧化折叠和分泌。对内质网蛋白质二硫键异构酶家族成员氧化状态的检查显示，其转变为更氧化的状态。这伴随着氧化型谷胱甘肽与总谷胱甘肽的比例升高超过5倍。这种“超氧化”表型可以通过用亲环蛋白抑制剂环孢素A处理细胞来复制，这种处理通过诱导亲环蛋白B和C分泌到培养基中，从而有效地耗尽内质网中的亲环蛋白。为了确定负责内质网超氧化的途径，我们分别耗尽了几种已知或怀疑向内质网传递氧化当量的酶：Ero1αβ、VKOR、PRDX4或QSOX1。值得注意的是，这些酶均未导致环孢素A处理诱导的氧化型谷胱甘肽与总谷胱甘肽比例升高。这些发现确定亲环蛋白C为内质网亲环蛋白，证明亲环蛋白B和C在内质网氧化还原稳态中具有新的作用，并表明存在一种由内质网亲环蛋白调节的额外内质网氧化途径。
