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Efficient intracellular degradation of tau in neurons through engineered nuclear transport
Author(s) -
Cho Yongku P.
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.044204
Subject(s) - nuclear transport , ubiquitin , microbiology and biotechnology , nuclear localization sequence , ubiquitin ligase , intracellular , nuclear export signal , protein degradation , nuclear protein , tau protein , hek 293 cells , biology , chemistry , biophysics , cell nucleus , cell culture , cytoplasm , biochemistry , genetics , transcription factor , medicine , disease , pathology , alzheimer's disease , gene
Background Targeted protein degradation is an attractive modality of therapeutic intervention for a wide range of neurodegenerative diseases. Intervention at the protein level is highly desired since pathogenic changes such as conformational change or post‐translational modifications can be directly targeted. Method We designed protein constructs that contain an intracellular binding domain for targeting tau fused to a domain for recruiting the ubiquitination machinery. We expressed these constructs in a cultured human cell line and dissociated cultures of mouse hippocampal neurons expressing human tau. We assessed the degradation of human tau using fluorescence microscopy and by tau quantification using immunoblotting. Result We found that the ubiquitin ligase substrate adaptor speckle‐type POZ protein (SPOP), when fused to camelid‐derived single‐domain antibody fragments (nanobodies), allows efficient degradation of nanobody binding proteins imported to the nucleus. We found that the endogenous SPOP domain contains a strong nuclear localization signal, resulting in nuclear accumulation and aggregation of tau. Based on this we hypothesized that the strong nuclear localization signal overloaded the intracellular protein degradation capacity, disrupting the cellular homeostasis. To test this, we engineered SPOP variants with altered nuclear transport efficiency. The engineered variants altered the degree of tau degradation and nuclear accumulation. Interestingly, this regulated degradation approach resulted in preferential degradation of tau dissociated from the microtubule, leading to nearly exclusive colocalization of tau to the microtubule in mammalian cells and primary mouse hippocampal neurons. Conclusion The new constructs developed here allow fine tuning of nuclear translocation rate and degradation efficiency of tau. This controlled degradation strategy may be beneficial in developing therapeutic approaches for regulated tau degradation without overloading the proteasomal capacity.