Investigation of the adaptor protein PLIC-2 in multiple pathways

- Pathway 1: Proteasomal Degradation Pathway - The two terminal domains of PLIC-2 UBA and UBL are involved in this pathway. The structure of UBL has been solved and its interaction with the proteasome has been established. In this paper we demonstrate the role of UBA in the degradation pathway: The two terminal domains UBL and UBA of PLIC-2, weakly interact with each other; the UBA domain interacts with mono-ubiquitin with an affinity that is several orders stronger than for the UBL domain. The binding interface of UBA to UBL or UBQ involves the conserved MGF loop and α3 helix consistent with the interactions seen among the family of its homologous proteins.
- Pathway 2: As an adaptor protein involved in cytoskeletal regulation - PLICs/Ubiquilins were discovered as a Protein Linking IAP (CD47) in the cell membrane to cell Cytoskeleton (PLIC). Here we try to determine the molecular details of this interaction: We tried to analyze this interaction through the cytoplasmic tail of CD47 with the different domains of PLIC-2. No perceptible interactions were observed, both through NMR and ITC. To make the investigation thorough, we also utilized the trans-membrane and cytoplasmic form of CD47 in nanodiscs to identify interactions occurring at the membrane water interface. The results again failed to show any interaction.
- Overall, the results support the role of PLIC-2 in the proteasomal degradation pathway while suggesting that its role in the cytoskeletal regulation pathway as a “PLIC” protein is unlikely.

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.

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