Updates from the Metabolism Community

As part of the new Morgridge Metabolism Initiative, we want to share news and publications from campus. We hope these communications familiarize you with new findings and increase the awareness of our experimental capabilities.

Please send along information on your latest published work in this format to metabolismdirector@morgridge.org to be included in the next update!

December 2020 Morphogenesis of the islets of Langerhans is guided by extra-endocrine Slit2/3 signals.

Gilbert JM, Adams MT, Sharon N, Jayaraaman H, Blum B.

Mol Cell Biol. 2020 Dec 14:MCB.00451-20. doi: 10.1128/MCB.00451-20. Online ahead of print.

The precise positioning of endocrine cells within the islets of Langerhans is required for optimal blood glucose homeostasis, however, the mechanisms underlying islet development remain unclear. We have previously identified a role for the Robo family of receptors in endocrine cell sorting. We further demonstrate the requirement of Slit-Robo signaling in islet development with Gilbert et al. We show that the canonical Robo-binding ligands Slit2 and Slit3 are expressed in non-endocrine tissue, and that simultaneous loss of both ligands results in islet fragmentation. Further, we find that Slit2/3 can prevent endocrine cell migration. Our data suggests that Slit2/3 interact with Robo receptors on the surface of islet endocrine cells to promote islet morphogenesis and cell sorting. These findings may benefit ongoing work to generate bona fide islets from stem cells in vitro.

  • Confocal imaging
  • Cell migration assays
  • Glucose tolerance tests
December 2020 A marine microbiome antifungal targets urgent-threat drug-resistant fungi.

Zhang F, Zhao M, Braun DR, Ericksen SS, Piotrowski JS, Nelson J, Peng J, Ananiev GE, Chanana S, Barns K, Fossen J, Sanchez H, Chevrette MG, Guzei IA, Zhao C, Guo L, Tang W, Currie CR, Rajski SR, Audhya A, Andes DR, Bugni TS.

Science 2020 Nov 20;370 (6519): 974-978. doi: 10.1126/science.abd6919

New antifungal drugs are urgently needed to address the emergence and transcontinental spread of infectious fungal diseases. Principal among these pathogens is the urgent-threat pandrug-resistant Candida auris. By employing cutting-edge metabolomics and genomic tools, we capitalized on the microbiomes of marine animals and their vast chemical diversity to identify antifungal molecules with promising in vivo efficacies. The most exciting lead, turbinmicin, displays potent in vitro and in vivo activities against not only C. auris, but also Aspergillus fumigatus. Turbinmicin shows excellent mouse-model efficacy against multiple-drug–resistant fungal pathogens and exhibits a wide safety index. Data strongly support the hypothesis that turbinmicin functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway. The efficacy, safety, and a mode of action distinct from other antifungal drugs make turbinmicin a highly promising antifungal drug lead to help address devastating global fungal pathogens such as C. auris and A. fumigatus.

  • Hierarchical Component Analysis (HCA) and Principal Component Analysis (PCA) applied to the metabolomes of > 1000 microbial strains of marine origin.
  • Nuclear Magnetic Resonance (NMR) and X-ray crystallographic structure elucidation of turbinmicin.
  • In vitro microbroth antifungal assays employing multidrug-resistant pathogens.
  • In vivo efficacy determination using a U.S. Food and Drug Administration (FDA) standard fungal model
  • Chemical genetics using a Saccharomyces cerevisiae DNA-barcoded knockouts/knockdowns
  • Fluorescence imaging of vesicle-mediated trafficking
December 2020 l-Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate.

Prasanth Kumar, Anthony Meza, Jonathan M Ellis, Grace A Carlson, Craig A Bingman, Andrew R Buller

ACS Chem Biol. 2021 Jan 15;16(1):86-95. doi: 10.1021/acschembio.0c00753. Epub 2020 Dec 18.

Important advances in the field of metabolic engineering have been guided by our understanding of the structure and mechanism of enzymes that catalyze novel reactions. ObiH, an L-threonine transaldolase, is a recently discovered enzyme that catalyzes a stereoselective C-C bond formation reaction using simple metabolic precursors to form diverse β-hydroxy amino acids. We have carried out detailed mechanistic and structural characterization of ObiH to decipher its unique reactivity. Strikingly, ObiH forms a remarkably persistent glycyl quinonoid intermediate, whose unusual stability enables its reactivity with diverse aldehydes to form a palette of β-hydroxy amino acids. These results explain the basis for the unique reactivity of ObiH and provide a foundation for targeted protein engineering towards developing ObiH as a biocatalyst.

  • UV-vis spectroscopic analysis of catalytic intermediates
  • Enzymatic synthesis of non-natural amino acids
  • Protein crystallography
  • MD simulations
December 2020 How the gut and liver hibernate.

Kurtz CC, Otis JP, Regan MD, Carey HV.

Comp Biochem Physiol A Mol Integr Physiol. 2020 Dec 25;253:110875. doi: 10.1016/j.cbpa.2020.110875. Online ahead of print.

Dr. Carey was invited to submit a graphical review as part of a special issue that celebrates the centennial of the 1920 Nobel Prize in Physiology or Medicine awarded to August Krogh, who is considered a foundational leader in the field of comparative physiology. Hibernation is an ideal topic for this special issue, as it beautifully fits with Krogh’s famous comment that “for a large number of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied”, and especially to reinforce his appreciation of animals that display “mechanisms and adaptations of exquisite beauty”. Dr. Carey invited three former trainees to develop this review with her to highlight their work on hibernation biology that was carried out in the Carey Lab. They were fortunate to have the assistance of an excellent scientific illustrator – Rush Dhillon, a former member of the UW-Madison Metabolism community - who generated our terrific figures.

December 2020 Multivalent interactions drive nucleosome binding and efficient chromatin deacetylation by SIRT6.

Liu WH, Zheng J, Feldman JL, Klein MA, Kuznetsov VI, Peterson CL, Griffin PR, Denu JM.

Nat Commun. 2020 Oct 16;11(1):5244. doi: 10.1038/s41467-020-19018-y.

The cellular enzyme SIRT6 maintains metabolic homeostasis through the control of specific gene expression. SIRT6 represses genes by deacetylation of chromatin proteins that wrap DNA. This study describes the molecular details by which SIRT6 binds to nucleosomes, the basic repeating unit of chromatin. SIRT6 uses multivalent interactions with nucleosomes that couple productive binding to efficient deacetylation of histones. This work provides fundamental knowledge that will help explain how mutations in SIRT6 lead to human disease, or alternatively, to increased lifespan.

  • Fluorescence-based assays
  • Use of reconstituted nucleosomes
  • Nuclease sensitivity and electrophoretic mobility shift assays
  • Protein NMR
December 2020 Agonist-independent Gα(z) activity negatively regulates β-cell compensation in a diet-induced obesity model of type 2 diabetes.

Schaid MD, Green CL, Peter DC, Gallagher SJ, Guthery E, Carbajal KA, Harrington JM, Kelly GM, Reuter A, Wehner ML, Brill AL, Neuman JC, Lamming DW, Kimple ME.

J Biol Chem. 2020 Nov 10:jbc.RA120.015585. doi: 10.1074/jbc.RA120.015585. Online ahead of print.

Type 2 diabetes occurs when the pancreatic beta-cells are unable to fully compensate for peripheral insulin resistance by increasing their insulin secretion capacity, replication rate, or both. G protein coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular signals to intracellular signaling partners to elicit biological changes. Many GPCRs are well-known to be critical regulators of beta-cell function and mass. In Schaid et al., we elucidate a previously unknown signal transduction mechanism mediated by Prostaglandin EP3 receptor (EP3), a receptor for the arachidonic acid metabolite, prostaglandin E2 (PGE2), in the beta-cell’s ability to compensate for insulin resistance in a mouse model of obesity and type 2 diabetes. EP3 was found to be solely coupled to the unique inhibitory G protein, Gz, down-regulating beta-cell cyclic AMP production and hormone-potentiated insulin secretion even in the absence of enhanced PGE2 availability. In overexpression experiments in primary pancreatic islets, we found the EP3ɣ variant is nearly fully constitutively active, explaining these disparate results. Our findings provide a strong premise for further study of EP3/Gz signaling pathway in developing new therapies for the beta-cell dysfunction of type 2 diabetes.

  • Employment of a diet-induced mouse model of type 2 diabetes and phenotypic characterization
  • Live-cell imaging of beta-cell calcium and cAMP in response to GPCR ligands
  • Real-time measurement of insulin release from intact islets in response to GPCR ligands
  • Quantification of beta-cell replication and mass by immunofluorescence
December 2020 Comparisons of the Sexual Cycles for the Coccidian Parasites Eimeria and Toxoplasma.

Martorelli Di Genova B, Knoll LJ.

Front Cell Infect Microbiol. 2020 Dec 14;10:604897. doi: 10.3389/fcimb.2020.604897. eCollection 2020.

This review examines advances in research to understand the sexual cycles of Coccidia parasites, especially Eimeria spp. and Toxoplasma gondii. The molecular basis of sex in these pathogens has been significantly unraveled by new findings in parasite differentiation along with transcriptional analysis of T. gondii and Eimeria spp. pre-sexual and sexual stages. Focusing on the metabolic networks, analysis of these transcriptome datasets shows enrichment for several different metabolic pathways. Specifically, glycolytic enzymes are consistently more abundant in T. gondii cat infection stages than the asexual stages and Eimeria spp. pre-sexual and gamete stages compared to sporozoites. As the cyst stages produced from these Coccidian sexual cycles are highly infectious, analyzing these sexual cycles will allow us to develop new methods to stop the spread of these parasites.

