BNIP3磷酸化/去磷酸化及其对低氧下线粒体自噬的调控作用

The mitochondrial outer membrane protein BNIP3 enhances the cell viability under stress by promoting mitophagy, which mechanism is probably related to the phosphorylation of BNIP3. However, it is unclear that the kinase and the phosphatase of BNIP3 and the upstream and downstream pathways of mitophagy regulated by BNIP3 phosphorylation. We found in the preliminary experiment that the modification of phosphorylation under hypoxia was involved in the regulation of BNIP3 on mitophagy. Furthermore, we screened and found the new sites S60/T66 of BNIP3 phosphorylation, and the kinase JNK1/2 and phosphatase PP1A/C for the two sites. We thus propose a hypothesis that JNK1/2 and PP1A/C may separately activates and inactivates the phosphorylaiton at S60/T66 sites and therefore modulate the mitophagy mediated by BNIP3 under hypoxia. To verify this hypothesis, we will carry out the following studies: 1. Confirm the regulation of JNK1/2 and PP1A/C on BNIP3 S60/T66; 2. Investigate the role and mechanism of regulation of BNIP3 phosphorylation/dephosphorylation on mitophagy; 3. Reveal the pathways of mitophagy regulated by BNIP3 phosphorylation/dephosphorylation under hypoxia. The above study is expected to provide a new target and intervention strategy for the prevention and treatment of related clinical diseases.

线粒体外膜蛋白BNIP3通过促进线粒体自噬可增强细胞在应激下的存活能力，其调控机制可能与BNIP3磷酸化有关。然而BNIP3磷酸化修饰的激酶和磷酸酶及其调节线粒体自噬的上下游通路尚不清楚。我们前期研究发现低氧下BNIP3的磷酸化修饰参与了BNIP3对线粒体自噬的调控，并进一步筛选到BNIP3磷酸化的新位点 S60/T66和针对这两个位点的激酶JNK1/2和磷酸酶PP1A/C。我们由此提出假设：JNK1/2和PP1A/C可能通过作用于BNIP3 S60/T66，调控低氧下BNIP3介导的线粒体自噬。为验证该假设，本项目拟开展以下研究：1.确认JNK1/2和PP1A/C对BNIP3 S60/T66的调控作用；2.探明BNIP3磷酸化/去磷酸化调节线粒体自噬的作用及机制；3.揭示低氧下BNIP3磷酸化/去磷酸调节线粒体自噬的上下游通路。通过上述研究有望为临床相关疾病的防治提供新的靶点与干预策略。

BNIP3（Bcl-2 and adenovirus E1B 19 kDa-interacting protein 3）是一种定位于线粒体外膜的BCL-2家族BH3-only成员，也是线粒体自噬的一种受体分子，已知研究发现低氧等多种应激条件可以通过上调BNIP3来促进线粒体自噬的发生。然而翻译后修饰，特别是磷酸化修饰在BNIP3介导线粒体自噬的调控中发挥怎样的作用及相关机制目前仍知之甚少。.在本研究中我们发现低氧可以通过JNK1/2（Jun N-terminal kinase 1/2）和PP1/2A（protein phosphatase 1/2A）调控BNIP3 S60/T66的磷酸化进而影响BNIP3的稳定性及其与自噬膜蛋白LC3（microtubule-associated protein 1 light chain 3）的结合，最终对线粒体自噬的发生产生调控作用。我们研究证实，适度低氧能增强JNK1/2活性，进而通过JNK1/2促进BNIP3 S60/T66的磷酸化，使BNIP3的稳定性得到加强，同时通过该磷酸化位点BNIP3与LC3发生相互作用，并因此诱导线粒体自噬；而严重低氧导致JNK1/2的活性受到抑制， BNIP3 S60/T66被PP1/2A去磷酸化，去磷酸化的BNIP3经泛素蛋白酶体途径被迅速降解，使其无法与LC3相结合，最终抑制线粒体自噬的发生。.总之，我们的研究表明低氧能调控BNIP3的磷酸化修饰，并进一步揭示了BNIP3新的磷酸化位点和相应的激酶和磷酸酶，以及BNIP3该位点磷酸化修饰对其自身稳定性和对线粒体自噬的调节作用。该研究成果有望为低氧相关疾病的干预提供更加精准的干预靶点。