Mätlik K, Yu Li-ying, Eesmaa A, Hellman M, Lindholm P, Peränen J, Galli E, Anttila J, Saarma M, Permi P, Airavaara M, Arumäe U
Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Viikki Biocenter, PO Box 65, Helsinki 00014, Finland.
Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PO Box 65, Helsinki 00014, Finland.
Cell Death Dis. 2015 Dec 31;6(12):e2032. doi: 10.1038/cddis.2015.371.
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. As an in vivo model for studying the effect of these mutants after their extracellular application, we used the rat model of cerebral ischemia. Even though we found no evidence for oxidoreductase activity of MANF, the mutation of CXXC motif completely abolished its protective effect, showing that this motif is crucial for both MANF's intracellular and extracellular activity. The RTDL motif was not needed for the neuroprotective activity of MANF after its extracellular application in the stroke model in vivo. However, in vitro the deletion of RTDL motif inactivated MANF in the sympathetic neurons where the mutant protein localized to Golgi, but not in the sensory neurons where the mutant localized to the ER, showing that intracellular MANF protects these peripheral neurons in vitro only when localized to the ER.
中脑星形胶质细胞衍生的神经营养因子（MANF）是一种促生存蛋白，在体外细胞内应用或体内细胞外应用时可保护细胞。其保护机制尚不清楚。在此，我们研究了MANF羧基（C）末端结构域内的两个短序列基序在其神经保护活性中的作用：可能参与氧化还原反应的CKGC序列（CXXC基序），以及作为内质网（ER）保留信号的C末端RTDL序列。我们对这些基序进行了突变，并分析了在培养的交感神经元或感觉神经元中过表达时这些MANF突变体的抗凋亡作用和细胞内定位。作为研究这些突变体在细胞外应用后效果的体内模型，我们使用了脑缺血大鼠模型。尽管我们没有发现MANF具有氧化还原酶活性的证据，但CXXC基序的突变完全消除了其保护作用，表明该基序对MANF的细胞内和细胞外活性都至关重要。在体内中风模型中，细胞外应用MANF后，RTDL基序对其神经保护活性并非必需。然而，在体外，在交感神经元中删除RTDL基序会使MANF失活，此时突变蛋白定位于高尔基体，而在感觉神经元中突变体定位于内质网时则不会，这表明细胞内的MANF仅在内质网定位时才能在体外保护这些外周神经元。
