Current & Upcoming

Cellular membranes are dynamic chemical systems whose lipid composition is tightly regulated across space and time, yet the mechanisms governing lipid metabolism, transport, and function remain incompletely understood. I will describe our eKorts in synthetic lipid biology to develop chemical and optogenetic approaches for interrogating membrane function in living cells. These include activity-based imaging strategies for visualizing lipid metabolism and transport, photoaKinity lipid probes for mapping lipid interactomes and uncovering hidden biological functions, and optogenetic membrane editing technologies that enable rapid and spatially precise manipulation of membrane lipid composition. By integrating these approaches with proximity labeling, chemoproteomics, and advanced imaging, we are beginning to reveal new mechanisms governing lipid homeostasis, inter-organelle communication, and the spatiotemporal organization of membrane metabolism and transport. More broadly, these studies highlight how engineered chemical tools can provide new insight into the architecture and function of cellular membranes.

Alterations in glycoprotein expression and composition are an undisputed corollary of developmental processes, host-pathogen interactions and cancer formation. Consequently, some of the most important tumor biomarkers are heavily glycosylated. Understanding cellular glycoproteome changes is paramount but hampered by experimental limitations. Protein glycosylation is mediated by the activities of >200 glycosyltransferases mainly located in the secretory pathway. Since these transferases are interdependent through compensation and competition, traditional methods of molecular cell biology fail to fully address the complexity of glycoprotein biosynthesis. Furthermore, workflows in mass spec-glycoproteome analysis are often restricted to isolated cell lines that do not adequately reflect the interactions within tissues or between tumor and microenvironment. Thus, we lack strategies to understand 1) the protein substrate specificities of individual glycosyltransferases and 2) which glycoproteins are made by cells in response to their microenvironment. We also 3) miss chemical probes to investigate and disrupt cancer-relevant glycosylation. Here, I describe our development of chemical “Precision Tools” to dissect cellular glycosylation. We employ bump-and-hole (BH) engineering to render glycosyltransferases receptive to a chemically modified nucleotide-sugar substrate that carries a bioorthogonal tag and is not used by wildtype transferases. Engineering individual transferases allows differential profiling of their protein substrate specificities. We found that establishing cellular BH systems required innovation in the delivery of corresponding nucleotide-sugarsto the secretory pathway. We have also taken initiative in the development of small molecule inhibitors against cancer-relevant glycosylation enzymes. Thus, chemical Precision Tools allow us to profile protein glycosylation as a key player in cancer biology.

TBA