Mutations and substrates of the E3 ubiquitin ligase adaptor Kelch-like 15 and link to neurodevelopmental disorders
Year: 2020
Type: dissertation
Abstract: Intellectual disability (ID) is a prevalent and devastating neurodevelopmental disorder (NDD) with high genetic heterogeneity. Widespread application of next-generation sequencing (NGS) has fostered our understanding of the molecular underpinning of ID, especially X-linked forms (XLID). In this study, we identified 18 affected males from 11 unrelated XLID families carrying de novo and maternally inherited mutations in KLHL15 gene. The encoded protein, Kelch-like 15 (KLHL15), is a substrate adaptor for cullin3 (Cul3)-based E3 ubiquitin ligase that selectively targets the brain-enriched B'beta regulatory subunit of protein phosphatase 2A (PP2A/B'beta) for ubiquitination and proteasomal degradation. Functional assessment characterizes both loss-of-function (LoF) and gain-of-function (GoF) mutations with regard to the regulation of B'beta via ubiquitin-proteasome system (UPS). Structural analysis reveals disturbance in the E3 ligase complex by both LoF and GoF mutations. Taken together, structural impairment and functional alteration point to a dysregulated machinery explaining KLHL15 pathogenicity.In an effort to explore PP2A-independent regulation of intracellular signaling by KLHL15, a sequence and structural search identifies a family of neuronal microtubule-associated proteins (MAPs) as KLHL15 substrates. Biochemical validation confirms doublecortin (DCX), an X-linked disease gene, and doublecortin-like kinase 1 and 2 (DCLK1/2) are bona fide KLHL15 interactors and KLHL15 docking regions are mapped to their microtubule-binding DCX-domain tandems. A FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and DCLK1/2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 stabilizes DCX proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of wild-type DCX reduces dendritic complexity, while a stabilizing mutation (Y259L) fails to counteract KLHL15, demonstrating the role of FRY motif in finetuning neuronal dendritogenesis. Collectively, our findings (1) offer insight into the possible link of perturbed homeostatic regulation of PP2A/B'beta by KLHL15 mutations and pathogenesis of XLID; and (2) highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal MAPs and identify a regulatory network important for development of the mammalian nervous system.
DOI: 10.17077/etd.005732
Authors: Jianing Song, Frederick Quelle, Hanna Stevens, Robert Piper, Stefan Strack, Yuriy M Usachev
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4238367762