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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2019)

The decision of whether to allocate resources toward cellular growth or toward quality control is a matter of cellular life and death; disruption of growth pathways is an emerging driving force in diseases ranging from cancer to neurodegeneration. In mammalian cells, the protein kinase activity of mTORC1 promotes cellular anabolism and impedes cellular catabolism, ultimately achieving a balance that dictates the rate of cell growth. In response to nutrient levels, mTORC1 is activated upon recruitment to the lysosome, an organelle whose role as a nutrient sensing integrator has recently come into focus. The Rag GTPases are required for mTORC1 recruitment to the lysosome, but the mechanisms via which the Rags sense nutrients and precisely couple the degree of mTORC1 lysosomal recruitment levels to the level of available nutrients were unknown.

I report new live-imaging and reconstitution approaches that enabled the discovery that when the Rags transition from their inactive nucleotide binding state to their active nucleotide state in response to nutrient stimulation, they also loosen their binding affinity for their lysosomal scaffold, Ragulator. The resulting spatial cycling between the lysosome and the cytoplasm ultimately limits mTORC1 accumulation on its Rag-Ragulator lysosomal scaffold, and promotes rapid responsiveness of mTORC1.

Next, I asked whether the nucleotide states of the two Rag GTPase domains are coordinated. Prior work had established that a complex of Folliculin (FLCN) and FLCN-interacting protein 2 (FLCN:FNIP2) serves as a RagC-specific GTPase-activating protein (GAP) and thus has a positive role in mTORC1 stimulation. However, genetic evidence placed FLCN as a tumor suppressor, suggesting a negative role. I reconstituted the “Lysosomal Folliculin Complex” (LFC), a supercomplex composed of Ragulator, inactive-loaded Rags, and FLCN:FNIP2 that localizes to lysosomes. I discovered that in the LFC, FLCN:FNIP2 clamps Rags in their inactive state (RagAGDP:RagCGTP) by directly inhibiting nucleotide exchange in RagA, concomitant with inhibition of its RagC GAP activity, a conclusion reinforced by a high-resolution (3.6 Å) structure of the LFC. Thus, when nutrients are low, FLCN:FNIP2 is able to maintain the Rag heterodimer in its inactive state, but, in response to a rise in nutrients, FLCN:FNIP2 is converted into a functional GAP.

Finally, by assessing newly available structures of active nucleotide-bound and inactive nucleotide-bound Rag heterodimers, along with recent structural information about Rag interactors, I was able to assemble an integrated structure-guided model of the Rag-mediated cycle of recruitment and activation of mTORC1 at the lysosome. My findings increase our understanding of the molecular logic of nutrient sensing and point to new opportunities for manipulating mTORC1 signaling in disease contexts.

    Cover page: Mechanisms of mTORC1 signal regulation by the Rag GTPases
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2020)
    The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2022)
      In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.
        Cover page: A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2021)
        Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2's dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five 'aromatic fingers'. NSs binding preserves eIF2B activity by maintaining eIF2B's conformation in its active A-State.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2018)
          A key step in nutrient sensing is activation of the master growth regulator, mTORC1 kinase, on the lysosomal membrane. Nutrients enable mTORC1 scaffolding by a complex composed of the Rag GTPases (Rags) and Ragulator, but the underlying mechanism of mTORC1 capture is poorly understood. Combining dynamic imaging in cells and reconstituted systems, we uncover an affinity switch that controls mTORC1 lifetime and activation at the lysosome. Nutrients destabilize the Rag-Ragulator interface, causing cycling of the Rags between lysosome-bound Ragulator and the cytoplasm, and rendering mTORC1 capture contingent on simultaneous engagement of two Rag-binding interfaces. Rag GTPase domains trigger cycling by coordinately weakening binding of the C-terminal domains to Ragulator in a nucleotide-controlled manner. Cancer-specific Rag mutants override release from Ragulator and enhance mTORC1 recruitment and signalling output. Cycling in the active state sets the Rags apart from most signalling GTPases, and provides a mechanism to attenuate mTORC1 signalling.
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2019)
            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.
              Cover page: Structural mechanism of a Rag GTPase activation checkpoint by the lysosomal folliculin complex
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2017)
              All pathogens must acquire nutrients from their hosts. The intracellular bacterial pathogen Legionella pneumophila, the etiological agent of Legionnaires' disease, requires host amino acids for growth within cells. The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved master regulator of host amino acid metabolism. Here, we identify two families of translocated L. pneumophila effector proteins that exhibit opposing effects on mTORC1 activity. The Legionella glucosyltransferase (Lgt) effector family activates mTORC1, through inhibition of host translation, whereas the SidE/SdeABC (SidE) effector family acts as mTORC1 inhibitors. We demonstrate that a common activity of both effector families is to inhibit host translation. We propose that the Lgt and SidE families of effectors work in concert to liberate host amino acids for consumption by L. pneumophila.
                Cover page: Positive and Negative Regulation of the Master Metabolic Regulator mTORC1 by Two Families of Legionella pneumophila Effectors
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2019)
                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.
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