September 2020 A large-scale genome-lipid association map guides lipid identification.

Linke V, Overmyer KA, Miller IJ, Brademan DR, Hutchins PD, Trujillo EA, Reddy TR, Russell JD, Cushing EM, Schueler KL, Stapleton DS, Rabaglia ME, Keller MP, Gatti DM, Keele GR, Pham D, Broman. KW, Churchill GA, Attie AD, Coon JJ.

Nat Metab 2, 1149–1162 (2020). doi: 10.1038/s42255-020-00278-3. ePub 2020 Sep 21.

Lipids are crucial actors in metabolism, but comprehensive annotation of their variety of identities and genetic basis remain bottleneck challenges to biological interpretation. We used mass spectrometry–based discovery lipidomics to globally survey plasma and liver lipids of 384 diversity outbred mice, quantifying 3,283 molecular features. These known and unknown lipid features were mapped to 5,622 genetic loci, creating a large-scale genome-lipid association resource, termed LipidGenie. Harnessing this resource, we identified gangliosides through their association with B4galnt1, found evidence for a potentially novel group of sex-specific phosphatidylcholines, and suggested acyl-chain-specific functions for proteins of the ABHD family.

  • Discovery lipidomics by mass spectrometry
  • Mouse husbandry and biopsies
  • Cell transfection for protein overexpression
  • Western blot analysis
  • Biostatistic and bioinformatic analysis
September 2020 Lysine oxidase exposes a dependency on the thioredoxin antioxidant pathway in triple-negative breast cancer cells.

Chepikova OE, Malin D, Strekalova E, Lukasheva EV, Zamyatnin AA Jr, Cryns VL.

Breast Cancer Res Treat. 2020 Oct;183(3):549-564. doi: 10.1007/s10549-020-05801-4. Epub 2020 Jul 21.

Cancer cells are dependent on several amino acids for their growth and survival. We have demonstrated that lysine oxidase, an enzyme that depletes lysine and generates hydrogen peroxide, kills breast tumor cells by causing oxidative stress, which is buffered by the thioredoxin antioxidant pathway. Notably, the combination of lysine oxidase and auranofin, an inhibitor of thioredoxin reductase, results in enhanced tumor cell death. Hence, lysine oxidase primes tumor cells to respond to thioredoxin reductase inhibitors, pointing to this combination as a novel metabolic strategy for breast cancer.

  • Cellular ROS assay
  • Reduced glutathione
  • (GSH)/oxidized glutathione (GSSG) assay

  • Thioredoxin reductase assay
September 2020 Phosphoproteome Response to Dithiothreitol Reveals Unique Versus Shared Features of Saccharomyces cerevisiae Stress Responses.

MacGilvray ME, Shishkova E, Place M, Wagner ER, Coon JJ, Gasch AP.
J Proteome Res. 2020 Aug 7;19(8):3405-3417. doi: 10.1021/acs.jproteome.0c00253. Epub 2020 Jul 13.

All cells must respond to stressful cellular situations, including those that result from toxins, cellular defect, or disease. How cells coordinate a multi-cellular response to environmental stress is not fully understood, in part because less is known about the upstream signaling networks activated by stress. This work, a collaboration between the Gasch and Coon labs at UW-Madison, used quantitative phospho-proteomics and regulatory network inference to understand the upstream signaling response in yeast activated by the reducing agent dithiothreitol (DTT). They also compared the implicated signaling network to the phosphoproteomic response to salt stress, revealing surprising similarities and differences in how cells respond to different types of stress. The results provide new information about how eukaryotic cells respond to suboptimal conditions with common versus specific responses.

  • phospho-proteomic mass spec in the Coon lab
  • molecular biology.
September 2020 Ovariectomy uncouples lifespan from metabolic health and reveals a sex-hormone-dependent role of hepatic mTORC2 in aging.

Arriola Apelo SI, Lin A, Brinkman JA, Meyer E, Morrison M, Tomasiewicz JL, Pumper CP, Baar EL, Richardson NE, Alotaibi M, Lamming DW.

Elife. 2020 Jul 28;9:e56177. doi: 10.7554/eLife.56177.

Inhibition of mTOR (mechanistic Target Of Rapamycin) signaling by rapamycin promotes healthspan and longevity in mice, but “off-target” inhibition of mTOR Complex 2 (mTORC2) activity by rapamycin results in many negative side effects that have precluded its use as an anti-aging intervention in humans. In 2014, we discovered that genetic inhibition of hepatic mTORC2 (mTOR Complex 2) specifically reduces the lifespan of males, but not females. We tested the role of sex hormones in this response using gonadectomy, and determined that the sex-specific impact of reduced hepatic mTORC2 is not reversed by depletion of sex hormones. Ovariectomy unexpectedly promoted the midlife survival of female mice lacking hepatic mTORC2, significantly increasing the survival of those mice that do not develop cancer. In addition to identifying a sex hormone-dependent role for hepatic mTORC2 in female longevity, our results demonstrate that metabolic health is not inextricably linked to lifespan in mammals, as ovariectomized females having improved survival despite paradoxically having increased adiposity and decreased control of blood glucose levels.

  • Gonadectomy surgery
  • Body composition analysis
  • Metabolic chambers for analysis of food consumption, activity and energy expenditure
  • Determination of mouse lifespan
  • Glucose and insulin tolerance tests
September 2020 Pyruvate Kinase Controls Signal Strength in the Insulin Secretory Pathway.

Sophie L Lewandowski, Rebecca L Cardone, Hannah R Foster, Thuong Ho, Evgeniy Potapenko, Chetan Poudel, Halena R VanDeusen, Sophia M Sdao, Tiago C Alves, Xiaojian Zha , Megan E Capozzi, Arnaldo H de Souza, Ishrat Jahan, Craig J Thomas, Craig S Nunemaker, Dawn Belt Davis, Jonathan E Campbell, Richard G Kibbey, Matthew J Merrins

Cell Metab. 2020 Nov 3;32(5):736-750.e5. doi: 10.1016/j.cmet.2020.10.007.

Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health

Abudukadier Abulizi, Rebecca L Cardone, Romana Stark, Sophie L Lewandowski, Xiaojian Zhao, Joelle Hillion, Lingjun Ma, Raghav Sehgal, Tiago C Alves, Craig Thomas, Charles Kung, Bei Wang, Stephan Siebel, Zane B Andrews, Graeme F Mason, Jesse Rinehart, Matthew J Merrins, Richard G Kibbey

Cell Metab. 2020 Nov 3;32(5):751-766.e11. doi: 10.1016/j.cmet.2020.10.006.

Pancreatic b-cells couple nutrient metabolism with appropriate insulin secretion. In a pair of papers recently published in Cell Metabolism, Lewandowski et al. demonstrate that pyruvate kinase, rather than oxidative phosphorylation, is the ATP/ADP generator that closes β-cell KATP channels to initiate insulin secretion. Small-molecule activators of pyruvate kinase were shown to potently amplify insulin secretion from human and rodent islets by switching mitochondria from oxidative phosphorylation to anaplerotic phosphoenolpyruvate biosynthesis. In the companion paper, Abulizi et al. show in preclinical models of diabetes that pyruvate kinase activators improve glucose homeostasis in vivo, by increasing insulin secretion and insulin sensitivity, decreasing gluconeogenesis, increasing red blood cell glycolysis, and reducing hepatic steatosis. Together, these papers suggest that targeting pyruvate kinase may aid type 2 diabetes treatment.

  • Single-channel recordings of the ATP-sensitive K+ channel (KATP) from pancreatic b-cells
  • Patch-clamp measurements of insulin exocytosis
  • Live-cell imaging of calcium, ATP/ADP, mitochondrial membrane potential, mitochondrial pH, glutamate, lactate, and pyruvate kinase activity
  • Simultaneous measurements of insulin and glucagon release from intact islets
September 2020 Classification of T-cell activation via autofluorescence lifetime imaging.

Walsh AJ, Mueller KP, Tweed K, Jones I, Walsh CM, Piscopo NJ, Niemi NM, Pagliarini DJ, Saha K, Skala MC.

Nat Biomed Eng. 2020 Jul 27. doi: 10.1038/s41551-020-0592-z. Online ahead of print.

Summary: The function of a T cell depends on its subtype and activation state. Here, we show that imaging the NAD(P)H lifetime of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2, CD3 and CD28 showed specific activation-state-dependent patterns of NAD(P)H lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99% accuracy, and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97% accuracy. NAD(P)H lifetime imaging can be used to non-destructively determine T-cell function.

  • Two-photon fluorescence lifetime imaging
September 2020 Sex- and region-specific differences in the transcriptomes of rat microglia from the brainstem and cervical spinal cord.

Ewald AC, Kiernan EA, Roopra AS, Radcliff AB, Timko RR, Baker TL, Watters JJ.

J Pharmacol Exp Ther. 2020 Jul 13;375(1):210-22. doi: 10.1124/jpet.120.266171. Online ahead of print.

