The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that becomes activated at the lysosome in response to nutrient cues. Here we identify cholesterol, an essential building block for cellular growth, as a nutrient input that drives mTORC1 recruitment and activation at the lysosomal surface. The lysosomal transmembrane protein, SLC38A9, is required for mTORC1 activation by cholesterol through conserved cholesterol-responsive motifs. Moreover, SLC38A9 enables mTORC1 activation by cholesterol independently from its arginine sensing function. Conversely, the Niemann-Pick C1 (NPC1) protein, which regulates cholesterol export from the lysosome, binds to SLC38A9 and inhibits mTORC1 signaling through its sterol transport function. Thus, lysosomal cholesterol drives mTORC1 activation and growth signaling through the SLC38A9-NPC1 complex.

The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagAGDP:RagCGTP GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo–electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling.

Precise regulation of lipid biosynthesis, transport, and storage is key to the homeostasis of cells and organisms. Cells rely on a sophisticated but poorly understood network of vesicular and non-vesicular transport mechanisms to ensure efficient delivery of lipids to target organelles. The lysosome stands at the crossroads of this network due to its ability to process and sort exogenous and endogenous lipids. The lipid sorting function of the lysosome is intimately connected to its recently discovered role as a metabolic command-and-control center, which relays multiple nutrient cues to the master growth regulator, mechanistic Target of Rapamycin Complex 1 (mTORC1) kinase. In turn, mTORC1 potently drives anabolic processes, including de novo lipid synthesis, while inhibiting lipid catabolism. Here we describe the dual role of the lysosome in lipid transport and biogenesis, and we discuss how integration of these two processes may play important roles both in normal physiology and in disease.

Autophagy is a conserved eukaryotic pathway critical for cellular adaptation to changes in nutrition levels and stress. The class III phosphatidylinositol (PI)3-kinase complexes I and II (PI3KC3-C1 and -C2) are essential for autophagosome initiation and maturation, respectively, from highly curved vesicles. We used a cell-free reaction that reproduces a key autophagy initiation step, LC3 lipidation, as a biochemical readout to probe the role of autophagy-related gene (ATG)14, a PI3KC3-C1-specific subunit implicated in targeting the complex to autophagy initiation sites. We reconstituted LC3 lipidation with recombinant PI3KC3-C1, -C2, or various mutant derivatives added to extracts derived from a CRISPR/Cas9-generated ATG14-knockout cell line. Both complexes C1 and C2 require the C-terminal helix of VPS34 for activity on highly curved membranes. However, only complex C1 supports LC3 lipidation through the curvature-targeting amphipathic lipid packing sensor (ALPS) motif of ATG14. Furthermore, the ALPS motif and VPS34 catalytic activity are required for downstream recruitment of WD-repeat domain phosphoinositide-interacting protein (WIPI)2, a protein that binds phosphatidylinositol 3-phosphate and its product phosphatidylinositol 3, 5-bisphosphate, and a WIPI-binding protein, ATG2A, but do not affect membrane association of ATG3 and ATG16L1, enzymes contributing directly to LC3 lipidation. These data reveal the nuanced role of the ATG14 ALPS in membrane curvature sensing, suggesting that the ALPS has additional roles in supporting LC3 lipidation.

Background: UDP-glucose dehydrogenase (UGDH) mutants were engineered to perturb hexamer:dimer quaternary structure equilibrium. Results: Dimeric species of UGDH have reduced activity in vitro and in supporting hyaluronan production by cultured cells. Conclusion: Only dynamic UGDH hexamers support robust cellular function. Significance: Manipulation of UGDH activity by hexamer stabilization may offer new therapeutic options in cancer and other pathologies.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD+-dependent oxidation steps. Human UGDH apoprotein purifies as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking was achieved by encoding p-benzoyl-L-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing to long wavelength UV in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% tetramer and higher molecular mass species. Activity of the crosslinked enzyme was reduced by almost 60% relative to the uncrosslinked UGDH mutant, and UV exposure had no effect on activity of the wildtype enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.

T cell therapy is a promising immunotherapy treatment option that uses genetically modified immune cells (T cells) to eliminate tumors. This approach uses the patients’ own immune cells to generate a “living drug” and can avoid the toxic side effects of other common therapies, such as radiation or chemotherapies. However, the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) can establish several obstacles to protect the tumor from T cells, including delivery of inhibitory and death signals that shut down T cells, usurping metabolic nutrients, and recruiting and/or converting immune cells to inhibitory phenotypes that block the T cell response. CD47 is upregulated in PDAC tumors, and patients with CD47hi tumors exhibit worse survival. CD47 serves as a “don’t eat me” signal by binding to the SIRPα receptor on macrophages to block phagocytosis. CD47 expression also inhibits T cell-mediated tumor rejection by blocking cross-presentation of tumor antigens by SIRPα+ dendritic cells. Reagents that interfere with SIRPα-CD47 signaling, such as monoclonal antibodies, are currently in clinical trials and have been shown to synergize with chemotherapy in PDA models, but native CD47 expression can produce erythrocyte and platelet depletion. Costimulatory signals delivered by surface-bound receptors can initiate gene expression programs that address multiple issues in the PDAC TME by mechanisms such as lowering the threshold of activation, altering metabolic programming, and reducing exhaustion. We develop engineering strategies to deliver costimulatory signals to engineered T cells. Immunomodulatory fusion proteins (IFPs) combine the ectodomain of an inhibitory T cell receptor with an intracellular costimulatory signaling domain and we have shown in proof-of-concept studies that this effectively “replaces a brake with an accelerator” in CD8 T cells. We have found that generating IFPs is not as simple as indiscriminately fusing any two proteins together and we have developed critical design concepts that inform the selection of the fusion site, ectodomain and costimulatory signaling domain pairing, and immunological synapse localization to enhance signaling. To develop a CD47-targeted IFP, we combined the SIRPa ectodomain with the CD28 signaling endodomain and tested this technology with our mesothelin-targeted TCR-T cell platform. In in vitrostudies, SIRPa-CD28 T cells exhibited enhanced proliferation, accumulation, and tumor cytotoxicity. SIRPa IFP-T cells exhibited increased intratumoral accumulation and therapeutic efficacy in the KrasLSL-G12D/+; Trp53LSL-R172H/+;p48Cre/+ (KPC) model, without toxicity or erythrocyte depletion. Here we describe how a thoughtfully engineered fusion protein can “armor” T cells against multiple obstacles and significantly improve therapeutic efficacy against PDAC. Citation Format: Shannon Oda, Leah Schmidt, Ashley Thelen, Cody Jenkins, Edison Chiu, Aitong Ruan, Philip Greenberg. Overcoming PDAC T cell therapy barriers with CD47-targeted costimulatory fusion proteins [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR008.