Session MOE. There are 4 abstracts in this session.

Session: Metabolism and Disease, time: 3:00 - 3:25 pm

Defining mitochondrial protein function through systems biochemistry

David J. Pagliarini1, 2
1Morgridge Institute for Research, Madison, WI; 2University of Wisconsin, Madison, WI

Despite their position as the iconic powerhouses of cellular biology, many aspects of mitochondria remain remarkably obscure—a fact that contributes to our poor ability to address mitochondrial dysfunction therapeutically. Such dysfunction contributes to a vast array of human diseases through distinct means. For instance, aberrant mitochondrial biogenesis can fail to properly set cellular mitochondrial content; dysregulated signaling processes can fail to calibrate mitochondrial activity to changing cellular needs; and malfunctioning proteins can render core bioenergetic processes ineffectual. A major bottleneck to understanding—and ultimately addressing—these processes is that the proteins driving them are often undefined. Concurrently, the functions of hundreds of mitochondrial proteins that may fulfill these roles are not known, or at best are poorly understood. Thus, the high-level goal of my research program is to help achieve a more complete, systems-level understanding of mitochondrial biology by systematically establishing the functions of orphan mitochondrial proteins and their roles within disease-related processes. We do so by first devising multi-dimensional analyses designed to make new connections between these proteins and established pathways and processes. We then employ mechanistic and structural approaches to define the functions of select proteins at biochemical depth. This ‘systems biochemistry’ strategy is helping us address three outstanding biological questions: Which orphan mitochondrial proteins fulfill the missing steps in classic mitochondrial processes, including the biosynthesis of coenzyme Q and other aspects of respiratory chain function? What proteins assist in the orchestrated assembly of lipids, metabolites, and proteins (from two genomes) to ensure proper mitochondrial biogenesis? And, which resident signaling proteins direct the post-translational regulation of mitochondrial activities? In answering these questions, we aim to help transform the mitochondrial proteome from a component list into a metabolic circuitry of connected functions, and to elucidate the biochemical underpinnings of mitochondrial dysfunction in human disease.

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Session: Metabolism and Disease, time: 3:25 - 3:50 pm

Metabolomics-Based Discovery of Metabolic Aspects of Cancer and Other Diseases

Anne Le
Johns Hopkins Medicine, Baltimore, MD

Dr. Anne Le’s research primarily focuses on cancer metabolism. Using metabolomics technologies, her work has led to breakthrough discoveries revealing several characteristic features of the metabolism of cancer.  In her talk, Dr. Le will discuss the complexity and diversity of cancer cell metabolism within the same tissue of origin, and even within individual tumor cell populations. Importantly, she will illustrate how to take advantage of metabolomics technologies for the discovery of metabolic aspects of cancer and other diseases.

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Session: Metabolism and Disease, time: 3:50 - 4:05 pm

An Optimized Data Independent Acquisition (DIA) Method for Peptide-Centric Analysis in Metabolism Studies

Christopher A. Barnes1; Lindsay K. Pino2; Bong J. Kim1; Brian C. Searle2; Michael J. MacCoss2
1Novo Nordisk Research Center Seattle, Inc., Seattle, WA; 2Department of Genome Sciences, University of Washi, Seattle, WA

Small, bioactive polypeptides including those derived from posttranslational prepropeptide hormone processing of longer translated gene products are often integral bioactive factors that control metabolic processes. Very well known examples such as insulin and glucagon are both short, processed polypeptides that act temporally to regulate glucose homeostasis. Even after decades studying these and other peptide hormones, there is still much unknown about the other peptide fragments that can be liberated from the same proprotein precursors. In the case of the proglucagon precursor, the polypeptide chain can be specifically cleaved into numerous bioactive peptides including both glucagon and the also therapeutically important glucagon-like peptide 1 (GLP-1). LC-MS/MS-based proteomics approaches using data-independent acquisition (DIA) are peptide-centric by nature and potentially suitable for this sort of novel peptide discovery. Using the recently published DIA-based chromatogram-library approach, we demonstrate optimization of precursor mass window size on the Thermo Lumos platform for both DIA-based peptide identifications and profiling of different biological samples. We show that relative quantitation is feasible with overlapping 8 m/z (“mzol”) windows allowing for an effective 4 m/z precursor range. In a comparison of liver proteomes generated from fasted, fed, and refed (after fasting) mice, we show that this DIA approach yielded a slight increase in quantified proteins compared to tandem mass tagging (TMT). Analysis of the same sample set with both techniques generating believable proteome changes in well-known proteins associated with fasting such as the observed increased levels of phosphoenolpyruvate carboxykinase (PCK1) or the observed decreased levels of fatty acid synthase (FASN) in fasted mice. We conclude that both TMT and DIA using on-column chromatogram libraries can yield high quality biologically-meaningful results with DIA offering improved modularity and a higher attention to individual peptide reproducibility that will be more easily ported to the peptide-centric nature of future discovery experiments.   

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Session: Metabolism and Disease, time: 4:05 - 4:20 pm

Peroxisome plasticity as a metabolic strategy for virus replication

Katelyn C Cook; Pierre M Jean Beltran; Yutaka Hashimoto; Ileana M Cristea
Princeton University, Princeton, NJ

Peroxisomes are cellular organelles with essential functions in human health, such as lipid production, fatty acid oxidation, and detoxification. However, peroxisomes are among the least studied organelles in biological contexts that rely on organelle remodeling, such as virus infections. Viruses cause broad alterations in organelle composition, structure, and localization in order to facilitate virus replication and spread. Here, we uncover a previously unrecognized function for peroxisomes in the replication of enveloped viruses. We initially examined human cytomegalovirus (HCMV), a beta-herpesvirus with nearly 90% worldwide seroprevalence and a significant health concern for pregnant women and immunocompromised patients. Using quantitative targeted mass spectrometry (parallel reaction monitoring, PRM), we found that peroxisome proteins increased in abundance as infection progressed, notably including biogenesis and lipid synthesis proteins. With mathematical modeling and microscopy structural analyses, we showed that infection triggers peroxisome growth and fission and the translocation of key host proteins to peroxisome membranes, interfering with peroxisome structure and increasing peroxisome biogenesis by nearly 4-fold. To determine the functional relevance of these changes, we generated a series of CRISPR knockouts in primary human fibroblasts and used pharmacological treatments to perturb peroxisome abundance and morphology. This analysis demonstrated that HCMV hijacks peroxisome metabolic pathways to facilitate virus production, which we further examined by lipidomics mass spectrometry. We discovered that infection enhances the synthesis of plasmalogen phospholipids at peroxisomes, and this is required for the assembly of new viruses. Moreover, by comparing other virus infections and analyzing samples from patients with genetic peroxisome disorders, we found this mechanism to be conserved across replication cycles of enveloped viruses, and likely relevant for a range of critical human diseases. Our integrative study illustrates the ability of human pathogens to manipulate subcellular organization for their replication and spread, and defines peroxisome regulation as a key aspect of virus replication.

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