Men and women display important sexual dimorphisms in breathing (and ultimately metabolism), controlled by key areas in the central nervous system (CNS) including brainstem and cervical spinal cord regions. While neurons in these regions are well-studied, the contributions of microglia, CNS-resident immune cells that are key cellular partners to neurons, have not been studied with regard to how they may contribute to some of these sex differences in respiration. We analyzed the transcriptomes of microglia from adult male and female rat brainstem and cervical spinal respiratory neural control CNS regions, and found that while brainstem microglia generally lacked sexual dimorphisms in gene expression, cervical spinal microglia isolated from regions where phrenic motor neurons reside (which innervate the diaphragm inspiratory muscle) had hundreds of genes that were differentially expressed between the sexes. While Gene Ontology and STRING analyses failed to identify functional gene categories into which the differentially upregulated genes in female microglia fell relative to males, the differentially down-regulated genes in female cervical spinal microglia segregated into categories including cellular and protein metabolism, ribosome and proteasome function, and oxidative phosphorylation. These data suggest that male spinal microglia may have higher protein synthesis and posttranslational protein regulatory capacity, as well as higher oxidative metabolism, than female cells, perhaps contributing to sexual dimorphisms in respiratory neural control. Interestingly, bioinformatics analysis using MAGICTRICKS identified the histone demethylase PHF8 (which demethylates H3K4 to repress gene transcription) as a major driver of sexually dimorphic down-regulated genes in female cervical spinal microglia versus male, and EZH2 (a component of the polycomb repressive complex that places methyl marks on H3K27) as a primary transcriptional driver of differentially down-regulated genes in cervical spinal microglia compared to brainstem microglia, in both sexes. These observations suggest that the cellular mechanisms controlling microglial transcriptomes differ both by CNS region and by sex, underscoring the need to better understand how the local CNS microenvironment and hormonal milieu impacts microglial function as it relates to how the brain controls breathing. This work sets the stage for future studies that will begin to dissect the molecular mechanisms underlying potential histone modifications in microglia, and how differentially expressed microglial genes contribute to respiratory motor neuron function in the context of physiologic systems that are integral to metabolic regulation.

  • RNA-sequencing
  • Immunomagnetic cell sorting
  • Computer-controlled gas delivery system for manipulating inspired oxygen and carbon dioxide levels
  • Novel MAGICTRICKS bioinformatics algorithm to identify common transcriptional gene drivers
July 2020 A Bacterial Biosynthetic Pathway for Methylated Furan Fatty Acids.

Lemke RA, Olson SM, Morse K, Karlen SD, Higbee A, Beebe ET, Ralph J, Coon JJ, Fox BG, Donohue TJ

J. Biol. Chem. 2020 Jul 17; 295(29):9786-9801 Epub 2020 May 20

Furan fatty acids (FuFAs), characterized by the presence of a furan ring within the molecule’s hydrophobic core, are found at low levels in many cells, and have properties that make them attractive in industrial, medical and other applications, but their biosynthetic origins are not clear. In this study, an interdisciplinary research team deployed a suite of genetic, genomics, biochemical, and high-resolution analytical techniques (GC-MS and NMR) to identify the chemical structures of intermediates in the FuFA acid biosynthetic pathway and isotopic studies to determine the source of the oxygen in the furan ring. These findings lay the foundation for detailed study of novel biosynthetic enzymes, obtaining new insights into cellular FuFA functions, and engineering cells that overproduce these fatty acids for various applications.

July 2020 21-plex DiLeu isobaric tags for high-throughput quantitative proteomics.

Frost DC, Feng Y, Li L.

Anal Chem. 2020 June 16; 92(12):8228-8234. doi: 10.1021acs.analchem.0c00473. ePub 2020 May 13.

Quantitative mass spectrometry has emerged as a central technology for biomedical research and provides deep insights into complex biological mechanisms and disease states. Multiplexed isobaric tagging is a particularly powerful tool that enables broad investigation of many samples in a single LC-MS/MS experiment, but the cost of employing commercial tagging reagents for large-scale, discovery-based studies is often prohibitive. As a practical alternative, we previously developed our own isobaric tags, dimethylated leucine (DiLeu), that we synthesize in-house at a fraction of the cost. In this work, we devise a novel synthetic avenue to generate additional isotopic variants that allow us to greatly increase multiplexing capability, beyond that of established isobaric tags, while preserving the existing design and its advantages. The result is a new generation of DiLeu tags that permits simultaneous identification and quantification of 21 samples in parallel on mainstream Orbitrap MS platforms. As such, the 21-plex DiLeu isobaric tags offer the potential to substantially facilitate the high-throughput analyses and large-scale experiments that are increasingly being conducted in discovery-based quantitative proteomics and metabolomics workflows.

  • Novel chemical tag design and synthesis
  • Stable isotope labeling of complex biological samples
  • Highly multiplexed quantitative analysis by mass spectrometry
July 2020 Postpartum Supplementation with Fermented Ammoniated Condensed Whey Altered Nutrient Partitioning to Support Hepatic Metabolism.

Caputo Oliveira R, Erb SJ, Pralle RS, Holdorf HT, Seely CR, White HM.

J Dairy Sci. 2020 Aug;103(8):7055-7067. doi: 10.3168/jds.2019-17790. Epub 2020 Jun 10.

General Background:
In ruminant animals, carbohydrates are fermented in the rumen and absorbed as volatile fatty acids rather than glucose and other monomers, therefore nearly all glucose must be generated via gluconeogenesis in the liver and then transported to the mammary gland as a lactose precursor. In the lactating dairy cow, this glucose need can range from 10 to 20 lb of glucose daily and lactose is a key limiter of milk production.

Study summary:
Shifting nutrient partitioning to support the glucose and energy needs during the peripartum period in lactating dairy cows can contribute to improved feed efficiency and improved metabolic health. While lactate is a known gluconeogenic precursor, previous attempts to feed lactate sources led to rumen acidosis. Supplementation of ammoniated lactate to postpartum dairy cows resulted in improved feed efficiency and decreased incidence of hyperketonemia and hepatic lipid content. Examining hepatic gene expression and protein abundance supported a multi-faceted mechanism of increasing gluconeogenic precursors to support glucose production and increasing the TCA cycle oxidative capacity which together may have supported the improvements in feed efficiency and metabolic health.

  • Bovine liver biopsies
  • Liver lipid quantification
  • Real-time quantitative PCR
  • Western blot analysis
  • Metabolite quantification
April 2020 Automated Extraction and Visualization of Protein-Protein Interaction Networks and Beyond: A Text-Mining Protocol.

Raja K, Natarajan J, Kuusisto F, Steill J, Ross I, Thomson J, Stewart R.

Methods Mol Biol. 2020;2074:13-34. doi: 10.1007/978-1-4939-9873-9_2.

Proteins perform their function by interacting with other proteins. Protein-protein interaction (PPI) analysis allows us to better understand the functions of individual proteins, the mechanisms of biological processes, and disease mechanisms. In many metabolic reactions, PPI is essential for electron transfer reactions (ex. electron transfer from ferredoxin to lipoic acid synthase). Information on PPI are abundant in published PubMed articles and their extraction is possible only through automated approaches. We describe the standard text-mining protocol that includes four major tasks, namely, recognizing protein mentions, normalizing protein names and aliases to unique identifiers such as gene symbol, extracting PPIs, and visualizing the PPI network using Cytoscape or other visualization tools. We show the application of KinderMiner, our recent text-mining tool on retrieving significant co-occurring protein pairs from ~30 million PubMed articles. KinderMiner retrieved known and new PPIs from PubMed articles and performed better than String and PolySearch2, the well-known PPI resources, on extracting mitochondrial PPIs. KinderMiner is a general text mining system applicable to a wide range of metabolic and other biological queries.

  • Automated approach to replace abbreviations with respective original text in ~30 million PubMed articles
  • Many new features were added to KinderMiner
  • For applications other than PPI using KinderMiner, we generated diseases lexicon, phenotypes lexicon, and drugs lexicon from existing resources.
April 2020 Systematically Sifting Big Data to Identify Novel Causal Genes for Human Traits.

Nicholas J. Hand and Daniel J. Rader

Cell Metab. 2020 Apr 7;31(4):658-659. doi: 10.1016/j.cmet.2020.03.013.

Identifying the causal gene(s) that connects genetic variation to a phenotype is a challenging problem in genome-wide association studies (GWASs). Here, we develop a systematic approach that integrates mouse liver co-expression networks with human lipid GWAS data to identify regulators of cholesterol and lipid metabolism. Through our approach, we pinpoint Sestrin1 as a causal gene associated with plasma cholesterol levels in humans. Our validation studies demonstrate that Sestrin1 influences plasma cholesterol in multiple mouse models and regulates cholesterol biosynthesis. Our results highlight the power of combining mouse and human datasets for prioritization of human lipid GWAS loci and discovery of lipid genes.

  • Co-expression network analysis with the Center for High Throughput Computing (CHTC)
  • Radio-label tracing of cholesterol biosynthesis
April 2020 Integrating Mouse and Human Genetic Data to Move beyond GWAS and Identify Causal Genes in Cholesterol Metabolism.

Zhonggang Li, James A. Votava, Gregory J.M. Zajac, Jenny N. Nguyen, Fernanda B. Leyva Jaimes, Sophia M. Ly, Jacqueline A. Brinkman, Marco De Giorgi, Sushma Kaul, Cara L. Green, Samantha L. St. Clair, Sabrina L. Belisle, Julia M. Rios, David W. Nelson, Mary G. Sorci-Thomas, William R. Lagor, Dudley W. Lamming, Chi-Liang Eric Yen, and Brian W. Parks

Cell Metab. 2020 Apr 7;31(4):741-754.e5. doi: 10.1016/j.cmet.2020.02.015. Epub 2020 Mar 19.

April 2020 Dual metabolomic profiling uncovers Toxoplasma manipulation of the host metabolome and the discovery of a novel parasite metabolic capability.

Olson WJ, Martorelli Di Genova B, Gallego-Lopez G, Dawson AR, Stevenson D, Amador-Noguez D, Knoll LJ.

PLoS Pathog. 2020 Apr 7;16(4):e1008432. doi: 10.1371/journal.ppat.1008432. eCollection 2020 Apr.

