Modulation of Proteostasis for the Efficient Intracellular Transport of the ΔF508 Mutant of Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) ΔF508 mutant (ΔF508CFTR) contributes to 70% cystic fibrosis cases, and it undergoes aberrant proteostasis, misfolding, intracellular retention and degradation. Targeting ΔF508CFTR proteostasis components has been successful in the partial rescue of ΔF508CFTR at the plasma membrane. Drug screening has identified correctors which rescue a fraction of the ΔF508CFTR at the plasma membrane. However, all these correctors are marginally effective and are not therapeutically viable. This thesis project used a systems-biology-based meta-analysis approach of gene expression induced by corrector drugs to infer their mechanisms of action, and this led to the identification of a group of genes that are commonly regulated by many of these drugs. These groups of genes were used to determine the networks/ pathways/ molecules that might be correlated with their correction. These include components of RNA processing, the cell cycle, ubiquitin ligases, and kinases, many of which have led to partial rescue of ΔF508CFTR when they have been depleted by RNA interference. Furthermore, two of these pathways are characterised here: the MAP3K11-JNK cascade and the CaM kinase (CAMKK2) cascade. Several members of these cascades rescued ΔF508CFTR. The MAP3K11-initiated pathway has a role in ER-associated degradation and plasma-membrane stability of ΔF508CFTR. MAP3K11 also appears to link oxidative stress and inflammation to intracellular proteostasis of ΔF508CFTR. Some upstream activators and downstream targets of MAP3K11 can also rescue ΔF508CFTR. Drugs that inhibit the MAP3K11-JNK cascade rescued functional ΔF508CFTR at the plasma membrane. Combinations of these drugs with the previously clinically studied corrector pharmacochaperone VX-809 led to a high level of correction of ΔF508CFTR, which was much greater than for VX-809 alone. Many candidate genes and drugs that are identified in the current study open the way to the development of the new efficient therapeutic agents for cystic fibrosis caused by the ΔF508 mutation

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

Phosphorylation of the N‐terminal domain of the huntingtin (HTT ) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD ) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK ‐binding kinase 1 (TBK 1), that efficiently phosphorylates full‐length and N‐terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo . TBK 1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans ) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK 1‐mediated neuroprotective effects are due to phosphorylation‐dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT . These findings suggest that upregulation and/or activation of TBK 1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

Phosphorylation of the N-terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that efficiently phosphorylates full-length and N-terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation