@article {67627, title = {In~vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {Cell Reports}, volume = {35}, year = {2021}, month = {2021 Jun 22}, pages = {109284}, abstract = {

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1\ μM to 10\ mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109284}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {64866, title = {In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor.}, journal = {bioRxiv}, year = {2019}, month = {2019 Mar 8}, abstract = {Glucose is arguably the most important molecule in metabolism, and its mismanagement underlies diseases of vast societal import, most notably diabetes. Although glucose-related metabolism has been the subject of intense study for over a century, tools to track glucose in living organisms with high spatio-temporal resolution are lacking. We describe the engineering of a family of genetically encoded glucose sensors with high signal-to-noise ratio, fast kinetics and affinities varying over four orders of magnitude (1 {\textmu}M to 10 mM). The sensors allow rigorous mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed \~{}3-fold higher glucose changes in astrocytes versus neurons. In larval Drosophila central nervous system explants, imaging of intracellular neuronal glucose suggested a novel rostro-caudal transport pathway in the ventral nerve cord neuropil, with paradoxically slower uptake into the peripheral cell bodies and brain lobes. In living zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized in real time. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory- and pharmacological perturbations gave rise to large but enigmatic changes in the brain. These sensors will enable myriad experiments, most notably rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.}, doi = {10.1101/571422}, author = {Keller, Jacob P and Marvin, Jonathan S and Lacin, Haluk and Lemon, William C and Shea, Jamien and Kim, Soomin and Lee, Richard T and Koyama, Minoru and Keller, Philipp J and Looger, Loren L} } @article {48732, title = {Genetically encoded biosensors.}, volume = {B2}, number = {14/350,199}, year = {2017}, month = {2017 Aug 01}, edition = {G01N 33/557 (20060101); G01N 33/58 (20060101); C07K 14/435 (20060101); C07K 14/245 (20060101); C07K 14/195 (20060101); G01N 33/68 (20060101)}, abstract = {

The present disclosure provides, inter alia, genetically encoded recombinant peptide biosensors comprising analyte-binding framework portions and signaling portions, wherein the signaling portions are present within the framework portions at sites or amino acid positions that undergo a conformational change upon interaction of the framework portion with an analyte.

}, url = {http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2\&Sect2=HITOFF\&u=\%2Fnetahtml\%2FPTO\%2Fsearch-adv.htm\&r=2\&p=1\&f=G\&l=50\&d=PTXT\&S1=(\%22Howard+Hughes+medical\%22.ASNM.)\&OS=AN/\%22Howard+Hughes+medical\%22\&RS=AN/\%22Howard+Hughes+medical\%22}, author = {Marvin, Jonathan S and Looger, Loren L and Lee, Richard T and Schreiter, Eric R} } @article {46414, title = {Common genetic variation at the IL1RL1 locus regulates IL-33/ST2 signaling.}, journal = {The Journal of Clinical Investigation}, volume = {123}, year = {2013}, month = {2013 Oct}, pages = {4208-18}, abstract = {

The suppression of tumorigenicity 2/IL-33 (ST2/IL-33) pathway has been implicated in several immune and inflammatory diseases. ST2 is produced as 2 isoforms. The membrane-bound isoform (ST2L) induces an immune response when bound to its ligand, IL-33. The other isoform is a soluble protein (sST2) that is thought to be a decoy receptor for IL-33 signaling. Elevated sST2 levels in serum are associated with an increased risk for cardiovascular disease. We investigated the determinants of sST2 plasma concentrations in 2,991 Framingham Offspring Cohort participants. While clinical and environmental factors explained some variation in sST2 levels, much of the variation in sST2 production was driven by genetic factors. In a genome-wide association study (GWAS), multiple SNPs within IL1RL1 (the gene encoding ST2) demonstrated associations with sST2 concentrations. Five missense variants of IL1RL1 correlated with higher sST2 levels in the GWAS and mapped to the intracellular domain of ST2, which is absent in sST2. In a cell culture model, IL1RL1 missense variants increased sST2 expression by inducing IL-33 expression and enhancing IL-33 responsiveness (via ST2L). Our data suggest that genetic variation in IL1RL1 can result in increased levels of sST2 and alter immune and inflammatory signaling through the ST2/IL-33 pathway.

}, keywords = {Amino Acid Substitution, Female, Gene Expression, Gene Expression Regulation, Genetic Association Studies, HEK293 Cells, Humans, Interleukins, Male, Middle Aged, Models, Molecular, Myelin and Lymphocyte-Associated Proteolipid Proteins, Myeloid Differentiation Factor 88, NF-kappa B, Polymorphism, Single Nucleotide, Promoter Regions, Genetic, Prospective Studies, Protein Structure, Tertiary, Receptors, Cell Surface, Signal Transduction, Transcription Factor AP-1}, issn = {1558-8238}, doi = {10.1172/JCI67119}, author = {Ho, Jennifer E and Chen, Wei-Yu and Chen, Ming-Huei and Larson, Martin G and McCabe, Elizabeth L and Cheng, Susan and Ghorbani, Anahita and Coglianese, Erin and Emilsson, Valur and Johnson, Andrew D and Walter, Stefan and Franceschini, Nora and O{\textquoteright}Donnell, Christopher J and Dehghan, Abbas and Lu, Chen and Levy, Daniel and Newton-Cheh, Christopher and Lin, Honghuang and Felix, Janine F and Schreiter, Eric R and Vasan, Ramachandran S and Januzzi, James L and Lee, Richard T and Wang, Thomas J} } @article {46410, title = {The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release.}, journal = {Journal of Cell Science}, volume = {123}, year = {2010}, month = {2010 Jul 1}, pages = {2308-18}, abstract = {

Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGF-like domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane pro-form from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HB-EGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

}, keywords = {Amino Acid Sequence, Amphiregulin, Animals, Cell Communication, Cell Proliferation, Cercopithecus aethiops, COS Cells, EGF Family of Proteins, Glycoproteins, Heparan Sulfate Proteoglycans, Heparin, Heparin-binding EGF-like Growth Factor, Intercellular Junctions, Intercellular Signaling Peptides and Proteins, Mice, Molecular Sequence Data, Mutation, Peptides, Protein Precursors, Protein Structure, Tertiary}, issn = {1477-9137}, doi = {10.1242/jcs.058321}, author = {Prince, Robin N and Schreiter, Eric R and Zou, Peng and Wiley, H Steven and Ting, Alice Y and Lee, Richard T and Lauffenburger, Douglas A} } @article {46409, title = {Thioredoxin-independent regulation of metabolism by the alpha-arrestin proteins.}, journal = {Journal of Biological Chemistry}, volume = {284}, year = {2009}, month = {2009 Sep 11}, pages = {24996-5003}, abstract = {

Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the alpha-arrestin protein family; the alpha-arrestins are related to the classical beta-arrestins and visual arrestins. Txnip is the only alpha-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved alpha-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related alpha-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast alpha-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of alpha-arrestin proteins.

}, keywords = {Amino Acid Sequence, Animals, Arrestin, Arrestins, Carrier Proteins, Fibroblasts, Gene Expression Regulation, Humans, Mice, Molecular Sequence Data, Mutation, Plasmids, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Thioredoxins}, issn = {1083-351X}, doi = {10.1074/jbc.M109.018093}, author = {Patwari, Parth and Chutkow, William A and Cummings, Kiersten and Verstraeten, Valerie L R M and Lammerding, Jan and Schreiter, Eric R and Lee, Richard T} } @article {46405, title = {IL-33 and ST2 comprise a critical biomechanically induced and cardioprotective signaling system.}, journal = {The Journal of Clinical Investigation}, volume = {117}, year = {2007}, month = {2007 Jun}, pages = {1538-49}, abstract = {

ST2 is an IL-1 receptor family member with transmembrane (ST2L) and soluble (sST2) isoforms. sST2 is a mechanically induced cardiomyocyte protein, and serum sST2 levels predict outcome in patients with acute myocardial infarction or chronic heart failure. Recently, IL-33 was identified as a functional ligand of ST2L, allowing exploration of the role of ST2 in myocardium. We found that IL-33 was a biomechanically induced protein predominantly synthesized by cardiac fibroblasts. IL-33 markedly antagonized angiotensin II- and phenylephrine-induced cardiomyocyte hypertrophy. Although IL-33 activated NF-kappaB, it inhibited angiotensin II- and phenylephrine-induced phosphorylation of inhibitor of NF-kappa B alpha (I kappa B alpha) and NF-kappaB nuclear binding activity. sST2 blocked antihypertrophic effects of IL-33, indicating that sST2 functions in myocardium as a soluble decoy receptor. Following pressure overload by transverse aortic constriction (TAC), ST2(-/-) mice had more left ventricular hypertrophy, more chamber dilation, reduced fractional shortening, more fibrosis, and impaired survival compared with WT littermates. Furthermore, recombinant IL-33 treatment reduced hypertrophy and fibrosis and improved survival after TAC in WT mice, but not in ST2(-/-) littermates. Thus, IL-33/ST2 signaling is a mechanically activated, cardioprotective fibroblast-cardiomyocyte paracrine system, which we believe to be novel. IL-33 may have therapeutic potential for beneficially regulating the myocardial response to overload.

}, keywords = {Animals, Biomechanical Phenomena, Cardiotonic Agents, Cells, Cultured, Fibroblasts, Humans, Interleukins, Membrane Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Myocytes, Cardiac, NF-kappa B, Rats, Receptors, Cell Surface, Receptors, Interleukin, Recombinant Proteins, Signal Transduction}, issn = {0021-9738}, doi = {10.1172/JCI30634}, author = {Sanada, Shoji and Hakuno, Daihiko and Higgins, Luke J and Schreiter, Eric R and McKenzie, Andrew N J and Lee, Richard T} } @article {46403, title = {Role of thioredoxin in cell growth through interactions with signaling molecules.}, journal = {Antioxidants and Redox Signaling}, volume = {8}, year = {2006}, month = {2006 Nov-Dec}, pages = {2143-51}, abstract = {

The thioredoxin system helps maintain a reducing environment in cells, but thioredoxin functions as more than simply an antioxidant. Thioredoxin functions depend on the protein{\textquoteright}s redox state, as determined by two conserved cysteines. Key biologic activities of thioredoxin include antioxidant, growth control, and antiapoptotic properties, resulting from interaction with target molecules including transcription factors. Mechanisms by which thioredoxin regulates cell growth include binding to signaling molecules such as apoptosis signal-regulating kinase-1 (ASK-1) and thioredoxin-interacting protein (Txnip). The molecular interplay between thioredoxin, ASK-1, and Txnip potentially influences cell growth and survival in diverse human diseases such as cancer, diabetes, and heart disease. In this review, we focus on the structure of thioredoxin and its functional regulation of cell growth through the interactions with signaling molecules.

}, keywords = {Carrier Proteins, Cell Growth Processes, Humans, MAP Kinase Kinase Kinase 5, Models, Molecular, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Signal Transduction, Thioredoxins}, issn = {1523-0864}, doi = {10.1089/ars.2006.8.2143}, author = {Yoshioka, Jun and Schreiter, Eric R and Lee, Richard T} }