Toxoplasma gondii is an obligate intracellular parasite that is auxotrophic for several key metabolites and must scavenge these from the host. It is unclear how Toxoplasma manipulates host metabolism for its overall growth rate and non-essential metabolites. To address this question, we measured changes in the joint host-parasite metabolome over a time course of infection. These experiments led to the discovery of a Toxoplasma sedoheptulose bisphosphatase, which funnels carbon from glycolysis into ribose synthesis through an energetically driven dephosphorylation reaction. This second route for ribose synthesis resolves a conflict between the Toxoplasma tricarboxylic acid cycle and pentose phosphate pathway, which are both NADP+ dependent. Sedoheptulose bisphosphatase represents a novel step in Toxoplasma central carbon metabolism that allows Toxoplasma to satisfy its ribose demand without using NADP+. Sedoheptulose bisphosphatase is not present in humans, highlighting its potential as a drug target.

  • Discovery mass spectrometry metabolomics and RNAseq
  • CRISPR-mediated genome edited Toxoplasma
  • Stable isotope labeling with mass spectrometry
April 2020 Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains.

Blachowicz A, Raffa N, Bok JW, Choera T, Knox B, Lim FY, Huttenlocher A, Wang CCC, Venkateswaran K, Keller NP

mBio. 2020 Feb 18;11(1):e03415-19

The opportunistic human pathogen Aspergillus fumigatus synthesizes spore specific secondary metabolites, several of which (DHN melanin, trypacidin) are virulence factors in invasive aspergillosis. This article demonstrates that DHN melanin and other spore metabolites serve as UV protectants for the fungus dependent on fungal isolate. This work supports previous hypotheses that secondary metabolites which evolved for protection against environmental stresses can serve as double edged swords in pathogenesis.

  • Creation of Aspergillus mutants
  • Zebrafish virulence assays
April 2020 Beta Cell Dedifferentiation Induced by IRE1α Deletion Prevents Type 1 Diabetes.

Lee H, Lee YS, Harenda Q, Pietrzak S, Oktay HZ, Schreiber S, Liao Y, Sonthalia S, Ciecko AE, Chen YG, Keles S, Sridharan R, Engin F.

Cell Metab. 2020 Apr 7;31(4):822-836.e5. doi: 10.1016/j.cmet.2020.03.002. Epub 2020 Mar 26.

Immune-mediated destruction of insulin-producing β-cells causes type 1 diabetes (T1D). However, how β-cells participate in their own destruction during the disease process is poorly understood. Here, we report that modulating the unfolded protein response (UPR) in β-cells of non-obese diabetic (NOD) mice by deleting the UPR sensor IRE1α prior to insulitis induced a transient dedifferentiation of the β-cells, resulting in substantially reduced islet immune cell infiltration and β-cell apoptosis. Single-cell and whole-islet transcriptomics analyses of immature β-cells revealed remarkably diminished expression of β-cell autoantigens, MHC class I components and upregulation of immune inhibitory markers. IRE1α-deficient mice exhibited significantly less cytotoxic CD8+ T-cells in their pancreata and adoptive transfer of their total T-cells did not induce diabetes in Rag1-/- mice. Our results indicate that inducing β-cell dedifferentiation, prior to insulitis, allows these cells to escape immune-mediated destruction and may be used as a novel preventive strategy for T1D in high-risk individuals.

  • Bulk-RNAseq
  • Single cell RNAseq
  • Adoptive transfer experiments
  • Immunophenotyping
  • Inflammation scoring
  • Metabolic assays
  • Islet histomorphometry
April 2020 A Genetic Toggle for Chemical Control of Individual Plk1 Substrates.

Johnson JM, Hebert AS, Drane QH, Lera RF, Wan J, Weaver BA, Coon JJ, Burkard ME.

Cell Chem Biol. 2020 Mar 19;27(3):350-362.e8. doi: 10.1016/j.chembiol.2020.01.007. Epub 2020 Feb 3.

Enzymes like kinases can often catalyze many molecular events, each setting off a separate signaling cascade. However, disentangling the complex diversity of the signaling events can be a challenge. James Johnson (Burkard lab) in collaboration with Alex Hebert (Coon lab) built and tested a new chemical genetic tool. By manipulating the kinase under study, Polo-like kinase 1, he was able to reprogram it to selectively identify either serine or threonine amino acids. Next, he recoded the substrate proteins to ‘toggle’ serine-and-threonine, thereby placing desired phosphorylation events under genetic control. This was validated with deep phosphoproteomics.

February 2020 The Rise of Physiologic Media.

Cantor JR.

Trends Cell Biol. 2019 Nov;29(11):854-861. doi: 10.1016/j.tcb.2019.08.009. Epub 2019 Oct 14. Review.

Conventional cell culture media has been a staple in scientific research for over 70 years. This media has remained relatively unchanged during this time. The major goal of this media is to keep cells alive and have them grow rapidly, but is this the best way? This review published by the Cantor looks at cell culture media from a different view point and articulates the importance of understanding how cells work in the human body. Further summary.

February 2020 Real-time health monitoring through urine metabolomics.

Miller IJ, Peters SR, Overmyer KA, Paulson BR, Westphall MS, Coon JJ.

NPJ Digit Med. 2019 Nov 11;2:109. doi: 10.1038/s41746-019-0185-y. eCollection 2019.

The advent of wearable devices for health monitoring is ushering in an era of more personalized and preventive medicine. However, most consumer-grade health data is currently limited to metrics such as step count and heart rate. We imagine technology that could enable the passive and continuous monitoring of metabolic health by measuring the diverse set of metabolites in urine. There are over 4,500 metabolites in human urine that are known to be associated with more than 600 human conditions. These metabolites can further reflect lifestyle choices, such as alcohol and tobacco consumption, in addition to exercise and physical activity. In this small pilot study, we collected every urine sample from two healthy volunteers for 10 days along with biohealth data (diet, sleep, exercise) from smart phone applications. Using gas chromatography and mass spectrometry we measured the relative levels of hundreds of individual metabolites and found patterns in the urine metabolites that reflected the ebb and flow of daily life. For instance, we could see associations with nutrition, OTC drug metabolism, exercise, and sleep. We plan to expand this type of study to a larger cohort and to develop technology that could be integrated into a “smart bathroom” and achieve passive measurements of metabolic health at scale.

  • Longitudinal metabolomics analysis using gas chromatography and mass spectrometry
  • Collection and integration of digital health data collected from smartphones and wearable devices.
February 2020 Hepatic stearoyl CoA desaturase 1 deficiency increases glucose uptake in adipose tissue partially through the PGC-1α-FGF21 axis in mice.

Aljohani A, Khan MI, Bonneville A, Guo C, Jeffery J, O'Neill L, Syed DN, Lewis SA, Burhans M, Mukhtar H, Ntambi JM

J Biol Chem. 2019 Dec 20;294(51):19475-19485. doi: 10.1074/jbc.RA119.009868. Epub 2019 Nov 5.

Increased carbohydrate consumption increases hepatic de novo lipogenesis, which has been linked to the development of chronic metabolic diseases, including obesity, hepatic steatosis, and insulin resistance. Stearoyl CoA desaturase 1 (SCD1) is a critical lipogenic enzyme that catalyzes the synthesis of two monounsaturated fatty acids, oleate and palmitoleate, from the saturated fatty acids stearate and palmitate, respectively. SCD1-deficient mouse models are protected against diet-induced adiposity, hepatic steatosis, and hyperglycemia. However, the mechanism of this protection by SCD1 deficiency is unclear. Using liver-specific SCD1 knockout (LKO) mice fed a high-carbohydrate, low-fat diet, we show that hepatic SCD1 deficiency increases systemic glucose uptake. Hepatic SCD1 deficiency enhanced glucose transporter type 1 (GLUT1) expression in the liver and also up-regulated GLUT4 and adiponectin expression in adipose tissue. The enhanced glucose uptake correlated with increased liver expression and elevated plasma levels of fibroblast growth factor 21 (FGF21), a hepatokine known to increase systemic insulin sensitivity and regulate whole-body lipid metabolism. Feeding LKO mice a triolein-supplemented but not tristearin-supplemented high-carbohydrate, low-fat diet reduced FGF21 expression and plasma levels. Consistently, SCD1 inhibition in primary hepatocytes induced FGF21 expression, which was repressed by treatment with oleate but not palmitoleate. Moreover, deletion of the transcriptional coactivator PPARγ coactivator 1α (PGC-1α) reduced hepatic and plasma FGF21 and white adipocyte tissue-specific GLUT4 expression and raised plasma glucose levels in LKO mice. These results suggest that hepatic oleate regulates glucose uptake in adipose tissue either directly or partially by modulating the hepatic PGC-1α-FGF21 axis.

  • Measurements of Plasma FGF21 levels using the FGF-21 Quantikine ELISA Kit.
  • in vivo 2-[3H] deoxyglucose uptake assays.
  • Positron Emission Tomography-computed tomography (PET/CT) imaging analysis.
February 2020 Quantitative Spatial Analysis of Metabolic Heterogeneity Across in vivo and in vitro Tumor Models.

Heaster TM, Landman BA, Skala MC.

Front Oncol. 2019 Nov 1;9:1144. doi: 10.3389/fonc.2019.01144. eCollection 2019.

The goal of this paper was to quantify the spatial distribution of tumor cells with distinct cell metabolism using multivariate spatial statistical tools. Autofluorescence images of tumors in vivo and in 3D culture revealed spatial patterns of cell metabolism specific to drug response and resistance. This quantitative analysis framework can be used to assess the effect of drugs on cell-level heterogeneity in tumors.

  • Two photon fluorescence lifetime imaging of NAD(P)H and FAD
  • Single cell segmentation
  • Multivariate spatial statistical analysis
November 2019 Evaporative cooling provides a major metabolic energy sink.

