Session TOG. There are 4 abstracts in this session.



Session: TECHNOLOGY: CHEMICAL PROTEOMICS AND DRUG DISCOVERY, time: 4:30 PM - 4:55 PM

Cysteine-Mediated Redox Signaling: Chemical Tools for Biological Discovery

Kate Caroll

The exploration of thiol-based redox regulation and signaling offers a grand challenge for achieving a molecular-level understanding of its unique role in physiology and pathology. Redox biology also represents a frontier for developing new therapeutics for cancer, neurodegenerative, and metabolic diseases. We are developing novel small-molecule probes as a way to identify and study the underlying chemistry that governs thiol-based redox signaling. This talk will present our latest results in the discovery and understanding of reactive oxygen species as emerging new chemical signals and their influence on protein function vis-à-vis the oxidative post-translational modification of pivotal cysteine residues.
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Session: TECHNOLOGY: CHEMICAL PROTEOMICS AND DRUG DISCOVERY, time: 4:55 PM - 5:20 PM

Lysine-targeted Covalent Inhibitors and Chemoproteomic Probes

Jack Tauton

Most targeted covalent drugs have been designed to react with cysteine. However, many ligand binding sites in protein targets of interest lack an accessible cysteine. An alternative strategy is to target lysine, which is more prevalent yet less reactive than cysteine. Here, I will describe our efforts to design chemoproteomic probes that selectively modify catalytic and noncatalytic lysines in living cells. We have developed a chemoproteomic workflow that enables direct identification of probe targets and modification sites by mass spectrometry. Our lysine-targeted probes have shown utility in cell biological and target engagement experiments.
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Session: TECHNOLOGY: CHEMICAL PROTEOMICS AND DRUG DISCOVERY, time: 5:20 PM - 5:35 PM

Mapping Cell Surface Lectin-Glycoprotein Interactions in situ using Oxidation Proteomics 

Yixuan (Axe) Xie; Ying Sheng; Qiongyu Li; Seunghye Ju; Carlito B. Lebrilla
University of California, Davis, Davis, California

    The cell membrane is composed of a network of glycoconjugates, including glycoproteins and glycolipids, that presents a dense matrix of carbohydrate playing critical roles in many biological processes. Microarray and confocal imaging with fluorescent lectins have been widely used to investigate these glycoconjugates. However, the detailed information regarding the protein targets of lectins in their native environment has generally not been studied due to the weak and transient nature of these interactions. 

    Here, we describe an oxidative mapping strategy for identifying cell surface lectin-targeted glycoproteins in situ. Nine commonly-used lectins from plants and humans have been modified with the oxidation probe containing Fe(III) and treated to the living cell culture. By introducing hydrogen peroxide, the proteins which were in close proximity to the lectins were oxidized and detected using mass spectrometry. As a result, more than 150 proteins were oxidized, and more than 70% of the proteins were confirmed with the respective glycosylation using glycoproteomic analysis. The specificity of each lectin was compared. Interestingly, the targets of sialic acid-binding lectin, including Sambucus Nigra (SNA) and Maackia Amurensis II (MAL-II), indicated a great deal of specificity of compared to other lectins such as Hippeastrum Hybrid (HHL). The non-glycosylated proteins were investigated and recognized as the glycoprotein-associated proteins using STRING software, and five central proteins, G3P, ANXA2, EF2, EF1A1, MYH9, and ACTB, were commonly oxidized across the experiments. This result reveals their pivotal roles in the cellular processes through association with glycoproteins such as integrins. Additionally, most of the oxidized proteins were found enriched in the lipid raft microdomain, which is consistent with the results from visualization studies using confocal imaging. By elucidating the details of lectin-glycoprotein interactions in situ, more effective therapeutics may be developed.

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Session: TECHNOLOGY: CHEMICAL PROTEOMICS AND DRUG DISCOVERY, time: 5:35 PM - 5:50 PM

Chemical modulation of ER proteostasis to inhibit Dengue and Zika virus propagation

Lars Plate; Katherine Almasy; Jonathan Davies
Vanderbilt University, Nashville, TN

The flavivirus family is responsible for some of the most common tropical and subtropical transmitted diseases. Development of effective antiviral therapies and safe vaccines against Dengue (DENV) and Zika (ZIKV) virus has been challenged by high mutation rates and antibody-dependent enhancement of secondary infection with related serotypes, which frequently leads to more severe disease progression. This motivates efforts to find alternative therapeutic approaches. Here, we focus on common host cell processes that are required for Dengue, Zika, Yellow Fever, West Nile or Hepatitis C virus infection and propagation. Flavivirus replication and assembly occurs at the endoplasmic reticulum (ER) membrane. The virus extensively remodels the organelle through activation of unfolded protein response (UPR)-associated transcription factors. The UPR induces expression of distinct ER chaperones and protein folding factors that maintain protein homeostasis and aid in the assembly and secretion of mature virions. To probe the individual roles of the UPR branches in the context of DENV infection, we take advantage of recently developed pharmacologic agents. We discovered that treatment with the small-molecule ER proteostasis regulator 147, a preferential activator of the ATF6 branch, results in a significant reduction of DENV propagation. Using chemoproteomics for target identification, we define that the molecule activates ATF6 through perturbation of ER redox signaling processes. Furthermore, we will discuss results from global proteomics profiling to determine other cellular pathways in DENV infected host cells that are impacted by the small molecule. Our ongoing efforts are directed at characterizing how modulation of these cellular redox processes is involved in regulating DENV propagation and the ability to assembly and secrete new virions. Our results suggest that treatment with 147 could be an effective strategy to impair proliferation of other flaviviruses, including Zika virus, offering a broadly-applicable therapeutic strategy to target essential host cell processes to impair viral infections.

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