BOTTOM-UP RECONSTITUTION AND CHEMICAL MANIPULATION OF LYSOSOMAL SIGNALING
As cell biologists and biochemists, we believe that one cannot truly understand a biological process unless some, or all, of its key features can be recreated in a test tube. This is especially true for lysosomal nutrient signaling, a process of remarkable complexity that involve the precise orchestration of multiple protein complexes, membrane trafficking steps, and metabolite transport rates, all in response to rapidly changing metabolic conditions. In order to progress toward the creation of a ‘synthetic lysosome’, we combine biochemical reconstitution of proteins and vesicles within in vitro systems that we characterize using advanced live microscopy (bulk and single molecule) (link). These systems are informed by structural insights (derived from X-ray crystallography and cryo-EM) that we developed through collaborative work, thus they take into account stoichiometric and topological constraints likely to occur in an intact cell (link and link).
A deep mechanistic understanding of the lysosomal nutrient-sensing system can also inform the design of innovative chemical screening platforms that can enable precise manipulation of nutrient signaling. This is especially critical in the context of mTORC1, given that currently available inhibitors are incomplete (rapamycin) or likely to cause significant toxicities (ATP-competitive). In collaborative work, we have employed covalent chemistry to identify novel mTORC1 inhibitors that operate by targeting the protein complexes that recruit mTORC1 to the lysosomal membrane (link). Ultimately, our reconstitution and chemical screening pipeline may lead to the identification of novel compounds to combat cancer, protein misfolding disease and metabolic syndrome.