The structure-function relationship of the ARISC complexes

The JAMM/MPN+ protein BRCA1-containing complex subunit 36 (BRCC36) forms two distinct complexes in the cell: the cytoplasmic BRISC-SHMT2 complex, and the related ARISC-RAP80 complex in the nucleus. The BRISC-SHMT2 complex plays a vital role in interferon signalling whereas the ARISC-RAP80 complex regulates DNA damage and repair. Both complexes contain shared subunits (BRCC36, BRCC45 and MERIT40) but differ in their targeting proteins (SHMT2 and RAP80, respectively) and in the MPN- domain containing proteins (Abraxas2 and Abraxas1, respectively). As an isolated protein, BRCC36 is catalytically inactive and partnering with Abraxas2 results in a BRCC36-Abraxas2 minimally active complex. Abraxas2 supports the activity of BRCC36 through direct interactions by inducing a conformational change within the MPN+ domain of BRCC36 and its active site. Additionally, BRCC36 and Abraxas form a super dimer that is required for deubiquitylase activity and assembly of higher order BRISC and ARISC complexes. The main goal of this thesis is to understand how higher order ARISC complexes assemble and how this affects DUB activity and interactions with the targeting subunit RAP80. Shortly after the start of this project, a low resolution model of the four membered ARISC complex was published. The model revealed that the complex forms a U-shape architecture, which consists of the BRCC36-Abraxas1 super dimer at the base of the U and BRCC45-MERIT40 at the arms, suggesting a 2:2:2:2 stoichiometry. In this thesis, the structure of the ARISC complexes (ARISC-RAP80 and ARISC) were explored by negative stain and cryo electron microscopy. These structural analyses revealed that the complex displays conformational heterogeneity and exists in higher ordered (ARISC dimer of octamer) and subcomplexes. These results were further supported by mass spectrometry and native PAGE analysis. Negative stain and cryo electron microscopy analysis of the complexes also indicate that at ARISC-RAP80 complex stability was concentration dependent. This phenomenon was also true for the related BRISC-SHMT2 cytoplasmic complex after cryo electron microscopy data was collected at protein concentrations higher than usual and in a molecular crowded environment. Results showed that despite particle overcrowding, data processing was possible and resulted in reduced particle heterogeneity. In addition, preliminary interactions studies to elucidate the function of RAP80 protein revealed that RAP80 is the major interacting subunit for ubiquitin the major contributor for ARISC-RAP80 complex ability to interact with DNA. These results provide a platform for future structural and mechanistic studies into the function of the ARISC-RAP80 complex in the nucleus.