Publication
Proteins are fundamental biomolecules in living organisms, performing diverse functions through intricate interactions with their environment. Understanding these interactions is crucial not only for elucidating normal biological processes, but also for unraveling the complexities of various diseases. Integrative structural biology emerges as a powerful method for studying these complex biomolecular interactions by combining techniques from structural biology, biochemistry, and computational modeling. In this thesis, we employ an integrative structural biology approach to investigate two proteins associated with human diseases: GOLPH3 and PREPL. The first part of this thesis focuses on GOLPH3, a key protein within the Golgi apparatus that orchestrates the sorting of Golgi resident enzymes into retrograde-bound COPI vesicles during cisternal maturation. Our research elucidates the precise molecular mechanisms underlying GOLPH3-mediated Golgi retention. We reveal that GOLPH3 engages with the cytosolic tails of its clients Golgi resident glycosylation enzymes through a negatively charged surface. We further elucidate the complex nature of GOLPH3's interaction with the Golgi membrane, which involves both non-specific and specific lipid interactions, as well as a newly discovered post-translational modification, S-acylation. Additionally, our study reveals that novel mutations in GOLPH3 identified in patients, compromise protein stability and function, suggesting its potential disease implications beyond its known oncogenic roles. We also explore therapeutic strategies aimed at disrupting critical GOLPH3 interactions to mitigate its overexpression and associated oncogenic pathways. In the second part, we investigate PREPL, a protein whose genetic alterations are linked to Congenital Myasthenic Syndrome-22 (CMS22), a rare genetic disorder. While previous studies have documented deletions and nonsense variants in PREPL, the impact of missense variants on CMS22 pathology remains unexplored. Our research focuses on characterizing missense variants in three CMS22 patients, each exhibiting characteristic clinical features of the disorder. Biochemical characterization of the mutants demonstrates that these missense variants do not impair the enzyme's hydrolase activity. Structural analyses reveal that these variants affect regions likely involved in intra-protein or protein-protein interactions, with differential impacts on binding to known interactors. Additionally, we investigate the non-hydrolytic functions of PREPL using catalytically inactive PREPL p.Ser559Ala cell lines and we reveal that while hydrolase activity is crucial for mitochondrial function, it is not essential for AP1-mediated transport within the trans-Golgi network. Finally, we propose a new model for the physiological function of PREPL, encompassing both its catalytic and non-catalytic functions. Altogether, this dissertation provides exemplary case studies demonstrating how integrative s