Investigation of the adaptor protein PLIC-2 in multiple pathways

Abstract

PLIC, Protein Linking IAP (CD47) to Cytoskeleton, have long since been implicated in connecting the extracellular membrane to the intracellular cell cytoskeleton. This phenomenon is supposedly achieved by bridging a receptor protein CD47 to vimentin, an intermediate filament, which in turn regulates integrin dependent cell spreading. Since the discovery of these proteins, the molecular details of the above-mentioned interactions and the underlying complexes are yet to be characterized. Several independent studies have together emphasized PLIC/Ubiquilin’s role in the proteasomal degradation pathway. This seems to be in contrast to the purported initial discovery of PLIC as a cytoskeletal adaptor protein. In an effort to reconcile the different roles associated with the ubiquitous PLIC proteins, we tested the involvement of PLIC-2 both in the proteasomal degradation pathway and as a protein linking the cell cytoskeleton to the cytoplasmic tail of CD47. This was achieved thorough an in vitro investigation of their binding interface using a combination of biophysical techniques. Our results show that the two terminal domains of PLIC-2 interact weakly with each other, while the C-terminal UBA domain interacts strongly with ubiquitin. Interestingly, no perceptible interaction was observed for PLIC-2 with the cytoplasmic tail of CD47 questioning its role as a “PLIC” protein linking the cell membrane to the cytoskeleton.

Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions

“…One major difference between FL and 379–624 constructs is the absence of the UBL domain in the latter. The UBL and UBA domains interact weakly (with a Kd of ~ 175 μM) (Nguyen et al, 2017). Removing the UBA-UBL interactions in UBQLN2 379–624 could enable the UBA domain to fully participate in promoting LLPS.…”

“…Mapping of the Ub binding site on UBQLN2 indicated that Ub binds to one face of the UBA domain (Figure 6B), with the largest CSPs observed for residues 592–594 and 610–620, the same residues exhibiting concentration-dependent CSPs in the absence of Ub (Figures 4D and4E). Interestingly, the UBL domain also binds the UBA domain at identical sites where Ub binds, albeit much more weakly (Nguyen et al, 2017). Therefore, we suspect that Ub binding to UBQLN2 eliminates at least two multivalent interaction sites, i.e., residues 592–594 and 610– 620, thus reducing the propensity for UBQLN2 to oligomerize and phase separate in the presence of Ub.…”

Ubiquitin‐Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?

“…We hypothesize that UBL effects on LLPS are mediated via weak UBA‐UBL interactions with a binding affinity around 175 µ m . [ 58 ] Therefore, we suggest that the STI1‐II is the main driver of UBQLN2 oligomerization and LLPS with further modulation by the PXX region and UBL and UBA domains. [ 59–61 ]…”

Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

“…It is well-established that UBL and UBA domains of Ub-binding shuttle proteins interact with each other on an intra- and intermolecular basis (Lee et al, 2013; Lowe et al, 2006; Nguyen et al, 2017). To validate this within our UBQLN2 constructs, we monitored the interactions between the isolated UBA and UBL domains of UBQLN2 using NMR spectroscopy (Figure 2), similar to the experiments employed in (Nguyen et al, 2017). We used UBQLN2 1-107 and UBQLN2 571-624 as our UBL and UBA constructs, respectively, with a C-terminal His-tag (see Methods and Table S1).…”

“…Ubiquitin-binding to the UBA significantly reduces the driving forces for UBQLN2 phase separation by decreasing the number of multivalent “stickers” needed for LLPS, thus driving the disassembly of UBQLN2 condensates in vitro . Importantly, it is well-established that UBL and UBA domains interact with each other (Lowe et al, 2006; Nguyen et al, 2017). How interplay among these folded domains and the middle region of UBQLN2 mediates phase separation remains poorly understood.…”