DOCSLIB.ORG

Site-Specific Labeling of Cellular Proteins with Unnatural Substrates of Biotin Ligases by Irwin Chen AB, Chemistry

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Site-Specific Labeling of Cellular Proteins with Unnatural Substrates of Biotin Ligases by Irwin Chen AB, Chemistry Site-specific Labeling of Cellular Proteins with Unnatural Substrates of Biotin Ligases by Irwin Chen A.B., Chemistry (2000) Harvard University M.S., Chemistry (2002) Stanford University Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology September 2007 © 2007 Massachusetts Institute of Technology All rights reserved Signature of the Author: ____________________________________________________ Department of Chemistry June 5, 2007 Certified by: _____________________________________________________________ Alice Y. Ting Pfizer-Laubach Associate Professor of Chemistry Thesis Supervisor Accepted by: _____________________________________________________________ Robert W. Field Chairman, Departmental Committee on Graduate Students This doctoral thesis has been examined by a committee of the Department of Chemistry as follows: ________________________________________________________________________ Timothy M. Swager John D. MacArthur Professor of Chemistry and Head of the Department Chair ________________________________________________________________________ Alice Y. Ting Pfizer-Laubach Associate Professor of Chemistry Thesis Supervisor ________________________________________________________________________ Barbara Imperiali Class of 1922 Professor of Chemistry and Professor of Biology 2 Site-specific Labeling of Cellular Proteins with Unnatural Substrates of Biotin Ligases by Irwin Chen Submitted to the Department of Chemistry on June 5, 2007 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy ABSTRACT E. coli biotin ligase (BirA) catalyzes the site-specific ligation of biotin to the lysine within its 15-amino acid peptide substrate (AP). We harnessed the high peptide substrate specificity of BirA to develop a general methodology for site-specific protein labeling with biophysical probes. We synthesized an isosteric ketone analog of biotin and discovered that BirA also ligates it to the AP with similar kinetics while retaining the high peptide specificity of the native reaction. Because ketones are absent from native cell surfaces, recombinant AP-fused cell surface proteins can be tagged with the ketone analog, and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrated this two-step protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe were incorporated, with total labeling times as short as 20 minutes. To develop new protein labeling methodology, we synthesized azide analogs of biotin, but found that BirA did not accept them as substrates. Our efforts to rationally design mutations in the active site to accommodate these analogs met with little success. We thus investigated in vitro compartmentalization as a platform for re-engineering the substrate specificity of BirA through in vitro evolution. Selections did not enrich for BirA mutants that could catalyze ligation of the azide analog to the AP, but instead highlighted the technical difficulties of our in vitro compartmentalization strategy. We used phage display to engineer a new and orthogonal biotin ligase-acceptor peptide pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not biotinylate the AP, but we discovered a new 15-amino acid substrate for yBL, which we call the yeast acceptor peptide (yAP). The yAP is not biotinylated by BirA, and thus we were able to specifically label AP and yAP fusion proteins co-expressed in the same cell with differently colored quantum dots. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is also very sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast- based applications. Thesis Supervisor: Alice Y. Ting Title: Pfizer-Laubach Associate Professor of Chemistry 3 Acknowledgements I would first like to thank all of the people who have helped me edit this thesis. I thank my advisor, Professor Alice Y. Ting, for taking the time out to read drafts of most of my chapters. I also thank Dr. Hemanta Baruah and Yoon-Aa Choi for proofreading Chapters 5 and 6, respectively. Lastly, I am deeply grateful to my fiancée, Nicole DeBenedictis, for bravely venturing into the scientific arena and providing an extremely detailed and careful reading of my thesis for spelling and grammatical mistakes. Any remaining mistakes in this thesis are solely my own fault. Next, I thank the National Science Foundation Graduate Research Fellowship, the American Chemical Society Organic Division Fellowship sponsored by Wyeth Research, and Alice’s many grants for allowing me to live comfortably (for a graduate student) in Cambridge over the past five years. Lastly and most importantly, I am deeply grateful to many people who have contributed to my intellectual and personal growth over the past five years. First and foremost, I would like to thank Alice for her mentorship. I could never have imagined that my decision to join Alice’s fledgling lab would turn out so well. In the beginning she patiently taught me, a complete biology neophyte, all of the basic molecular biology techniques and knowledge that have finally become routine after over four years of practice. Towards the end of my graduate career, I appreciated her switch to a more hands-off approach, allowing me to try out my own ideas to solving problems. Throughout my time in her lab, her tireless enthusiasm and devotion to the members of her laboratory has been inspirational to me. From Alice I have learned how to