Research Group Biosynthesis of Neural Structures, Zentrum für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.
Department of Cell Biology and Neuroscience, Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, New Jersey, USA.
FASEB J. 2022 Jan;36(1):e22074. doi: 10.1096/fj.202100816R.
Cell adhesion molecule L1 regulates multiple cell functions and L1 deficiency is linked to several neural diseases. Proteolytic processing generates functionally decisive L1 fragments, which are imported into the nucleus. By computational analysis, we found at L1's C-terminal end the chromo shadow domain-binding motif PxVxL, which directs the binding of nuclear proteins to the heterochromatin protein 1 (HP1) isoforms α, β, and ɣ. By enzyme-linked immunosorbent assay, we show that the intracellular L1 domain binds to all HP1 isoforms. These interactions involve the HP1 chromo shadow domain and are mediated via the sequence KDET in the intracellular domain of murine L1, but not by L1's C-terminal PxVxL motif. Immunoprecipitation using nuclear extracts from the brain and from cultured cerebellar and cortical neurons indicates that HP1 isoforms interact with a yet unknown nuclear L1 fragment of approximately 55 kDa (L1-55), which carries ubiquitin residues. Proximity ligation indicates a close association between L1-55 and the HP1 isoforms in neuronal nuclei. This association is reduced after the treatment of neurons with inhibitors of metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1), or ɣ-secretase, suggesting that cleavage of full-length L1 by these proteases generates L1-55. Reduction of HP1α, -β, or -ɣ expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons and decreases the L1-dependent migration of L1-transfected HEK293 cells in a scratch assay. These findings indicate that the interaction of the novel fragment L1-55 with HP1 isoforms in nuclei affects L1-dependent functions, such as neurite outgrowth and neuronal migration.
细胞黏附分子 L1 调节多种细胞功能，L1 缺乏与多种神经疾病有关。蛋白水解处理生成具有决定功能的 L1 片段，这些片段被导入细胞核。通过计算分析，我们在 L1 的 C 端末端发现了染色质阴影域结合基序 PxVxL，该基序指导核蛋白与异染色质蛋白 1（HP1）同工型α、β和γ的结合。通过酶联免疫吸附试验，我们表明细胞内 L1 结构域与所有 HP1 同工型结合。这些相互作用涉及 HP1 染色质阴影域，并且通过细胞内 L1 结构域中的序列 KDET 介导，而不是通过 L1 的 C 末端 PxVxL 基序介导。使用来自大脑和培养的小脑和皮质神经元的核提取物进行免疫沉淀表明，HP1 同工型与大约 55 kDa 的未知核 L1 片段（L1-55）相互作用，该片段携带泛素残基。接近连接表明 L1-55 与神经元核中的 HP1 同工型密切相关。在用金属蛋白酶抑制剂、β-淀粉样前体蛋白裂解酶（BACE1）或 γ-分泌酶处理神经元后，这种关联减少，表明这些蛋白酶对全长 L1 的裂解生成了 L1-55。通过 siRNA 降低 HP1α、β或 γ 的表达会减少来自培养的皮质神经元的 L1 依赖性神经突生长，并减少划痕试验中 L1 转染的 HEK293 细胞的 L1 依赖性迁移。这些发现表明，新型片段 L1-55 与核中 HP1 同工型的相互作用影响 L1 依赖性功能，例如神经突生长和神经元迁移。
