Context-dependent regulation of the human ubiquitin ligase HACE1

• Applicant: Dr. Sonja Gisela Lorenz
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 567443487

Ubiquitination is a pervasive posttranslational modification in eukaryotes that governs myriad signaling pathways by determining protein abundances, lifetimes, interactions, activities, and trafficking. It is thus pivotal for homeostasis that the ubiquitination machinery is specific and stringently controlled. In particular, the specificity-determining ubiquitin ligases (E3s) were shown to be modulated by the dynamic interplay of posttranslational modifications, oligomerization, and interaction partners. A still underexplored layer of regulation is the subcellular localization and associated macromolecular context of E3s. Here, I propose to illuminate this concept structurally and functionally, focusing on the human HECT-type ligase HACE1, which selectively associates with small GTPases of the RAB family (‘RABs’). RABs provide central hubs for membrane trafficking by associating with distinct subcellular membranes and coordinating the biogenesis, localization, and dynamics of organelles and vesicles. In line with this, HACE1 coordinates various aspects of membrane dynamics, such as cell adhesion and migration, autophagy, and the maintenance of the Golgi apparatus. My laboratory has recently uncovered structural principles of regulation and substrate specificity in HACE1, demonstrating that the ligase is autoinhibited by dimerization. Yet, it has remained elusive how the autoinhibition of HACE1 is released at relevant subcellular sites. Our preliminary data suggest that HACE1 can be activated by selective interactions with active, GTP-loaded RABs on liposomes. To understand how membrane-bound RABs program the localization, catalytic activity, and substrate specificity of HACE1 is the overarching goal of this research proposal. Using in-vitro reconstitution approaches, we will first elucidate the requirements of HACE1 recruitment to liposome-bound active RAB1, of the associated ligase stimulation and RAB1 ubiquitination. In particular, we will dissect the significance of two distinct small GTPase binding sites we identified in HACE1 and the influence of membrane lipids. Next, we will determine structures of HACE1-RAB1 complexes on liposomes with and without ubiquitin by cryo-electron tomography, visualizing membrane-associated catalytic HACE1 assemblies. A second line of research is directed at broadly deriving selectivity determinants in the HACE1-RAB interplay on liposomes, aiming to identify mutations that disrupt or enhance interactions of the ligase with specific RABs. Such mutations will provide powerful entry points for cell-based analyses of the RAB-dependent substrate spectrum of HACE1 by quantitative ubiquitinomics. Taken together, our results will illuminate the putative role of HACE1 as a selective RAB effector and provide unprecedented insight into the fundamental question of how ubiquitin ligase specificities can be programmed spatially.

• DFG Programme: Research Grants