Session TOB. There are 4 abstracts in this session.



Session: CELL BIOLOGY: SYSTEMS APPROACHES FOR CELLULAR SIGNALING, time: 09:50 AM - 10:15 AM

Proximity dependent sensors define a role for HOPS in macropinocytosis-dependent control of cell growth

Anne-Claude Gingras

The mechanistic Target of Rapamycin Complex 1 (mTORC1) couples nutrient sufficiency to cell growth, and is an important therapeutic target, notably in cancers. Amino acids can be up-taken in free form from transporters, and detected by different protein complexes that control the nucleotide-binding status of small GTPases of the Rag families. Nucleotide-bound Rag proteins recruit mTORC1 to the surface of the lysosome to mediate its full activation. A less well-characterized pathway of amino acid uptake is through macropinocytosis, in which extracellular proteins are endocytosed and delivered to the lysosome where they are degraded to amino acids, which also results in mTORC1 activation. Macropinocytosis is importantly enhanced by oncogenic KRAS mutations, and is thought to contribute to cancer survival in conditions of low free nutrient supply. Leveraging a proximity-dependent map of a human cell (Go, Knight et al., submitted), we generated proximity-dependent biotinylation “sensors” to study the recruitment of mTORC1 components to the surface of the endolysosomes (Hesketh et al., submitted). This revealed an unsuspected role for the HOPS trafficking complex in the activation of mTORC1 specifically in macropinocytic contexts. We also define a mechanism by which free nutrient availability represses macropinocytic mTORC1 activation. Together, our results help understanding the mechanisms of growth regulation by macropinocytosis, and may offer new therapeutic avenues for cancers in which this pathway is upregulated.
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Session: CELL BIOLOGY: SYSTEMS APPROACHES FOR CELLULAR SIGNALING, time: 10:15 AM - 10:40 AM

Identification of ligand-dependent GPCR protein interaction networks with temporal and spatial resolution

Ruth Huttenhain

G protein-coupled receptors (GPCRs) represent the largest family of signaling receptors and drug targets. Following ligand-induced activation of GPCRs signal transduction is mediated by protein interaction networks operating on short timescales and across multiple cellular locations. While temporal dynamics of protein interactions have been previously characterized, a major challenge remains largely unmet: how to interrogate the protein interaction networks engaged by GPCRs while capturing both their spatial and temporal context. Previously, we have developed an analytical approach combining APEX-based proximity labeling with quantitative proteomics and a system of spatial references, which delivers, with sub-minute temporal resolution, protein interaction networks and subcellular location of the receptor. We not only validated capture of proteins known to interact with the receptors, including those with transient or low affinity interactions, but demonstrated that our approach can be used to discover new network components regulating receptor function. We recently extended the approach to understand how chemically distinct agonists targeting the same GPCR produce different receptor based effects. Specifically, we determined cellular location and protein interaction networks engaged by the mu-type opioid (MOR) after stimulation with full, partial, and G protein biased agonists. The data revealed distinct intracellular trafficking comparing the agonists, while we observed rapid endocytosis for the full, unbiased agonist, the G protein biased agonist did not provoke any endocytosis. Interestingly, as interactors of MOR we discovered EYA4 and KCTD12, which were common for all three agonists, and have not been linked previously with MOR. These two novel interactors might play a role in regulating signaling downstream of receptor activation.
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Session: CELL BIOLOGY: SYSTEMS APPROACHES FOR CELLULAR SIGNALING, time: 10:40 AM - 10:55 AM

R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies 

Mario Leutert; Ricard Rodriguez-Mias; Noelle Fukuda; Judit Villén
University of Washington, Seattle,

Recent developments in proteomics have enabled signaling studies where >10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid-robotic-phosphoproteomics (R2-P2), an end-to-end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry-ready peptides for proteome and phosphoproteome analyses. R2-P2 is robust, versatile, high-throughput, and achieves higher sensitivity than classical protocols. To showcase the method, we applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large-scale signaling studies involving hundreds of perturbations opening the door to systems-level studies aiming to capture signaling complexity.

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Session: CELL BIOLOGY: SYSTEMS APPROACHES FOR CELLULAR SIGNALING, time: 10:55 AM - 11:10 AM

Identification and characterization of lactate-mediated histone lactylation pathway by proteomics approaches 

Di Zhang1; Zhanyuan Tang2; He Huang1; Guolin Zhou1; Chang Cui1; Wenchao Liu1; Mathew Perez Neut1; Robert G.Roeder2; Becker Lev1; Yingming Zhao1
1the university of chicago, Chicago, IL; 2the rockefeller university, new york, NY

The Warburg effect, originally describing augmented lactogenesis in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, macrophage polarization, and T-cell activation. This phenomenon is intimately linked with multiple diseases including neoplasia, sepsis, and autoimmune diseases. Lactate, a compound generated during Warburg effect, is widely known as an energy source and metabolic byproduct. However, its non-metabolic functi­ons in physiology and disease remain unknown. In this presentation, we will report identification of histone lysine lactylation by mass spectrometric analysis as a new epigenet­ic modification (Nature, 2019, Oct;574(7779):575-580). The histone mark was extensively verified by mass spectrometry, HPLC coelution, and biochemical methods. We identified and quantified histone lysine lactylation sites among diverse cells and under variant cancer-associated environments. In total, we identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce production of lactate through glycolysis that in turn serves as precursor for stimulating histone lactylation. Using bacterially-exposed M1 macrophages as a model system, we demonstrate that histone lactylation has different temporal dynamics from acetylation. We demonstrate that histone lactylation directly stimulates gene transcription from chromatin. Histone lactylation is a new type of epigenetic mechanism that links metabolism with gene activity, representing a new avenue for understanding the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.

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