improve my oral and written communication skills, which are critical to the success of any 4 scientific career. In particular, I thank her for the opportunities to present my work in front of her colleagues; those experiences have greatly helped me find a job and prepare my thesis defense. I remain deeply grateful to my former advisor at Stanford, Professor Barry Trost. His passion for science and devotion to being an educator continue to influence me. I am also grateful for the periodic kind words from Professor Justin Du Bois at Stanford. I thank Professor Stuart Licht for helpful discussions on kinetics and Professor K. Dane Wittrup for his help in troubleshooting yeast display. I also thank Professors Tim Swager and Barbara Imperiali for their helpful comments on this thesis. I am equally grateful to all past and present members of the Ting laboratory for teaching me valuable lessons in science and life. I wish you all the best of luck in your future endeavors. I thank Dr. Mark Howarth for his intellectual contributions to my first paper. I also thank Yoon-Aa for her enormous contributions to my last paper and Sarah Slavoff for enlightening me with her fascinating studies of other biotin ligases. I thank Hemanta, Yoon-Aa, Chi-wang Lin, Dr. Dan Chinnapen, Dr. Sujiet Puthenveetil, Dr. Yi Zheng, and Marta Fernandez-Suarez for their friendship and countless words of wisdom. I would like to give a special acknowledgement to the IVC subgroup (Hemanta, Dan, Yoon-Aa, Dr. Guobin Luo, E. Ken Hamill). I fondly remember our character-building struggles with “bands or no bands,” “the big dude,” and “Natasha.” I have also been blessed with the opportunity to work closely with a talented group of undergraduate researchers. I thank Eric McNeill for his great work on the Pauson-Khand route to “ketone biotin” and Lara Collazo for her invaluable help and 5 sunny disposition. I would also like to thank Jeremy Baskin, Karen Riesenburger, and Sean Liu for the fun times when we worked in Building 56. I would also like to thank my friends outside the MIT community for helping me maintain a life and perspective outside of the lab. In particular, I would like to thank Shai Sahay, Jon Man, Justin Saif, Mike Dillon, and Jimmy Young for their friendship. I am deeply grateful to my mother, father, and brother Ray for their kindness, empathy, generosity, and love over the years. Watching the Philadelphia Eagles play on Sunday afternoons with Ray was a great way to unwind after a long week of work. Finally, I am most grateful to my fiancée, Nicole. I am the lucky beneficiary of her patience, kindness, generosity, and love. I also thank her for stressing the importance of standing up for what I believe in, being proud of who I am, and adopting healthier dietary habits. My survival of the graduate school experience has greatly depended on her emotional support. I cannot imagine my future without her by my side. 6 Table of Contents Title Page ............................................................................................................................1 Signature Page....................................................................................................................2 Abstract...............................................................................................................................3 Acknowledgements ............................................................................................................4 Table of Contents ...............................................................................................................7 List
Load more

Recommended publications
  • B1–Proteases As Molecular Targets of Drug Development
    Abstracts B1–Proteases as Molecular Targets of Drug Development B1-001 lin release from the beta cells. Furthermore, GLP-1 also stimu- DPP-IV structure and inhibitor design lates beta cell growth and insulin biosynthesis, inhibits glucagon H. B. Rasmussen1, S. Branner1, N. Wagtmann3, J. R. Bjelke1 and secretion, reduces free fatty acids and delays gastric emptying. A. B. Kanstrup2 GLP-1 has therefore been suggested as a potentially new treat- 1Protein Engineering, Novo Nordisk A/S, Bagsvaerd, Denmark, ment for type 2 diabetes. However, GLP-1 is very rapidly degra- 2Medicinal Chemistry, Novo Nordisk A/S, Maaloev, Denmark, ded in the bloodstream by the enzyme dipeptidyl peptidase IV 3Discovery Biology, Novo Nordisk A/S, Maaloev, DENMARK. (DPP-IV; EC 3.4.14.5). A very promising approach to harvest E-mail: [email protected] the beneficial effect of GLP-1 in the treatment of diabetes is to inhibit the DPP-IV enzyme, thereby enhancing the levels of The incretin hormones GLP-1 and GIP are released from the gut endogenously intact circulating GLP-1. The three dimensional during meals, and serve as enhancers of glucose stimulated insu- structure of human DPP-IV in complex with various inhibitors 138 Abstracts creates a better understanding of the specificity and selectivity of drug-like transition-state inhibitors but can be utilized for the this enzyme and allows for further exploration and design of new design of non-transition-state inhibitors that compete for sub- therapeutic inhibitors. The majority of the currently known DPP- strate binding. Besides carrying out proteolytic activity, the IV inhibitors consist of an alpha amino acid pyrrolidine core, to ectodomain of memapsin 2 also interacts with APP leading to which substituents have been added to optimize affinity, potency, the endocytosis of both proteins into the endosomes where APP enzyme selectivity, oral bioavailability, and duration of action.
