Roberto Zoncu, Ph.D. - US grants

DESCRIPTION (provided by applicant): A cell executes well over 10E8 simultaneous biochemical reactions at hundreds of different locations during every second of its lifetime. Coordinating these innumerable processes is a formidable task that is essential to the correct functioning of each tissue and the entire organism. In response to ever changing internal and external cues, cells have evolved mechanisms that can sense nutrient and energy status and, in response, prompt fine-tuned changes in metabolic activity that buffer these variations. Our recent work has highlighted the role of one organelle, the lysosome, as a gate-keeper of metabolic homeostasis. The lysosome can sense and relay variations in cellular nutrient levels to the master growth regulatory protein kinase mTORC1. In turn, mTORC1 governs catabolic reactions within the lysosome that supply the cell with metabolic building blocks and help maintain global nutrient supply. Importantly, dysregulated lysosomal function is the cause of hereditary metabolic disorders, and is emerging as a contributing factor to the progression of some aggressive cancers. Current technologies that employ mass spectrometers or fluorescent biosensors in whole cells or cell populations cannot reach inside the lysosome to identify and measure the hundreds of metabolites that are generated inside it over time. In order to build a comprehensive, systems-level model of how the lysosome regulates cellular metabolism, a reductionist approach that captures essential spatial and temporal features of lysosomal function in a simplified context is needed. Our goal is to develop a novel in vitro system that wil enable the study of metabolism at the single organelle level. In the current proposal, our system will reconstitute the fusion of lysosomes with organelles known as autophagosomes in test tubes or on the surface of coverslips. Imaging at high spatial and temporal resolution, we will dissect the participation of the lysosome in autophagy, a cellular 'self-eat' process that is essential to the homeostasis of both normal and cancer cells. By scaling up this preparation, and coupling it to high throughput metabolite profiling, we will generate a spatial and temporal 'metabolic map' that profiles hundreds of nutrients generated within the lysosome, describes the time course of their buildup and export, and identifies the transport mechanisms that release these nutrients to the cell. Leveraging the spatial and temporal resolution of our system, we will address a major challenge in present-day cancer research- how highly lethal pancreatic ductal adenocarcinoma (PDAC) exploits autophagy to gain a growth and survival advantage in nutrient-poor microenvironments. Using our 'inside knowledge' of the lysosomal metabolome, we will test the hypothesis that autophagy may allow PDAC cells to maintain homeostasis by tapping into large intracellular reservoirs of nutrients, and we will devise novel strategies to deplete these internal nutrient stores. This project will generate novel tools to illuminate the subcellular organization of metabolism, and lay the foundations for innovative ways to rewire cancer cell metabolism.

PROJECT SUMMARY The molecular mechanisms through which cells sense nutrients remain largely unknown, but their elucidation is key to our understanding of metabolic regulation both in normal and disease states. At the center of nutrient sensing and growth regulation is an ancient protein kinase known as the mechanistic Target of Rapamycin Complex 1 (mTORC1). In response to the combined action of metabolic inputs such as nutrients, growth factors, energy and oxygen, mTORC1 translocates from the cytoplasm to the surface of lysosomes, where its kinase function becomes activated. Accumulating evidence indicates that aberrant mTORC1 activation at the lysosome could be a driving force in diseases ranging from cancer to type-2 diabetes to neurodegeneration. Thus, a deep mechanistic understanding of how mTORC1 is activated in response to nutrients could point the way to novel therapeutic strategies in these diseases. The current proposal investigates a central aspect of mTORC1 function that has so far remained understudied and poorly understood, namely, its ability to sense lipids. We will build on our recent discovery that mTORC1 senses an important lipid, cholesterol, at the lysosome. Using innovative approaches both in cells and in vitro, we will address and elucidate key aspects of newly identified signaling pathway. In particular, we will determine i) the cellular location of the cholesterol pools that regulate mTORC1 ii) the transport circuits that make cholesterol available to mTORC1 and iii) the molecular mechanisms through which cholesterol induces mTORC1 recruitment to the lysosomal surface. Moreover, we will investigate how cholesterol sensing by mTORC1 depends on the Niemann-Pick C1 protein, loss of which causes a fatal metabolic and neurodegenerative disease. We will address these research aims via innovative and highly complementary approaches recently optimized in our lab, including measurement and targeted manipulations of the lipid content of selected organelle populations, combined with reconstitution-based assays of mTORC1 regulation. Our findings will impact the current understanding of the molecular mechanisms of cellular lipid homeostasis, and will point the way to novel approaches to manipulate mTORC1 signaling in disease settings.

PROJECT SUMMARY The molecular mechanisms through which cells sense nutrients remain largely unknown, but their elucidation is key to our understanding of metabolic regulation both in normal and disease states. At the center of nutrient sensing and growth regulation is an ancient protein kinase known as the mechanistic Target of Rapamycin Complex 1 (mTORC1). In response to the combined action of metabolic inputs such as nutrients, growth factors, energy and oxygen, mTORC1 translocates from the cytoplasm to the surface of lysosomes, where it becomes activated. Accumulating evidence indicates that aberrant mTORC1 activation at the lysosome could be a driving force in diseases ranging from cancer to type-2 diabetes to neurodegeneration. Lysosomal translocation and activation of mTORC1 requires the heterodimeric Rag guanosine triphosphatases (GTPases), which together with the pentameric Ragulator complex, form a nutrient-regulated scaffolding complex that physically anchors mTORC1 to the lysosomal surface. Combining dynamic imaging in cells with biochemical reconstitution and structural approaches, we recently discovered that the Ragulator-Rag complex is not static but is rather actively remodeled by nutrients, leading to spatial cycling of the Rag GTPases between the lysosomal surface and the cytoplasm. In turn, Rag cycling places a limit on the efficiency of mTORC1 capture and may facilitate its inactivation when nutrient levels fall. Importantly, Rag cycling is altered by cancer-specific mutations that affect mTORC1 signaling. Based on these findings, we hypothesize that spatial-temporal regulation of mTORC1 scaffolding is a novel and unrecognized mechanism to modulate the potency and selectivity of mTORC1 signaling responses, and that its disruption may drive the aberrant growth of mTORC1-driven cancers, including renal cell carcinoma and lymphoma. We will test this hypothesis via two highly complementary and innovative research aims. First, we will employ structure-guided mutagenesis to dissect the mechanisms that govern the assembly of the mTORC1- scaffolding complex in response to changing nutrient inputs. Second, we will characterize the mechanism of action of new-generation compounds we recently discovered, which block the assembly of the lysosomal mTORC1 scaffolding complex, and determine their ability to inhibit the metabolism and growth of mTORC1- driven cancers. Collectively, the proposed studies will generate new knowledge on the spatial-temporal regulation of mTORC1 signaling, and point the way to novel strategies to manipulate mTORC1 signaling in both normal and disease states.