Jingshi Shen, Ph.D. - US grants

Insulin facilitates glucose uptake into adipocytes and muscle cells by relocating glucose transporter 4 {GLUT4) from intracellular reservoirs to the plasma membrane. The translocation involves a vesicle fusion step that is mediated by three SNAREs - syntaxin 4, SNAP-23 and VAMP2 - and a number of regulatory proteins including MunciSc, synip and tomosyn. While the physiological importance of the SNARE regulatory proteins is clear, their molecular mechanisms of action and functional interactions among themselves are not known due to the complexity of the cellular environment. Recently, we reconstituted the SNARE-dependent GLUT4 vesicle fusion in both liposome (synthetic bilayers) and "flipped" SNARE cell-cell (native membranes) fusion systems. Here we propose to capitalize on these unique developments to ask key mechanistic questions about GLUT4 vesicle fusion, especially questions concerning how regulatory proteins act, alone or in concert, to control exocytosis at the molecular level. The specific hypothesis behind this proposed research is that regulatory proteins control different stages of the SNARE assembly cycle and contribute to the temporal and spatial Vegulation of GLUT4 exocytosis. Regulatory proteins will be added either as pure recombinant proteins or expressed as flipped proteins on th& cell surface. Kinetic effects of each regulator can be assessed when the regulator is added (alone or in combination) to the core fusion machinery of SNAREs. Two specific aims are proposed: 1) Define how the concerted action of SNAREs and regulatory factors controls GLUT4 vesicle fusion; 2) Characterize the fusion pore dynamics and transition state of GLUT4 exocytosis in the "flipped" SNARE fusion system. Our long-temn goal is to work our way up, protein by protein, until we can reconstitute the basic properties and fine-tuning of GLUT4 exocytosis. Insulin-regulated GLUT4 transport is crucial for glucose homeostasis, and imbalances in this process may lead to type 2 diabetes. Knowledge of how SNARE regulators work may Identify novel targets for therapeutic intervention. Since many components of GLUT4 transport are conserved, our work can also dhed light upon other exocytic pathways such as platelet and lung epithelial secretion.

PROJECT SUMMARY As the epidemic of insulin resistance and type 2 diabetes emerges worldwide, there is an urgent need to understand how insulin maintains blood glucose homeostasis at the molecular level. A major function of insulin is to promote glucose uptake into muscle and adipose tissues, a process mediated by the glucose transporter GLUT4. Upon insulin stimulation, GLUT4 is relocated from intracellular storage vesicles to the cell surface through regulated exocytosis. The exocytosis of GLUT4 vesicles requires the SNARE proteins as the core fusion machinery, as well as a group of fusion regulators. Loss-of-function mutations of the SNAREs or fusion regulators abrogate insulin-triggered GLUT4 exocytosis and disrupt blood glucose homeostasis. Moreover, imbalances in the GLUT4 vesicle fusion proteins have been implicated in obesity-associated insulin resistance. While the physiological importance of the SNAREs and fusion regulators is clear, it remains poorly understood how they act in concert to mediate and regulate GLUT4 vesicle fusion. The overall goal of this proposal is to answer this key question using novel and complementary approaches. We will first define the molecular mechanisms and functional interactions of the vesicle fusion proteins using a novel reconstituted fusion system. We will use both recombinant proteins and native proteins isolated from mouse adipocytes. We will then characterize GLUT4 vesicle fusion proteins in 3T3-L1 adipocytes and in adipocytes isolated from knockout mice. Finally, we will determine whether and how the activities of the vesicle fusion proteins are impaired in insulin resistance, using high fat diet-fed mice as a model system. If successfully accomplished, this research will substantially broaden our knowledge about the regulatory mechanisms of GLUT4 exocytosis. The findings will also shed light upon the diseases associated with glucose imbalances such as insulin resistance and type 2 diabetes, and will facilitate the development of novel strategies for therapeutic intervention.

DESCRIPTION (provided by applicant): Regulated exocytosis is a stimulus-dependent membrane fusion event of fundamental importance to a range of physiological processes. The membrane fusion reaction involves substantial lipid rearrangements and is opposed by the powerful hydrophobic force. For fusion to occur, a high energy barrier must be overcome. The overall goal of this proposed research is to determine the functional roles of membrane bilayer remodeling in overcoming the energy barrier of exocytic vesicle fusion, using the trafficking of the glucose transporter GLUT4 as a model system. To achieve this goal, we will employ a unique combination of complementary approaches including biochemical reconstitution, electron microscopic imaging, and biophysical measurements. First, we will define how two membrane bilayers are brought into close apposition by the SNARE-SM complex to prepare for the subsequent steps of membrane remodeling and merging. Next, we will assess the functional role of vesicle membrane bending in the fusion reaction. Finally, we will examine how the fusion reaction is regulated by local remodeling of the plasma membrane. We hypothesize that the fusion of exocytic vesicles with the plasma membrane involves a novel dual-curvature-induction mechanism: while the amphipathic motif of the v-SNARE bends the vesicle membrane, the hydrophobic loops of C2-domain molecules penetrate into the plasma membrane and induce local membrane curvature. Together, these membrane-bending activities are expected to create curvature stresses at the fusion sites to overcome the energy barrier for the fusion reaction. Successful completion of this proposed research will provide key insights into the molecular mechanisms of exocytic vesicle fusion. This work will also serve as a paradigm for understanding the general principles of intracellular membrane fusion. Ultimately, our findings will facilitate the development of novel therapeutic strategies for diseases associated with dysfunctional regulated exocytosis including diabetes, epilepsy, and immune disorders.