Kasza I, Adler D, Nelson DW, Eric Yen CL, Dumas S, Ntambi JM, MacDougald OA, Hernando D, Porter WP, Best FA, Alexander CM.

Mol Metab. 2019 Sep;27:47-61. doi: 10.1016/j.molmet.2019.06.023. Epub 2019 Jul 1. PMID: 31302039

Heat production (thermogenesis) in mammalian bodies creates the so-called b-adrenergic lipolytic environment which also serves to modify disease progression and inflammatory conditions. Furthermore, stimulation of thermogenesis could be used to combat obesity. However, the total demand for heat production is offset by heat losses through skin; this manuscript therefore focuses on the key properties of skin that determine heat loss. This interdisciplinary project relied on space science engineering collaborators to calculate the total energy sink through all modes of heat transfer. We found that a large fraction of total energy expenditure in mice can be accounted for by evaporative cooling, especially for mice with genetic lesions in skin lipogenesis. These mice are also resistant to diet-induced obesity, prompting us to speculate that skin cooling is a significant player in calorie disposition.

  • Analysis of heat transfer processes in biological systems, using infrared thermography (FLIR) and assay of trans-epidermal water loss (TEWL)
  • Assay of skins of genetically- and environmentally- modified mice
  • Evaluation of dermal white adipose tissues by immunofluorescent stains
  • Energy expenditure assays
November 2019 Metabolic Remodeling during Biofilm Development of Bacillus subtilis.

Pisithkul T, Schroeder JW, Trujillo EA, Yeesin P, Stevenson DM, Chaiamarit T, Coon JJ, Wang JD, Amador-Noguez D.

MBio. 2019 May 21;10(3). pii: e00623-19. doi: 10.1128/mBio.00623-19.

The ability to form biofilms, multicellular communities encapsulated by an extracellular matrix, is a widespread trait in bacteria. Using time-resolved metabolomic, fluxomic, and proteomic analyses we discovered a surprisingly widespread and dynamic remodeling of both primary and secondary metabolism during biofilm development in Bacillus subtilis. This work represents the most comprehensive and systematic characterization of metabolic alterations during bacterial biofilm development ever undertaken. Our results demonstrate that dynamic remodeling of metabolism is a critical, and heretofore unreported, component of the highly coordinated response that leads to biofilm development.

  • Targeted metabolomics
  • Shotgun proteomics
  • 13C isotope tracer experiments
November 2019 Gene loci associated with insulin secretion in islets from non-diabetic mice.

Keller MP, Rabaglia ME, Schueler KL, Stapleton DS, Gatti DM, Vincent M, Mitok KA, Wang Z, Ishimura T, Simonett SP, Emfinger CH, Das R, Beck T, Kendziorski C, Broman KW, Yandell BS, Churchill GA, Attie AD.

J Clin Invest. 2019 Jul 25;130:4419-4432. doi: 10.1172/JCI129143.

Type 2 diabetes occurs when pancreatic ß-cells are unable to keep up with the demand for insulin, often a consequence of insulin resistance. Human genetic studies show that most of the genetic predisposition to T2D results in a reduction of ß-cell insulin secretion or ß-cell mass. We used a genetically divers outbred population of mice to screen for genes that affect ß-cell insulin secretion. This was a heroic effort carried out by Mary Rabaglia, Kiki Schueler, Donnie Stapleton, and Shane Simonett and carefully supervised by Mark Keller. We collaborated with Gary Churchill at the Jackson Lab. The work identified multiple loci associated with insulin secretion. In three cases, we derived knockout mouse models and all showed an insulin secretion phenotype. With Josh Coon, we carried out proteomic and lipidomic analysis and have identified gene loci controlling protein and lipid abundance. With Federico Rey, we have connected the intestinal microbiome with genetic differences in serum bile acids. We now have multiple projects testing interesting candidate genes that have emerged from this screen.

  • insulin secretion measurements in isolated islets
  • mass spectrometry of proteins, lipids, and metabolites
  • systems biology analysis of pathways associated with metabolic disease
November 2019 Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction.

Martorelli Di Genova B, Wilson SK, Dubey JP, Knoll LJ.

PLoS Biol. 2019 Aug 20;17(8):e3000364. doi: 10.1371/journal.pbio.3000364. eCollection 2019 Aug.

It has long been a puzzle why felines are the only mammal permissive of the sexual cycle of Toxoplasma gondii. Cats lack intestinal delta-6-desaturase activity and as a consequence, they have systemic increases in linoleic acid. In this study, Morgridge Metabolism Interdisciplinary (MMI) fellow Bruno Martorelli Di Genova determined that inhibition of murine delta-6-desaturase and dietary supplementation with linoleic acid allowed Toxoplasma sexual development to occur in mice.

  • Intestinal organoid culture of cat and mouse cells
  • CRISPR-mediated genome edited mouse model
  • High resolution microscopy for Toxoplasma sexual stages 
November 2019 Hypothalamic mTORC2 is essential for metabolic health and longevity.

Chellappa K, Brinkman JA, Mukherjee S, Morrison M, Alotaibi MI, Carbajal KA, Alhadeff AL, Perron IJ, Yao R, Purdy CS, DeFelice DM, Wakai MH, Tomasiewicz J, Lin A, Meyer E, Peng Y, Arriola Apelo SI, Puglielli L, Betley JN, Paschos GK, Baur JA, Lamming DW.

Aging Cell. 2019 Oct; 18(5): e13014.

The mechanistic target of rapamycin (mTOR) is a conserved protein kinase that regulates growth and metabolism. Rapamycin, a drug that extends lifespan, promotes longevity through inhibition of mTORC1, but we have found that rapamycin also inhibits mTORC2. We find that hypothalamic mTORC2 signaling normally increases with age, and that mice genetically engineered to lack hypothalamic mTORC2 signaling display higher fat mass and impaired glucose homeostasis throughout life, become more frail with age, and have decreased overall survival. We conclude that hypothalamic mTORC2 is essential for the normal metabolic health, fitness, and lifespan of mice.

  • Phenotyping of glucoregulatory control, including in vivo glucose and insulin tolerance tests
  • Determination of body composition, activity and energy expenditure using metabolic chambers
  • Determination of lifespan and longitudinal assessment of frailty
November 2019 Pptc7 is an essential phosphatase for promoting mammalian mitochondrial metabolism and biogenesis.

Niemi NM, Wilson GM, Overmyer KA, Vogtle FN, Myketin L, Lohman DC, Schueler KL, Attie AD, Meisinger C, Coon JJ, and DJ Pagliarini

Nat Commun. 2019 Jul 19;10(1):3197. doi: 10.1038/s41467-019-11047-6.

Mitochondrial proteins are replete with phosphorylation, yet its functional relevance remains largely unclear. The presence of multiple resident mitochondrial phosphatases, however, suggests that protein dephosphorylation may be broadly important for calibrating mitochondrial activities. Here, we use CRISPR to delete the mitochondrial phosphatase Pptc7 in Mus musculus and find these mice exhibit hypoketotic hypoglycemia, elevated acylcarnitines and serum lactate, and die soon after birth. Pptc7−/− tissues have markedly diminished mitochondrial size and protein content despite normal transcript levels, and aberrantly elevated phosphorylation on select mitochondrial proteins. Overall, our data define Pptc7 as a protein phosphatase essential for proper mitochondrial function and biogenesis during the extrauterine transition.

  • Generation of a knockout mouse using CRISPR
  • Acylcarnitine profiling coupled with unbiased metabolomic and lipidomic analysis of perinatal mouse tissue
  • Phosphoproteomic and proteomic analysis of perinatal mouse tissue
  • Electron microscopy of mitochondria in perinatal mouse tissue
  • Mitochondrial import assays
November 2019 Acetyl-CoA flux regulates the proteome and acetyl-proteome to maintain intracellular metabolic crosstalk.

Dieterich IA, Lawton AJ, Peng Y, Yu Q, Rhoads TW, Overmyer KA, Cui Y, Armstrong EA, Howell PR, Burhans MS, Li L, Denu JM, Coon JJ, Anderson RM, Puglielli L.

Nature Communications. 2019; 10: 3929 (doi: 10.1038/s41467-019-11945-9).

AT-1/SLC33A1 is a key member of the endoplasmic reticulum (ER) acetylation machinery, transporting acetyl-CoA from the cytosol into the ER lumen where acetyl-CoA serves as the acetyl-group donor for Nε-lysine acetylation. Dysfunctional ER acetylation, as caused by heterozygous or homozygous mutations as well as gene duplication events of AT-1/SLC33A1, has been linked to both developmental and degenerative diseases. Here, we used two mouse models of AT-1 dysregulation to study how the cytosol-to-ER flux of acetyl-CoA promotes functional crosstalk between different intracellular organelles and compartments.

  • New mouse models
  • Biochemistry, histology, EM and diet manipulation
  • Quantitative proteomics
  • Stoichiometry of acetylation
  • Metabolomics
  • Primary cell isolation and culture
  • SIM microscopy
June 2019 Growth factor stimulation promotes multivesicular endosome biogenesis by prolongingrecruitment of the late-acting ESCRT machinery.

Quinney KB, Frankel EB, Shankar R, Kasberg W, Luong P, Audhya A.

Proc Natl Acad Sci U S A. 2019 Apr 2;116(14):6858-6867. doi: 10.1073/pnas.1817898116. Epub 2019 Mar 20.

Growth factor signaling plays a key role in directing metabolic flux within cells. In this work, we demonstrate a mechanism by which the mammalian ESCRT machinery acts to attenuate growth factor driven changes in metabolism using a combination of CRISPR-mediated genome editing, lattice light sheet microscopy, and super resolution STED microscopy.

