Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly

Abstract

UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein’s ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2’s role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.

Global proteomics ofUbqln2-based murine models of ALS

“…For example, UBQLN4 possesses a non-canonical LC3-binding motif (Lee et al, 2013) which is thought to confer a unique ability of the Ubqln family to promote autophagic protein degradation (N'Diaye et al, 2009a;2009b;Rothenberg et al, 2010;Şentürk et al, 2019). In addition, UBQLN2 contains a proline-rich repeat (the 'PXX-domain'), which may regulate client specificity (Gilpin et al, 2015), proteasome binding , and liquid-liquid phase separation into stress granules (Alexander et al, 2018;Dao et al, 2018;Sharkey et al, 2018). Mutations in UBQLN2 cause a heritable, X-linked, dominant form of ALS with frontotemporal dementia (fALS/FTD) in humans . This discovery has been corroborated through the use of mutant transgenic mouse models that mimic in vivo symptoms of motor neuron disease and exhibit protein aggregates in the brain Hjerpe et al, 2016).…”

“…Furthermore, UBQLN2 colocalizes with TDP-43 inclusions of familial ALS not only when UBQLN2 is mutated , but also in familial ALS characterized by an ALSassociated FUS mutation (Williams et al, 2012) or mutant TDP-43 overexpression . Like other Ubqlns, UBQLN2 oligomerizes (Ford and Monteiro, 2006) and is capable of phase separation which could potentially contribute to disease by influencing protein degradation pathways and even nucleating protein aggregation within the cell (Alexander et al, 2018;Dao et al, 2018;Sharkey et al, 2018). Thus, perturbation of UBQLN2 function may promote neurodegeneration by compromising the proteasomal degradation of proteins known to cause ALS, such as TDP-43.…”

Shared and divergent phase separation and aggregation properties of brain-expressed ubiquilins

“…Like many other proteins associated with neurodegenerative diseases [13][14][15][16][17][18] , UBQLN2 spontaneously phase separates to form condensates, or liquid droplets, in which proteins are concentrated yet remain mobile 13,19,20 . Liquid-like condensates are tightly regulated by cells and can provide sites for specific cellular functions [21][22][23][24] .…”

Uncovering active modulators of native macroautophagy through novel high-content screens

“…Automated stage movements, filter turret rotation, and image acquisition were controlled via µManager with original code written in BeanShell. In-house software was used to assign a barcode for each neuron, measure its fluorescent intensity, and register time of death as described previously 61, 62, 63, 98, 99 . For optical pulse labeling experiments a 1.5s pulse of 405nm light was used for photoconversion.…”