Functionality of genetic interaction networks complemented by proteomic and biochemical analyses of the deubiquitinase-sphingolipid axis

Abstract

Deubiquitinating enzymes (DUBs) play a crucial role in various cellular processes via regulating levels of the sorting signal ubiquitin. Sphingolipids are signalling molecules as well as structural components in all eukaryotic cells that are well studied; however, deubiquitinase­dependent sphingolipid regulation is not fully understood. Since dysregulation of the two crucial systems, deubiquination and sphingolipid metabolism, underlie many diseases, I hypothesize that these two systems are highly interactive. My thesis therefore aims to study the genetic complexity of the deubiquination/sphingolipid system using the genetically tractable model organism, Saccharomyces cerevisiae, as an approach to understand deubiquitinase­dependent sphingolipid regulation. In Chapter 2, I showed the connection between deubiquitination and sphingolipids by establishing the synthetic lethality of the proteasomal­ associated deubiquitinase gene UBP6 with myriocin, a drug known to inhibit the first and rate­limiting step in sphingolipid biosynthesis. Myriocin sensitivity of ubp6∆ was dependent on free ubiquitin levels, catalytic activity of Ubp6, as well as dihydrosphingosine and phytosphingosine levels. Thin­layer chromatography analyses revealed specific defects in sphingolipids, phospholipids and neutral lipids associated with myriocin sensitivity of ubp6∆, notably with a deficiency in the signalling sphingolipid sphingosine­1­phosphate. To investigate the genetic basis of these alterations, overexpression of lipid metabolism genes was investigated, which resulted in the identification of the glycerolipid biosynthetic SCT1 gene as a suppressor of myriocin sensitivity specifically in ubp6∆. Fluorescent microscopy analyses were then conducted to determine that myriocin sensitivity of ubp6∆ was associated with increased lipid droplet biogenesis and induction of the unfolded protein response. In Chapter 3, I constructed and analyzed growth of a ubp6∆xxx∆ library in different conditions, resulting in the identification of genetic interactions mediating five phenotypes (synthetic lethal interactions independent of myriocin, synthetic sick interactions independent of myriocin, suppression of myriocin sensitivity independent of genotype, suppression of myriocin sensitivity of ubp6∆, and an exacerbation of myriocin sensitivity of ubp6∆). For each phenotype, a specific genetic interaction network was constructed, communities were determined, and three measurements of network centrality (betweenness, closeness and degree) were measured to identify genes and processes integral to each phenotype. Community analysis of these networks revealed enrichment for suspected biological processes related to proteolysis as well as unsuspected processes such as ergosterol biosynthesis.

To complement the functional genomic analyses in Chapter 3, here in Chapter 4, I conducted functional proteomic analyses that measured protein abundance and localization in response to UBP6­deficiency and/or myriocin treatment. Among all DUBs, only Ubp3 was decreased in abundance in ubp6∆. Pathway analysis of 277 altered proteins revealed myriocin sensitivity of ubp6∆ is mediated via proteins involved in lipid droplet biogenesis, heat stress response, amino acid metabolism and endocytosis. Indeed, mislocalization of endocytic proteins in ubp6∆ was dependent on ubiquitin­dependent endocytosis, ubiquitin ligase expression and catalytic activity of Ubp6. Overall, these results provide insight into the genetic interaction between the Ubp6 deubiquitinase and sphingolipid metabolism. Notably, experimental results validated the predictions from genetic interaction network topological analyses. Key genes and processes provide mechanistic insight into the interconnectedness of major biological functions such as proteasomal activity, lipid metabolism, ER stress, endocytosis and translation machinery mediated by deubiquitinase­sphingolipid axis.