  • CRISPR-mediated genome editing using mammalian cell lines
  • FACS sorting to identify clonal edited cell populations
  • Stimulated emission depletion (STED) microscopy at the UW-Madison Optical Imaging Core
  • Thin section electron microscopy in collaboration with the SMPH EM facility
  • Electron tomography using the TF30 transmission electron microscope
June 2019 Capturing site-specific heterogeneity with large-scale N-glycoproteome analysis.

Riley NM, Hebert AS, Westphall MS, Coon JJ.

Nat Commun. 2019 Mar 21;10(1):1311. doi: 10.1038/s41467-019-09222-w.

New technology is needed to advance the characterization of protein glycosylation, which is a prevalent, chemically complex, and biologically diverse post-translational modification that plays particularly important roles at the cell surface. Here we applied a new technology we developed called activated ion electron transfer dissociation (AI-ETD) to improve the identification of intact N-glycopeptides, and we also designed new visualization approaches to capture the heterogeneity of the glycoproteome at a systems scale.

  • Mass spectrometer instrumentation modifications
  • MS-based glycoproteomics
  • Development of new visualization tools
June 2019 A heterodimeric glutathione S-transferase that stereospecifically breaks lignin's β(R)-aryl ether bond reveals the diversity of bacterial β-etherases.

Kontur WS, Olmsted CN, Yusko LM, Niles AV, Walters KA, Beebe ET, Vander Meulen KA, Karlen SD, Gall DL, Noguera DR, Donohue TJ.

J Biol Chem. 2019 Feb 8;294(6):1877-1890. doi: 10.1074/jbc.RA118.006548. Epub 2018 Dec 12.

In this study, GLBRC researchers identified and characterized a new type of enzyme that specifically cleaves β-aryl ether bonds. This work demonstrates that there is more variability in the bacterial pathway for cleaving the β-aryl ether bond than previously thought, and illustrates the importance of studying this pathway – as well as other pathways involved in metabolizing lignin-derived compounds – in multiple species to better understand and be able to capitalize on this diversity. Characterizing microbial strategies for lignin breakdown is important for understanding plant biomass turnover in nature, and could aid in developing industrial systems for producing commodity chemicals from this abundant renewable resource.

  • Expression and purification of proteins from recombinant hosts and cell free systems
  • Kinetic analysis of wild type and mutant proteins (including determine of Km, Vmax and kcat)
  • Modeling of enzyme active site
  • HPLC of aromatic compounds and glutathione-aromatic conjugates
  • Phylogenetic analysis of protein databases
June 2019 Evolution of a novel chimeric maltotriose transporter in Saccharomyces eubayanus from parent proteins unable to perform this function.

Baker EP, Hittinger CT.

PLoS Genet. 2019 Apr 4;15(4):e1007786. doi: 10.1371/journal.pgen.1007786. eCollection 2019 Apr.

Here the Hittinger lab showed how the genes encoding maltose transporters could undergo ectopic gene conversion to produce genes encoding transporters for the bulkier sugar maltotriose, a critical sugar in wort or the malt extract used to brew beer. This adaptive laboratory evolution experiment showed how Saccharomyces eubayanus, the wild, cold-tolerant parent of hybrid lager yeasts, could gain this industrially important function.

  • Illumina sequencing (Biotechnology Center)
  • Bulk segregant analysis (Hittinger Lab)
  • Adaptive laboratory evolution (Hittinger Lab)
  • Phylogenetic analysis (Hittinger Lab)
June 2019 Fungal secondary metabolism: regulation, function and drug discovery.

Keller NP.

Nat Rev Microbiol. 2019 Mar;17(3):167-180. doi: 10.1038/s41579-018-0121-1.

The fungal secondary metabolome is both a primary source of new pharmaceuticals and an endogenous source of secondary metabolites important for fungal warfare, defense and development. This review provides up-to-date insights into how to harvest bioactive fungal secondary metabolites and to elucidate their roles in fungal ecology.

  • Fungal molecular biology (deletion, over-expression and mutation of genes)
  • Culture growth and extraction of fungal metabolites
  • Liquid chromatography, high resolution mass spectrometry, NMR analysis
  • Bioinfomatics
June 2019 The Metabolic Response to a Low Amino Acid Diet is Independent of Diet-Induced Shifts in the Composition of the Gut Microbiome.

Heidi H. Pak, Nicole E. Cummings, Cara L. Green, Jacqueline A. Brinkman, Deyang Yu, Jay L. Tomasiewicz, Shany E. Yang, Colin Boyle, Elizabeth N. Konon, Irene M. Ong & Dudley W. Lamming.

Sci Rep. 2019 Jan 11;9(1):67. doi: 10.1038/s41598-018-37177-3.

Low protein diets promote metabolic health in both humans and mice, but the mechanisms that drive the physiological benefits of these diets have not been determined. Here, we find that reducing dietary protein significantly alters the taxonomic composition of the gut microbiome, but that this shift does not mediate the metabolic benefits of a low protein diet.

  • 16S rRNA profiling, RNA-seq and all gene expression analysis
  • Phenotyping of glucoregulatory control, including in vivo glucose and insulin tolerance tests
  • Determination of body composition, activity and energy expenditure using metabolic chambers
June 2019 TAT1 and TAT2 tyrosine aminotransferases have both distinct and shared functions in tyrosine metabolism and degradation in Arabidopsis thaliana.

Wang M, Toda K, Block A, Maeda HA.

J Biol Chem. 2019 Mar 8;294(10):3563-3576. doi: 10.1074/jbc.RA118.006539. Epub 2019 Jan 10.

This study demonstrated that two tyrosine aminotransferases play key roles in tyrosine degradation and metabolism in Arabidopsis thaliana, by combining reverse genetics, metabolite profiling, and 13C-precursor time course feeding experiments.

  • 13C labeling measurement using GC-MS
  • Dark respiration measurement using LI-COR LI-6400XT
June 2019 Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic β-cells.

Gregg T, Sdao SM, Dhillon RS, Rensvold JW, Lewandowski SL, Pagliarini DJ, Denu JM, Merrins MJ.

J Biol Chem. 2019 Mar 22;294(12):4656-4666. doi: 10.1074/jbc.RA118.006085. Epub 2019 Jan 30.

Beta cell mitochondria play a central role in coupling glucose metabolism with insulin secretion. These studies identify a novel role for complex I in mediating the effects of obesity on the beta cell secretory pathway, and implicate cyclin-dependent kinase 1 (CDK1) signaling as the mechanism driving this effect. Two graduate students in the Merrins lab, Trilly Gregg and Sophie Sdao, spearheaded the project (https://www.merrinslab.org/publications).

  • Live-cell imaging of the beta cell secretory pathway, including real-time measurements of cytosolic citrate, ATP/ADP, and calcium
  • 2-photon fluorescence lifetime imaging of mitochondrial NAD(P)H
  • Direct measurements of electron transport chain fluxes using oxygen consumption rate measurements
June 2019 An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

Lohman DC, Aydin D, Von Bank HC, Smith RW, Linke V, Weisenhorn E, McDevitt MT, Hutchins P, Wilkerson EM, Wancewicz B, Russell J, Stefely MS, Beebe ET, Jochem A, Coon JJ, Bingman CA, Dal Peraro M, Pagliarini DJ.

Mol Cell. 2019 Feb 21;73(4):763-774.e10. doi: 10.1016/j.molcel.2018.11.033. Epub 2019 Jan 17.

Coenzyme Q is among the most hydrophobic molecules in nature, which presents challenges for its biosynthesis and delivery. In this paper, we describe how COQ9 uses accesses, binds, and presents CoQ precursors to another member of the biosynthesis machinery.

  • MS-based lipidomics with Josh Coon’s group
  • Protein purification
  • Crystallography with Craig Bingman and Bob Smith from the crystallography core
  • Liposome flotation assays (to assess protein binding to membranes of varying lipid composition)
  • Yeast growth assays
March 2018 Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys.

Rhoads TW, Burhans MS, Chen VB, Hutchins PD, Rush MJP, Clark JP, Stark JL, McIlwain SJ, Eghbalnia HR, Pavelec DM, Ong IM, Denu JM, Markley JL, Coon JJ, Colman RJ, Anderson RM.

Cell Metabolism. 2018 Mar 6;27(3):677-688.e5. doi: 10.1016/j.cmet.2018.01.014. PMCID: PMC5844481

A multi-disciplinary research team has uncovered new clues about calorie restriction and how it works to delay aging and age-related diseases. The researchers used a range of molecular profiling techniques to catalog over 20,000 molecules in rhesus liver. Data were analyzed using complex statistical approaches including machine learning techniques. The team showed that CR reprogrammed metabolism by harnessing distinct control mechanisms including RNA processing.

**Special Note: This publication was highlighted on the cover of the March 6, 2018 issue of Cell Metabolism.

    Next Generation Sequencing - Biotech Center
  • Mass Spectrometry Metabolomics/Proteomics - Coon Lab/Denu Lab
  • Nuclear Magnetic Resonance John Markely (NMR Fam)
  • Higher Order Statistical Modeling - Ong/McIlwain CPCP
March 2018 Ist1 regulates ESCRT-III assembly and function during multivesicular endosome biogenesis in Caenorhabditis elegans embryos.

Frankel EB, Shankar R, Moresco JJ, Yates JR 3rd, Volkmann N, Audhya A.

Nature Communications. 2017 Nov 13;8(1):1439. doi: 10.1038/s41467-017-01636-8. PMCID: PMC5682282.

The normal turnover of many integral membrane proteins is mediated by the formation of multivesicular endosomes, which sequester cargoes destined for degradation within intralumenal vesicles that bud away from the cytoplasm. In this work, we demonstrate a direct role for the ESCRT-III complex during membrane bending that is necessary to create intralumenal vesicles and define a new regulatory mechanism that preserves the continual action of the ESCRT machinery in repetitive cycles of vesicle biogenesis.

  • Immunogold labeling for electron microscopy
  • Super resolution (stimulated emission depletion) fluorescence microscopy
  • Electron tomography
March 2018 Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

Schilke BA, Ciesielski SJ, Ziegelhoffer T, Kamiya E, Tonelli M, Lee W, Cornilescu G, Hines JK, Markley JL, Craig EA.

PLoS Genet. 2017 Oct 30;13(10):e1007084. doi: 10.1371/journal.pgen.1007084. eCollection 2017 Oct. PMCID: PMC5679652.

This publication describes the isolation and analysis of gain-of-function mutations that overcome the requirement for an essential, conserved molecular chaperone. Results of analyses of isolated suppressor variants point to the idea that fine-tuning of Hsp70’s interaction cycle with substrate proteins can have major, unexpected consequences in vivo.

  • Yeast gain-of-function genetic selections
  • Structural analysis using NMR through National Magnetic Resonance Facility at Madison (NMRFAM)
  • Interaction studies using fluorescence anisotropy
March 2018 NRPS-Derived Isoquinolines and Lipopetides Mediate Antagonism between Plant Pathogenic Fungi and Bacteria.

Khalid S, Baccile JA, Spraker JE, Tannous J, Imran M, Schroeder FC, Keller NP.

ACS Chem Biol. 2018 Jan 19;13(1):171-179. doi: 10.1021/acschembio.7b00731. Epub 2017 Dec 18. PMID: 29182847

This work identifies antagonistic small molecule communication between two agricultural pathogens, the bacterium Ralstonia solanacearum and the fungus Aspergillus flavus. The fungal alkaloid product enhances fungal germination and slows bacterial growth but is inhibited in production by a bacterial lipopeptide.

  • Creation of genetic mutant strains of bacteria and fungi
  • RNA-seq and all gene expression analysis
  • Microbial physiology and statistical analysis
March 2018 Restoration of metabolic health by decreased consumption of branched-chain amino acids.

Cummings NE, Williams EM, Kasza I, Konon EN, Schaid MD, Schmidt BA, Poudel C, Sherman DS, Yu D, Arriola Apelo SI, Cottrell SE, Geiger G, Barnes ME, Wisinski JA, Fenske RJ, Matkowskyj KA, Kimple ME, Alexander CM, Merrins MJ, Lamming DW.

J Physiol. 2017 Dec 19. doi: 10.1113/JP275075. [Epub ahead of print]. PMID: 29266268

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in the blood of obese, insulin-resistant humans and rodents. In this work, we show that specifically reducing dietary BCAAs rapidly reverses diet-induced obesity and improves glucose tolerance and insulin sensitivity in diet-induced obese mice. Significantly, this occurs even in mice continuing to eat an otherwise unhealthy high-calorie, high-sugar “Western” diet. Reducing dietary levels of the BCAAs promotes leanness by increasing energy expenditure. Our results link dietary BCAAs with the regulation of metabolic health and energy balance in obese animals, and suggest that specifically reducing dietary BCAAs may represent a highly translatable option for the treatment of obesity and insulin resistance.

  • Feeding of amino acid defined diets to diet-induced obese mice
  • Phenotyping of glucoregulatory control, including in vivo glucose and insulin tolerance tests, and ex vivo characterization of pancreatic islet function and metabolism
  • Determination of body composition, activity and energy expenditure using metabolic chambers
March 2018 UCP1 deficiency increases adipose tissue monounsaturated fatty acid synthesis and trafficking to the liver.

Bond LM, Ntambi JM.

J Lipid Res. 2018 Feb;59(2):224-236. doi: 10.1194/jlr.M078469. Epub 2017 Dec 3. PMCID: PMC5794418.

This publication demonstrates that brown adipose tissue thermogenesis regulates whole body lipid homeostasis. Using mice lacking uncoupling protein-1, a protein critical for heat production, we revealed that loss of brown adipose tissue thermogenesis elevates the synthesis of monounsaturated fatty acids in white adipose tissue and causes triglyceride accumulation in the liver.

  • Analysis of fatty acid composition using gas chromatography
  • in vivo lipid synthesis and export assays
March 2018 Multi-omic mitoprotease profiling defines a role for Oct1p in coenzyme Q production.

Veling MT, Reidenbach AG, Freiberger EC, Kwiecien NW, Hutchins PD, Drahnak MJ, Jochem A, Ulbrich A, Rush JPR, Russell JD, Coon JJ, and Pagliarini DJ

Molecular Cell, 2017 doi: 10.1016/j.molcel.2017.11.023

March 2018 Multi-Omics Reveal Specific Targets of the RNA-Binding Protein Puf3p and Its Orchestration of Mitochondrial Biogenesis.

Lapointe CP, Stefely JA, Jochem A, Hutchins PD, Wilson G, Kwiecien NW, Coon JJ, Wickens MP, and Pagliarini DJ

Cell Systems, 2017 doi: 10.1016/j.cels.2017.11.012

March 2018 Conserved lipid and small molecule modulation of COQ8 reveals regulation of the ancient kinase-like UbiB family.

Reidenbach AG, Kemmerer ZA, Aydin D, Jochem A, McDevitt MT, Hutchins PD, Stark JL, Stefely, Reddy T, Hebert AS, Wilkerson EM, JA, Johnson IE, Bingman CA, Markley JL, Coon JJ, Dal Peraro M, and Pagliarini DJ

Cell Chemical Biology, 2017 doi.org/10.1016/j.chembiol.2017.11.001

In a three-paper series, we established new post-transcriptional regulatory processes for coenzyme Q (CoQ) metabolism. First, we discovered small- molecule and lipid activators for the atypical kinase-like protein COQ8 and developed an analog- sensitive version that can be selectively inhibited in vivo. Second, we used a multi-omic approach that incorporates measurements of mRNAs, proteins, lipids, and metabolites to identify endogenous targets for the mRNA binding protein, Puf3p, including the CoQ-related methyltransferase, COQ5. Finally, using a similar approach, we reveal numerous connections between mitoproteases and their biological functions, including a direct role for Oct1p in processes COQ5.

UW Collaborators: Josh Coon’s group, Marv Wicken’s group, John Markley’s group, and Craig Bingman

Further summary: https://morgridge.org/story/cracking-the-code-of-coenzyme-q-biosynthesis/

  • One-dimensional (1D) 1H nuclear magnetic resonance (NMR) ligand-affinity screen
  • Mass spec-based proteomics, metabolomics, and lipidomicsa
  • Liposome flotation analyses (i.e., protein interactions with liposomes)
  • Molecular modeling
  • Recombinant protein purification and various yeast growth assays
  • RNA binding protein analyses (RNA Tagging and HITS/CLIP)
October 2017 TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

Hanna MG 4th, Block S, Frankel EB, Hou F, Johnson A, Yuan L, Knight G, Moresco JJ, Yates JR 3rd, Ashton R, Schekman R, Tong Y, Audhya A.
Proc Natl Acad Sci U S A. 2017 Sep 12;114(37):E7707-E7716. doi: 10.1073/pnas.1709120114. Epub 2017 Aug 29. PMID: 28851831; PMCID: PMC5604033.

The endoplasmic reticulum (ER) serves as a platform for the packaging of most secretory proteins into conserved COPII-coated transport carriers destined for ER-Golgi intermediate compartments (ERGIC) in animal cells. In this work, we demonstrate that Trk-fused gene (TFG) simultaneously captures and concentrates COPII transport carriers at the ER/ERGIC interface to enable the rapid movement of secretory cargoes to the ERGIC.

  • CRISPR-mediated genome editing
  • Super resolution (stimulated emission depletion) fluorescence microscopy
  • In vitro reconstitution of vesicle tethering
October 2017 Chemical Genomics, Structure Elucidation, and in Vivo Studies of the Marine-Derived Anticlostridial Ecteinamycin.

Wyche TP, Alvarenga RFR, Piotrowski JS, Duster MN, Warrack SR, Cornilescu G, De Wolfe TJ, Hou Y, Braun DR, Ellis GA, Simpkins SW, Nelson J, Myers CL, Steele J, Mori H, Safdar N, Markley JL, Rajski SR, Bugni TS.
ACS Chem. Biol. 2017 Sep 15; 12(9):2287-2295 doi:10.1021/acschembio.7b00388. PMID: 28708379

This paper describes the isolation and structural characterization of the natural product ecteinamycin, the product of a marine-derived bacterium. Additionally, this paper strongly supports the hypothesis that ionophore antibiotics such as ecteinamycin and other more well established ionophores such as commonly used feed additives have tremendous potential as therapeutics for treating Clostridium difficile infections in humans. Ecteinamycin showed exceptional potency toward C. difficile, and yeast chemical genomics suggested that ecteinamycin disrupts vesicular trafficking pathways in eukaryotic cells that are required for C. difficile toxin maturation; impairment of such pathways appears to protect humans from the toxin effects of C. difficile.

  • LCMS-based metabolomics
  • Yeast chemical genomics using a DNA-barcoded knockout library
  • E. coli chemical genomics using DNA-barcoded knockout library
  • Residual Dipolar Couplings (RDCs) to assist with configurational analysis
October 2017 WBSCR16 is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion.

