Unravelling of Hidden Secrets: The Tumour Suppressor Lethal (2) Giant Discs (Lgd)/CC2D1, Notch Signalling and Cancer.

The endosomal pathway plays a pivotal role upon signal transduction in the Notch pathway. Recent work on lethal (2) giant discs (lgd) points to an additional critical role in avoiding uncontrolled ligand-independent signalling during trafficking of the Notch receptor through the endosomal pathway to the lysosome for degradation. In this chapter, we will outline the journey of Notch through the endosomal system and present an overview of the current knowledge about Lgd and its mammalian orthologs Lgd1/CC2D1b and Lgd2/CC2D1a. We will then discuss how Notch is activated in the absence of lgd function in Drosophila and ask whether there is evidence that a similar ligand-independent activation of the Notch pathway can also happen in mammals if the orthologs are inactivated.

Structural Analysis of the ESCRT-III Regulator Lethal(2) Giant Discs/Coiled-Coil and C2 Domain-Containing Protein 1 (Lgd/CC2D1).

Members of the LGD/CC2D1 protein family contain repeats of the family-defining DM14 domains. Via this domain, they interact with members of the CHMP family, which are essential for the ESCRT machinery-mediated formation of intraluminal vesicles during endosome maturation. Here, we investigate the requirement of the DM14 domains for the function of Lgd in detail. We found that although both odd-numbered DM14s can act in a functionally redundant manner, the redundancy is not complete and both contribute to the full function of Lgd. Our analysis indicates that some of the AAs that form the KARRxxR motif of the onDM14s are not exchangeable by similarly charged AAs without loss of function, indicating that they not only provide charge, but also fulfil structural roles. Furthermore, we show that the region of Lgd between DM14-4 and the C2 domain as well as its C-terminal region to the C2 domain are important for protein stability/function. Moreover, we analysed the importance of AAs that are conserved in all DM14 domains. Finally, our analysis of the C. elegans ortholog of Lgd revealed that it has only one DM14 domain that is functionally equivalent to the onDM14s. Altogether, the results further the understanding of how Lgd family members regulate the ESCRT machinery.

Structural Basis for Regulation of ESCRT-III Complexes by Lgd.

The ESCRT-III complex induces outward membrane budding and fission through homotypic polymerization of its core component Shrub/CHMP4B. Shrub activity is regulated by its direct interaction with a protein called Lgd in flies, or CC2D1A or B in humans. Here, we report the structural basis for this interaction and propose a mechanism for regulation of polymer assembly. The isolated third DM14 repeat of Lgd binds Shrub, and an Lgd fragment containing only this DM14 repeat and its C-terminal C2 domain is sufficient for in vivo function. The DM14 domain forms a helical hairpin with a conserved, positively charged tip, that, in the structure of a DM14 domain-Shrub complex, occupies a negatively charged surface of Shrub that is otherwise used for homopolymerization. Lgd mutations at this interface disrupt its function in flies, confirming functional importance. Together, these data argue that Lgd regulates ESCRT activity by controlling access to the Shrub self-assembly surface.

Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins.