Tales Of Phosphoinositide-Binding Enzymes: Unraveling The Physiological Role Of Human Sac2 And Legionella Effectors, Sidf & Sidc
Phosphoinositides (PIs), a group of key signaling and structural lipids, are involved in a myriad of biological processes of cell physiology. Their intracellular concentration and localization must be tightly regulated for proper cell function and survival. This is achieved by specific PI phosphatases and kinases, which are responsible for the spatial and temporal conversion of distinct phosphorylated species. Mutations in several of these metabolic enzymes have been found in a range of neurological diseases, and human cancers, further underscoring the crucial role of PIs. In recent years, it has also become evident that many intracellular pathogens have exploited host PIs for their pathogenesis by secreting eukaryotic-like factors such as PI phosphatases or PIbinding effector proteins. Thus, elucidating the role of mammalian/bacterial PI phosphatases and PI-binding effectors is paramount to the understanding of the host and host-bacterial signaling mechanism. The first part of the dissertation focuses on the structure, function and disease-associated mutations of mammalian PI phosphatases with emphasis on a family of enzymes containing a conserved enzymatic domain called Sac. We determined that one of the Sac domain-containing proteins, Sac2, is a PI 4-phosphatase that is highly expressed in the brain. Using overexpressed and CRISPR knockout Sac2 neuronal cell lines, we found that Sac2 is an important regulator in the endocytic recycling of surface receptors such as transferrin and integrin receptors, and plays an essential role in maintaining cell migration in neurons. This work uncovered a previously unidentified role of Sac2 and implicated Sac2 in neuronal functions and brain development. The second part of this dissertation discusses the role of bacterial PI phosphatases in infec- tion, and investigates in detail the role of an effector called SidF from the intracellular pathogen Legionella pneumophila. We found that SidF is a PI 3-phosphatase that specifically hydrolyzes PI(3,4)P2 and PI(3,4,5)P3 to PI(4)P and PI(4,5)P2, respectively. We solved the crystal structure of SidF in complex with its substrate, PI(3,4)P2 and revealed the structural basis for substrate recognition. Finally, we demonstrated that the catalytic activity of SidF is important for sculpting the lipid composition of the Legionella-containing vacuole (LCV) during infection. Our work revealed the first PI phosphatase encoded by Legionella pneumophila and elucidated its function during infection. In addition, the second half of Part II addresses specifically the function of the Legionella effector SidC in vitro and in vivo, and how the PI(4)P-binding property of SidC facilitates the LCV maturation process. Unexpectedly, we discovered that SidC is an E3 ubiquitin ligase that uses a cysteine in the catalytic triad to generate polyubiquitin chains. The enzymatic activity is necessary for recruiting ubiquitin conjugates and host ER vesicles to the LCV. Our findings illustrated a powerful strategy employed by the Legionella, and potentially other intracellular pathogens, to simultaneously utilize both the host PI and ubiquitination system for their pathogenesis. The final chapter summarizes our current understanding of both the mammalian/bacterial PI phosphatases and PI-binding effectors in Legionella, and explores future directions in further expanding our knowledge on host signaling and bacterial infection mechanisms.
Feigenson,Gerald W; Cerione,Richard A; Emr,Scott David
Molecular & Cell Biology
Ph.D. of Molecular & Cell Biology
Doctor of Philosophy
dissertation or thesis