Huang G, Massoudi D, Muir AM, Joshi DC, Zhang C-L, Chiu, SY, Greenspan DS.
Cell Reports. 2017 July 25; 20(4): 923-934. doi: 10.1016/j.celrep.2017.06.090. PMID:28746876

This study identifies WBSCR16 as a protein that co-localizes with OPA1 on the outer surface of the inner mitochondrial membrane. It also shows WBSCR16 to act as a GEF for OPA1, a dynamin-like GTPase essential to mitochondrial fusion, which is in turn essential to mitochondrial function.

  • Gene mapping and deep sequencing to identify a spontaneous mutation in the Wbscr16 gene in mice
  • Mitochondrial subfractionation to localize proteins within different mitochondrial compartments
  • Use of a proximity ligation assay to demonstrate close enough proximity of WBSCR16 and OPA1 in situ for direct protein-protein interactions
  • GEF activity assays, in vitro and for intact mitochondria, to demonstrate a role for WBSCR16 as an intramitochondrial GEF with specific activity for OPA1
October 2017 A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation..

Meyer MB, Benkusky NA, Kaufmann M, Lee SM, Onal M, Jones G, Pike JW.
J Biol Chem. 2017 Aug 14. pii: jbc.M117.806901. doi: 10.1074/jbc.M117.806901. PMID: 28808057

In this paper, we describe the functional identification of a kidney-specific genomic regulatory region in the mouse that controls the expression of the Cyp27b1 gene whose enzymatic product is responsible for the final synthesis of 1α,25-dihydroxyvitamin D3, the biologically active hormonal form of vitamin D3.

  • ChIP-seq analysis of genetic and epigenetic features of mouse kidney tissue to define the locations and activity of novel regulatory regions of the Cyp27b1 gene
  • CRISPR/Cas9 gene-editing methods to delete potential regulatory regions in the mouse for subsequent loss-of-function analyses
  • Extensive systemic, regulatory and skeletal phenotyping to describe the consequence of aberrant Cyp27b1 regulation by mineral regulating hormones
July 2017 Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

Sato TK, Tremaine M, Parreiras LS, Hebert AS, Myers KS, Higbee AJ, Sardi M, McIlwain SJ, Ong IM, Breuer RJ, Avanasi Narasimhan R, McGee MA, Dickinson Q, La Reau A, Xie D, Tian M, Reed JL, Zhang Y, Coon JJ, Hittinger CT, Gasch AP, Landick R.
PLoS Genetics. 2016 Oct 14;12(10):e1006372. doi: 10.1371/journal.pgen.1006372. eCollection 2016 Oct. PMID: 27741250; PMCID: PMC5065143.

This publication describes the identification and molecular characterization of mutations in yeast that were engineering and evolved to ferment xylose, a pentose sugar that the organism does not normally metabolize. Using genetic, proteomic and metabolomic comparisons, it was determined that inactivating mutations in Fe-S cluster mitochondrial biogenesis, cAMP/Protein Kinase A and MAP Kinase signaling pathways enabled the conversion of xylose to ethanol.

  • High throughput genome resequencing (UW Biotech Center) and sequence analysis
  • Yeast genetic engineering and directed evolution
  • Proteome quantification
  • Intracellular and extracellular metabolite quantification
July 2017 Dual interaction of scaffold protein Tim44 of mitochondrial import motor with channel-forming translocase subunit Tim23.

Ting SY, Yan NL, Schilke BA, Craig EA.
Elife. 2017 Apr 25;6. pii: e23609. doi: 10.7554/eLife.23609. PMID: 28440746; PMCID: PMC5422074.

This publication reveals functional attributes of Tim44 the "hub" protein of the Hsp70 chaperone-based “import motor”, which is located in the matrix of mitochondria and required for the translocation of proteins into that subcompartment from the cytosol.

  • Site-specific in vivo crosslinking using photoactivatable alternative amino acid Bpa
  • in organellar mitochondria import assays
  • Yeast genetic suppressor analysis
July 2017 Inference and Evolutionary Analysis of Genome-Scale Regulatory Networks in Large Phylogenies.

Koch C, Konieczka J, Delorey T, Lyons A, Socha A, Davis K, Knaack SA, Thompson D, O'Shea EK, Regev A, Roy S.
Cell Systems. 2017 May 24;4(5):543-558.e8. doi: 10.1016/j.cels.2017.04.010. PMID: 28544882.

In this paper, we developed a new computational method to infer gene regulatory networks in multiple non-model species from global transcriptomic profiles. We used this approach to study how regulatory networks, associated with stress response, evolve across six yeast species.

  • Design and implementation of the multi-species network inference algorithm
  • Analytical measures used to study regulatory network evolution at the structure and function level.
July 2017 SCnorm: robust normalization of single-cell RNA-seq data.

Bacher R, Chu LF, Leng N, Gasch AP, Thomson JA, Stewart RM, Newton M, Kendziorski C.
Nat Methods. 2017 Jun;14(6):584-586. doi: 10.1038/nmeth.4263. Epub 2017 Apr 17. PMID: 28418000; PMCID: PMC5473255.

This publication presents a statistical method for normalizing data from single-cell RNA sequencing experiments. Normalization is a critical step required prior to downstream analysis to ensure that technical artifacts are removed and that gene expression profiles can be compared across cells.

  • Quality control, normalization, and downstream analysis of bulk and single-cell RNA-sequencing data.
July 2017 Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.

Schenck C.A., Holland C.K., Schneider M., Men Y., Lee S.G., Jez J. and Maeda H.A.
Nature Chemical Biology 2017 Jun 26. doi: 10.1038/nchembio.2414. PMID: 28671678.

This study conducted phylogeny-guided structure-function analyses of key regulatory enzymes of tyrosine biosynthesis (prephenate and arogenate dehydrogenases), discovered a single amino acid residue that confers their substrate specificity and feedback inhibition, and provided molecular basis of long-known two alternative tyrosine biosynthetic pathways.

  • Structure-guided phylogenetic analysis of proteins from distantly-related organisms.
  • Prephenate and arogenate dehydrogenase assays using a plate reader and LC-fluorescence detection.
  • Key amino acid residue identification by defining a precise evolutionary timing of enzyme neofunctionalization, followed by primary sequence and structure comparisons, and site-directed mutagenesis.
April 2017 Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network.

Kuang MC, Hutchins PD, Russell JD, Coon JJ, Hittinger CT. Elife. 2016 Sep 30;5. pii: e19027. doi: 10.7554/eLife.19027. PubMed PMID: 27690225; PubMed Central PMCID: PMC5089864.

In this publication, we showed that more active metabolic networks also require more robust repression systems. The paper also showcases some of the metabolomic capabilities of the LBMS facility.

  • Liquid chromatography-mass spectrometry (LC-MS) assays to measure central carbon metabolites by the Laboratory of Biomolecular Mass Spectrometry (LBMS)
  • Custom galactose-1-phosphatse assay developed by LBMS
April 2017 Ptc7p dephosphorylates select mitochondrial proteins to enhance metabolic function.

Guo X*, Niemi NM*, Hutchins PD, Condon SGF, Jochem A, Ulbrich A, Higbee AJ, Russell JD, Senes A, Coon JJ, and Pagliarini DJ,
Cell Reports, 2017 18: 1–7

This publication reveals that disruption of the poorly characterized mitochondrial phosphatase, Ptc7p, perturbs mitochondrial phosphorylation of ~20 matrix proteins, partially inactivates citrate synthase, and decreases mitochondrial respiratory function.

  • LC-MS/MS-based phosphoproteomics
  • Recombinant protein purification, including site-specific phospho-incorporation
  • Citrate synthase assays
  • Blue native PAGE
  • Yeast liquid culture and plate-based assays
  • Molecular modeling of point mutations
April 2017 Dietary Conjugated Linoleic Acid-c9t11 Prevents Collagen-Induced Arthritis, Whereas Conjugated Linoleic Acid-t10c12 Increases Arthritic Severity.

Muhlenbeck JA, Butz DE, Olson JM, Uribe-Cano D, Cook ME.
Lipids. 2017 Mar 15. doi: 10.1007/s11745-017-4241-6. [Epub ahead of print] PubMed PMID: 28299528.]

This publication shows slight alteration in the positioning and geometric configuration of double bonds in isomers of linoleic acid have substantial effects on the onset of arthritis. Tissue fatty acids and joint cytokines are also presented.

  • GC fatty acid analysis conducted in ME Cook lab
  • Lumines Systems Cytokine array analysis conducted in the shared Pharmacokinetics, Pharmacodynamics, Pharmacogenetics Laboratory (3Plab)
  • Arthritic mouse experiment conducted in Animal Sciences Vivarium

Measuring the molecules of life – Q&A with Josh Coon

Proteins are the workhorse molecules that perform all the functions in the cell and the body. Being able to detect and measure proteins is critical to figuring out basic biology, and the signature of diseases such as Alzheimer’s, cancer and diabetes. Josh Coon is creating technologies to do exactly that.

Regulating iron in the blood for optimal health – Q&A with Rick Eisenstein

Iron is an essential nutrient to human life, the element by which we regenerate red blood cells. Too little iron can cause serious problems such as anemia. But too much can be toxic, potentially causing blood clotting. Rick Eisenstein studies iron metabolism, with the goal of helping humans achieve the optimal balance for health.

Creating the blueprints for new biofuels – Q&A with Tim Donohue

Earlier this month, the Great Lakes Bioenergy Research Center (GLBRC) received another major boost from the U.S. Department of Energy, receiving more than $250 million to conduct another five years of groundbreaking work on alternative fuels.

What we can learn from hibernation – Q&A with Hannah Carey

Hannah Carey, a UW-Madison professor of comparative biosciences, uses hibernating mammals as models to study extreme changes in physiology and nutrition that occur on a seasonal basis. Carey discusses this remarkable process and its potential to impact human health by improving trauma care.