a National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics , Chinese Academy of Sciences , Beijing , China.
b College of Life Sciences , University of Chinese Academy of Sciences , Beijing , China.
Autophagy. 2019 Jun;15(6):1017-1030. doi: 10.1080/15548627.2019.1569928. Epub 2019 Jan 27.
As a master regulator of the macroautophagy/autophagy-lysosomal pathway, TFEB (transcription factor EB) plays a prominent role in regulating neurodegenerative diseases and cancer. The transcription activity of TFEB is tightly controlled by phosphorylation and dephosphorylation. Phosphorylated S211 (p-S211) of TFEB can be recognized by YWHA/14-3-3 proteins for TFEB cytoplasmic localization. Here, we characterized the interactions between phosphorylated TFEB and YWHA/14-3-3 proteins and determined the structures of YWHA/14-3-3 proteins in complex with a TFEB p-S211-peptide. Although the critical arginine for YWHA/14-3-3 recognition is missing in the N terminus of the TFEB p-S211-peptide, the C-terminal additional hydrophobic residues of the peptide unexpectedly occupy nearly half of the target-binding groove of YWHA/14-3-3 proteins, which compensates for the N-terminal defect and is distinct from the canonical YWHA/14-3-3-binding mode. Mutations of essential residues in the interaction interface between TFEB and YWHA/14-3-3 proteins disrupted their interactions and severely impaired the cytoplasmic localization of TFEB, which altered the expression of TFEB target genes and affected autophagy. Thus, YWHA/14-3-3 proteins recognize phosphorylated TFEB by a noncanonical mode for controlling TFEB cytoplasmic localization and its activity. Abbreviation: ACTB: actin beta; ALP: autophagy-lysosomal pathway; ATP6V1H: ATPase H transporting V1 subunit H; bHLH: basic helix-loop-helix; CLEAR: coordinated lysosomal expression and regulation; Co-IP: co-immunoprecipitation; CTSB: cathepsin B; CTSD: cathepsin D; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MITF: melanocyte inducing transcription factor; NLS: nuclear localization signal; TFEB: transcription factor EB; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein.
作为宏观自噬/自噬溶酶体途径的主要调节因子，TFEB（转录因子 EB）在调节神经退行性疾病和癌症方面发挥着重要作用。TFEB 的转录活性受到磷酸化和去磷酸化的紧密控制。TFEB 的磷酸化 S211（p-S211）可被 YWHA/14-3-3 蛋白识别，用于 TFEB 细胞质定位。在这里，我们描述了磷酸化 TFEB 与 YWHA/14-3-3 蛋白之间的相互作用，并确定了 YWHA/14-3-3 蛋白与 TFEB p-S211-肽复合物的结构。尽管 TFEB p-S211-肽的 N 端缺少关键的精氨酸，但该肽的 C 端额外的疏水性残基出人意料地占据了 YWHA/14-3-3 蛋白靶结合槽的近一半，这弥补了 N 端缺陷，并与典型的 YWHA/14-3-3 结合模式不同。TFEB 与 YWHA/14-3-3 蛋白相互作用界面的必需残基突变破坏了它们的相互作用，严重阻碍了 TFEB 的细胞质定位，改变了 TFEB 靶基因的表达，并影响了自噬。因此，YWHA/14-3-3 蛋白通过非典型模式识别磷酸化 TFEB，从而控制 TFEB 的细胞质定位及其活性。缩写：ACTB：肌动蛋白 β；ALP：自噬溶酶体途径；ATP6V1H：ATP 酶 H 转运 V1 亚基 H；bHLH：碱性螺旋-环-螺旋；CLEAR：协调溶酶体表达和调节；Co-IP：共免疫沉淀；CTSB：组织蛋白酶 B；CTSD：组织蛋白酶 D；LAMP1：溶酶体相关膜蛋白 1；MAP1LC3/LC3：微管相关蛋白 1 轻链 3；MITF：黑素细胞诱导转录因子；NLS：核定位信号；TFEB：转录因子 EB；YWHA/14-3-3：酪氨酸 3-单加氧酶/色氨酸 5-单加氧酶激活蛋白。
