Doctor of Philosophy, Cornell University (2016)
Bachelor of Science, Peking University (2016)
Peter Kim, Postdoctoral Faculty Sponsor
Immune checkpoint blockade of programmed death-1 (PD-1) by monoclonal antibody drugs has delivered breakthroughs in the treatment of cancer. Nonetheless, small-molecule PD-1 inhibitors could lead to increases in treatment efficacy, safety, and global access. While the ligand-binding surface of apo-PD-1 is relatively flat, it harbors a striking pocket in the murine PD-1/PD-L2 structure. An analogous pocket in human PD-1 may serve as a small-molecule drug target, but the structure of the human complex is unknown. Because the CC' and FG loops in murine PD-1 adopt new conformations upon binding PD-L2, we hypothesized that mutations in these two loops could be coupled to pocket formation and alter PD-1's affinity for PD-L2. Here, we conducted deep mutational scanning in these loops and used yeast surface display to select for enhanced PD-L2 binding. A PD-1 variant with three substitutions binds PD-L2 with an affinity two orders of magnitude higher than that of the wild-type protein, permitting crystallization of the complex. We determined the X-ray crystal structures of the human triple-mutant PD-1/PD-L2 complex and the apo triple-mutant PD-1 variant at 2.0 Å and 1.2 Å resolution, respectively. Binding of PD-L2 is accompanied by formation of a prominent pocket in human PD-1, as well as substantial conformational changes in the CC' and FG loops. The structure of the apo triple-mutant PD-1 shows that the CC' loop adopts the ligand-bound conformation, providing support for allostery between the loop and pocket. This human PD-1/PD-L2 structure provide critical insights for the design and discovery of small-molecule PD-1 inhibitors.
View details for DOI 10.1073/pnas.1916916116
View details for PubMedID 31727844
Self-assembly of ESCRT-III complex is a critical step in all ESCRT-dependent events. ESCRT-III hetero-polymers adopt variable architectures, but the mechanisms of inter-subunit recognition in these hetero-polymers to create flexible architectures remain unclear. We demonstrate in vivo and in vitro that the Saccharomyces cerevisiae ESCRT-III subunit Snf7 uses a conserved acidic helix to recruit its partner Vps24. Charge-inversion mutations in this helix inhibit Snf7-Vps24 lateral interactions in the polymer, while rebalancing the charges rescues the functional defects. These data suggest that Snf7-Vps24 assembly occurs through electrostatic interactions on one surface, rather than through residue-to-residue specificity. We propose a model in which these cooperative electrostatic interactions in the polymer propagate to allow for specific inter-subunit recognition, while sliding of laterally interacting polymers enable changes in architecture at distinct stages of vesicle biogenesis. Our data suggest a mechanism by which interaction specificity and polymer flexibility can be coupled in membrane-remodeling heteropolymeric assemblies.
View details for DOI 10.7554/eLife.46207
View details for PubMedID 31246173
Budding yeast Saccharomyces cerevisiae is an ideal model organism to study membrane trafficking pathways. The ESCRT (endosomal sorting complexes required for transport) pathway was first identified in this organism. Upon recognition of endocytosed ubiquitinated membrane proteins at endosomes, ESCRTs assemble at these organelles to catalyze the biogenesis of multivesicular bodies (MVBs). Formation of MVBs leads to the trafficking of these membrane proteins to vacuoles for degradation. Here, we describe genetic and biochemical approaches to study ESCRT function. We outline in vivo endocytosis assays using two model cargoes in Saccharomyces cerevisiae and also describe an in vitro approach to analyze ESCRT-III polymerization on lipid monolayers.
View details for DOI 10.1007/978-1-4939-9492-2_8
View details for PubMedID 31250297
Malignant mesothelioma has a poor prognosis for which there remains an urgent need for successful treatment approaches. Infection with the Edmonston vaccine strain (MV-Edm) derivative of measles virus results in lysis of cancer cells and has been tested in clinical trials for numerous tumor types including mesothelioma. Many factors play a role in MV-Edm tumor cell selectivity and cytopathic activity while also sparing non-cancerous cells. The MV-Edm receptor CD46 (cluster of differentiation 46) was demonstrated to be significantly higher in mesothelioma cells than in control cells. In contrast, mesothelioma cells are not reliant upon the alternative MV-Edm receptor nectin-4 for entry. MV-Edm treatment of mesothelioma reduced cell viability and also invoked apoptotic cell death. Forced expression of eIF4E or translation stimulation following IGF-I (insulin-like growth factor 1) exposure strengthened the potency of measles virus oncolytic activity. It was also shown that repression of cap-dependent translation by treatment with agents [4EASO, 4EGI-1] that suppress host cell translation or by forcing cells to produce an activated repressor protein diminishes the strength of oncolytic viral efficacy.