PROJECT SUMMARY Inflammation is an immune response protecting us from infection and injury. However, unchecked or improperly activated inflammation can result in chronic diseases such as arthritis, cardiovascular diseases and insulin resistance. Insulin resistance is a metabolic condition in which tissues no longer respond to insulin. A major function of insulin is to lower blood glucose levels by promoting glucose uptake into peripheral tissues including skeletal muscles and adipocytes. Insulin-stimulated glucose uptake is mediated by GLUT4, a facilitative glucose transporter enriched in insulin-responsive tissues. Under basal conditions, GLUT4 is sequestered in intracellular storage vesicles. Upon insulin stimulation, GLUT4 is relocated from intracellular vesicles to the cell surface where it facilitates the uptake of excess blood glucose into the cells for disposal. Defects in GLUT4 exocytosis disrupt blood glucose balance and are a hallmark of insulin resistance. Commonly associated with obesity, insulin resistance is a characteristic feature of type 2 diabetes (T2D). Laboratory and clinical studies have established that inflammation plays a central role in the pathogenesis of insulin resistance. In particular, the two proinflammatory cytokines interleukin 1? (IL-1?) and tumor necrosis factor alpha (TNF?) directly interfere with insulin responses in adipocytes, hepatocytes and skeletal muscles. While the connection of proinflammatory cytokines to insulin resistance is well established, the molecular mechanisms of cytokine-induced insulin resistance remains poorly understood. The major goal of this exploratory project is to help bridge this gap by globally identifying mediators of cytokine-induced insulin resistance, using insulin-dependent GLUT4 exocytosis as a model system. In this work, we will first determine whether and how GLUT4 trafficking regulators are impaired by IL-1? and TNF?. We will also perform new unbiased genome-wide genetic screens to identify suppressors of cytokine-induced impairment in GLUT4 exocytosis. These exploratory studies will pave the path for a full understanding of cytokine-associated insulin resistance and will likely identify novel therapeutic targets for treating insulin resistance and T2D. This work will also serve as a springboard to understanding other functions of proinflammatory cytokines in the immune system.

PROJECT SUMMARY Cytotoxic T lymphocytes (CTLs) eliminate target cells primarily through polarized secretion of the contents of membrane-enclosed cytolytic granules, specialized secretory lysosomes loaded with the pore-forming protein perforin and the serine proteases granzymes. When a CTL recognizes its target, the contact area between the CTL and the target cell forms a highly organized and stable structure known as the immunological synapse. Subsequently, cytolytic granules migrate from their dispersed locations in the cytosol toward the immunological synapse, where they dock and fuse with the plasma membrane to secrete their contents. Released cytolytic molecules then enter the target cell and initiate programmed cell death (apoptosis). The regulation of cytolytic granule secretion is still poorly understood at the molecular level. The major goal of this pilot project is to dissect cytolytic granule secretion in CTLs using unbiased genome-wide CRISPR-Cas9 genetic screens. In our preliminary studies, we developed novel platforms to genetically dissect mammalian vesicle transport pathways using genome-wide CRISPR screens. Furthermore, we developed assays to measure cytolytic granule secretion in CTLs. Here, we will take strategic advantage of these systems to dissect cytolytic granule secretion using an unbiased CRISPR genetic screen. We will then validate the identified genes using a pooled secondary screen as well as individual gene editing. Finally, we will characterize the knockout phenotype of selected candidate genes in CTLs. If successfully accomplished, this proposed research will provide the first genome-scale view of cytolytic granule secretion, and will substantially expand our knowledge of how CTLs destroy target cells. Insights gleaned from these studies will help improve the efficacy and safety of cancer immunotherapy, and will facilitate the development of new therapeutics for immune disorders caused by defective cytolytic granule secretion.

Defective insulin-stimulated glucose uptake is a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). Insulin promotes glucose uptake by triggering the relocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. To develop effective and safe treatments for IR and T2D, it is crucial to gain a comprehensive understanding of insulin-stimulated GLUT4 exocytosis at the molecular level. GLUT4 exocytosis ? the fusion of GLUT4 vesicles with the plasma membrane ? requires the membrane-anchored SNAREs, the soluble SM proteins, and the C2-domain factor Doc2b. In our previous research, we reconstituted GLUT4 vesicle fusion in vitro, for the first time, using defined components, which overcame the limitations of conventional approaches and enabled us to attack the problem from a fundamentally new angle. Using this unique reconstitution system, we discovered a stimulatory function of the SM protein Munc18c in SNARE zippering and a membrane-remodeling role of Doc2b in GLUT4 vesicle fusion. In our preliminary studies, we substantially expanded our reconstitution experiments and uncovered new molecular functions of GLUT4 vesicle fusion proteins. In addition, our CRISPR genetic analyses revealed Munc18b as another SM protein involved in GLUT4 exocytosis. Based on these major advances, we will first define the detailed molecular mechanisms by which Munc18b and Munc18c control GLUT4 vesicle fusion using our reconstitution system. Next, we will establish how Doc2b cooperates with Munc18b and Munc18c to control each stage of the membrane fusion reaction. We will then validate the findings of the reconstitution studies in adipocytes and muscle cells, using both cultured cell lines and primary tissues isolated from genetically engineered mice. Finally, we will examine whether and how GLUT4 vesicle fusion proteins are altered in insulin-resistant human adipocytes isolated from biopsies of subcutaneous abdominal fat. Completion of this proposed research will fill major gaps in our knowledge of the GLUT4 exocytic pathway. Our findings will also shed light upon the pathogenesis of IR and T2D, and will facilitate the development of novel strategies for therapeutic intervention.