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

Abstract

The brain-expressed ubiquilins, UBQLNs 1, 2 and 4, are highly homologous proteins that participate in multiple aspects of protein homeostasis and are implicated in neurodegenerative diseases. Studies have established that UBQLN2 forms liquid-like condensates and accumulates in pathogenic aggregates, much like other proteins linked to neurodegenerative diseases. However, the relative condensate and aggregate formation of the three brain-expressed ubiquilins is unknown. We report that the three ubiquilins differ in aggregation propensity, revealed by in-vitro experiments, cellular models, and analysis of human brain tissue. UBQLN4 displays heightened aggregation propensity over the other ubiquilins and, like amyloids, UBQLN4 forms ThioflavinT-positive fibrils in vitro. Measuring fluorescence recovery after photobleaching (FRAP) of puncta in cells, we report that all three ubiquilins undergo liquid-liquid phase transition. UBQLN2 and 4 exhibit slower recovery than UBQLN1, suggesting the condensates formed by the brain-expressed ubiquilins have different compositions and undergo distinct internal rearrangements. We conclude that while all brain-expressed ubiquilins exhibit self-association behavior manifesting as condensates, they follow distinct courses of phase-separation and levels of aggregation. This variability among ubiquilins along the continuum from liquid-like to solid likely informs both the normal ubiquitin-linked functions of ubiquilins and their accumulation and potential contribution to toxicity in various neurodegenerative diseases.

Disrupting the Balance of Protein Quality Control Protein UBQLN2 Accelerates Tau Proteinopathy

“…To further determine the physiological relevance of UBQLN2 to tau homeostasis, we extended our analysis to tau transgenic mouse models. The intrinsic property of UBQLN2 to aggregate (Hjerpe et al, 2016;Sharkey et al, 2018;Gerson et al, 2021), combined with our results showing that UBQLN2 knockdown increased levels of wild-type tau, suggested that UBQLN2 aggregation might promote tau pathology. We also sought to determine whether tau pathology, in turn, could contribute to a cellular environment that enhances the tendency of UBQLN2 to aggregate and hence become dysfunctional.…”

“…In addition to sequestering other aggregating proteins, UBQLN2 itself is prone to aggregate and undergo liquid-liquid phase separation (LLPS) in vitro (Hjerpe et al, 2016;Dao et al, 2018;Sharkey et al, 2018). While both UBQLN1 and UBQLN4 share the ability to undergo LLPS with UBQLN2, the three UBQLN proteins diverge in liquid droplet characteristics and aggregation behavior, implying that, despite their similarity, functional differences may exist (Gerson et al, 2021).…”

Mechanistic insights into the enhancement or inhibition of phase separation by polyubiquitin chains of different lengths or linkages

“…Interestingly, pulldowns of K48-and K63-linked chains using GST-tagged UBQLN1, a close UBQLN2 homolog that also undergoes LLPS (Gerson et al, 2021), show that FL UBQLN1…”

“…Interestingly, pulldowns of K48- and K63-linked chains using GST-tagged UBQLN1, a close UBQLN2 homolog that also undergoes LLPS (Gerson et al, 2021), show that FL UBQLN1 only interacts with longer K63-linked chains (Harman and Monteiro, 2019). However, UBQLN1 and UBQLN2 UBAs have no significant preference for either chain (Fig.…”

RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control

“…The copyright holder for this preprint (which this version posted April 22, 2021. ; https://doi.org/10.1101/2021.04.21.440788 doi: bioRxiv preprint RTL8 binding to and nuclear recruitment of UBQLN2, RTL8 binding to UBQLN2 could modulate ubiquitin binding by UBQLN2. Intriguingly, all three UBQLNs undergo liquid-liquid-phase separation and are inherently aggregate-prone [52], and the UBA domain itself may seed UBQLN oligomerization and aggregation [22]. A systematic study of structural motifs and disease mutations in UBQLN2 will be required to determine which regions are critical for binding and whether fALS mutations impact RTL8 binding or RTL8-dependent properties of UBQLN2.…”