View details for DOI 10.18632/oncotarget.18656
View details for PubMedID 28968974
View details for PubMedCentralID PMC5609906
The endosomal sorting complexes required for transport (ESCRT) pathway facilitates multiple fundamental membrane remodeling events. Previously, we determined X-ray crystal structures of ESCRT-III subunit Snf7, the yeast CHMP4 ortholog, in its active and polymeric state (Tang et al., 2015). However, how ESCRT-III activation is coordinated by the upstream ESCRT components at endosomes remains unclear. Here, we provide a molecular explanation for the functional divergence of structurally similar ESCRT-III subunits. We characterize novel mutations in ESCRT-III Snf7 that trigger activation, and identify a novel role of Bro1, the yeast ALIX ortholog, in Snf7 assembly. We show that upstream ESCRTs regulate Snf7 activation at both its N-terminal core domain and the C-terminus ?6 helix through two parallel ubiquitin-dependent pathways: the ESCRT-I-ESCRT-II-Vps20 pathway and the ESCRT-0-Bro1 pathway. We therefore provide an enhanced understanding for the activation of the spatially unique ESCRT-III-mediated membrane remodeling.
View details for DOI 10.7554/eLife.15507
View details for Web of Science ID 000376391800001
View details for PubMedID 27074665
The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30 Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling.
View details for DOI 10.7554/eLife.12548
View details for Web of Science ID 000374007500001
View details for PubMedID 26670543
Vesicular stomatitis virus (VSV) is a potent oncolytic virus for many tumors. VSV that produces interferon-? (VSV-IFN?) is now in early clinical testing for solid tumors. Here, the preclinical activity of VSV and VSV-IFN? against non-small cell lung cancer (NSCLC) is reported. NSCLC cell lines were treated in vitro with VSV expressing green fluorescence protein (VSV-GFP) and VSV-IFN?. VSV-GFP and VSV-IFN? were active against NSCLC cells. JAK/STAT inhibition with ruxolitinib re-sensitized resistant H838 cells to VSV-IFN? mediated oncolysis. Intratumoral injections of VSV-GFP and VSV-IFN? reduced tumor growth and weight in H2009 nude mouse xenografts (p < 0.01). A similar trend was observed in A549 xenografts. Syngeneic LM2 lung tumors grown in flanks of A/J mice were injected with VSV-IFN? intratumorally. Treatment of LM2 tumors with VSV-IFN? resulted in tumor regression, prolonged survival (p < 0.0001), and cure of 30% of mice. Intratumoral injection of VSV-IFN? resulted in decreased tumor-infiltrating regulatory T cells (Treg) and increased CD8+ T cells. Tumor cell expression of PDL-1 was increased after VSV-IFN? treatment. VSV-IFN? has potent antitumor effects and promotes systemic antitumor immunity. These data support further clinical investigation of VSV-IFN? for NSCLC.
View details for DOI 10.18632/oncotarget.5320
View details for Web of Science ID 000363186600080
View details for PubMedID 26431376
Malignant mesothelioma is a devastating disease with a poor prognosis for which there is a clear need for more successful therapeutic approaches. Triptolide, a diterpenoid triepoxide, is a highly effective agent against several cancer types in animal models. Owing to triptolide's poor solubility in water, a water-soluble analog, minnelide, was synthesized. Minnelide is a prodrug of triptolide and is activated by exposure to phosphatases that are found in all body tissues, including blood. Mesothelioma cells were treated in vitro with minnelide or its parent compound, triptolide. Minnelide and triptolide were both found to significantly reduce mesothelioma cell viability and induce apoptosis. The level of Hsp70, a protein that promotes cancer cell survival, was measured in mesothelioma cells before and after treatment with triptolide. Hsp70 levels were decreased in a dose-dependent manner. In addition, triptolide sensitized cells to gemcitabine and pemetrexed as measured by cell viability. Mice bearing mesothelioma flank tumors were treated with daily injections (28 d) of minnelide or saline solution and xenograft tumor growth recorded. Mice displayed significantly reduced tumor burden. These findings support the clinical evaluation of minnelide therapy for mesothelioma.
View details for PubMedID 26000097
The hydrophobically assisted switching phase (HASP) method was applied in the assembly of oligomannosides. A new mannosyl donor with high reactivity was selected after a series of optimization studies, which was suitable for the synthesis of oligomannosides via the HASP method. The practicability of the HASP method towards the synthesis of branched oligosaccharides was explored and two branched penta-mannosides were assembled efficiently.
View details for DOI 10.1039/c5ob00730e
View details for Web of Science ID 000355985800007
View details for PubMedID 25967589
The endosomal sorting complexes required for transport (ESCRTs) have emerged as key cellular machinery that drive topologically unique membrane deformation and scission. Understanding how the ESCRT-III polymer interacts with membrane, promoting and/or stabilizing membrane deformation, is an important step in elucidating this sculpting mechanism. Using a combination of genetic and biochemical approaches, both in vivo and in vitro, we identify two essential modules required for ESCRT-III-membrane association: an electrostatic cluster and an N-terminal insertion motif. Mutating either module in yeast causes cargo sorting defects in the MVB pathway. We show that the essential N-terminal insertion motif provides a stable anchor for the ESCRT-III polymer. By replacing this N-terminal motif with well-characterized membrane insertion modules, we demonstrate that the N terminus of Snf7 has been tuned to maintain the topological constraints associated with ESCRT-III-filament-mediated membrane invagination and vesicle formation. Our results provide insights into the spatially unique, ESCRT-III-mediated membrane remodeling.
View details for DOI 10.1016/j.devcel.2013.09.009
View details for Web of Science ID 000326913900010
View details for PubMedID 24139821