    [Show full text]
  • Gpi-Anchored Proteins in Reconstituted Lipid Bilayers
    GPI-ANCHORED PROTEINS IN RECONSTITUTED LIPID BILAYERS: STRUCTURE, FUNCTION, AND CLEAVAGE BY PI-SPECFIC PHOSPHOLIPASE C A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by MARTY T. LEHTO In partial fulfilrnent of requirements for the degree of Doctor of Philosophy August 2001 O Marty T. Lehto, 2001 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON Kt A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre r4f8me Our file Notre réfdrence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfonq vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels May be printed or othenuise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. GPI-ANCHORED PROTEINS IN RECONSTITUTED LEPID BILAYERS: STRUCTURE, FUNCTION AND CLEAVAGE BY PI-SPECFIC PHOSPHOLPASE C Marty T. Lehto Advisor: University of Guelph, 200 1 Professor F.J. Sharom Many eukaryotic proteins are anchored to the ce11 surface by a glycosylphosphatidylinositol (GPI) moiety.
    [Show full text]
  • Shedding and Uptake of Gangliosides and Glycosylphosphatidylinositol-Anchored Proteins ⁎ Gordan Lauc A,B, , Marija Heffer-Lauc C
    Biochimica et Biophysica Acta 1760 (2006) 584–602 http://www.elsevier.com/locate/bba Review Shedding and uptake of gangliosides and glycosylphosphatidylinositol-anchored proteins ⁎ Gordan Lauc a,b, , Marija Heffer-Lauc c a Department of Chemistry and Biochemistry, University of Osijek School of Medicine, J. Huttlera 4, 31000 Osijek, Croatia b Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia c Department of Biology, University of Osijek School of Medicine, J. Huttlera 4, 31000 Osijek, Croatia Received 30 September 2005; received in revised form 22 November 2005; accepted 23 November 2005 Available online 20 December 2005 Abstract Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins have very different biosynthetic origin, but they have one thing in common: they are both comprised of a relatively large hydrophilic moiety tethered to a membrane by a relatively small lipid tail. Both gangliosides and GPI-anchored proteins can be actively shed from the membrane of one cell and taken up by other cells by insertion of their lipid anchors into the cell membrane. The process of shedding and uptake of gangliosides and GPI-anchored proteins has been independently discovered in several disciplines during the last few decades, but these discoveries were largely ignored by people working in other areas of science. By bringing together results from these, sometimes very distant disciplines, in this review, we give an overview of current knowledge about shedding and uptake of gangliosides and GPI-anchored proteins. Tumor cells and some pathogens apparently misuse this process for their own advantage, but its real physiological functions remain to be discovered.
    [Show full text]
  • Progress in Lipid Research Progress in Lipid Research 46 (2007) 297–314 Review Non-Vesicular Sterol Transport in Cells
    Progress in Lipid Research Progress in Lipid Research 46 (2007) 297–314 www.elsevier.com/locate/plipres Review Non-vesicular sterol transport in cells William A. Prinz * Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services, Bethesda, MD 20892, USA Abstract Sterols such as cholesterol are important components of cellular membranes. They are not uniformly distributed among organelles and maintaining the proper distribution of sterols is critical for many cellular functions. Both vesicular and non- vesicular pathways move sterols between membranes and into and out of cells. There is growing evidence that a number of non-vesicular transport pathways operate in cells and, in the past few years, a number of proteins have been proposed to facilitate this transfer. Some are soluble sterol transfer proteins that may move sterol between membranes. Others are inte- gral membranes proteins that mediate sterol efflux, uptake from cells, and perhaps intracellular sterol transfer as well. In most cases, the mechanisms and regulation of these proteins remains poorly understood. This review summarizes our cur- rent knowledge of these proteins and how they could contribute to intracellular sterol trafficking and distribution. Published by Elsevier Ltd. Keywords: Cholestrol; Transport; Non-vesicular; Membranes; Lipid transport proteins Contents 1. Introduction ...................................................................... 298 2. Non-vesicular sterol transport pathways .................................................. 299 2.1. Transfer of cholesterol to the outer and inner mitochondrial membranes . .................... 300 2.2. PM to ERC sterol transport . ........................................................ 300 2.3. Sterol movement to and from lipid droplets (LDs). ........................................ 300 2.4.
    [Show full text]
  • NIH Public Access Author Manuscript Trends Biochem Sci
    NIH Public Access Author Manuscript Trends Biochem Sci. Author manuscript; available in PMC 2011 March 1. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Trends Biochem Sci. 2010 March ; 35(3): 150±160. doi:10.1016/j.tibs.2009.10.008. The Sec14-superfamily and mechanisms for crosstalk between lipid metabolism and lipid signaling Vytas A. Bankaitis1,*, Carl J. Mousley1, and Gabriel Schaaf2,* 1Department of Cell & Developmental Biology, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27526-7090, USA 2ZMBP, Plant Physiology, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany Abstract Lipid signaling pathways define central mechanisms of cellular regulation. Productive lipid signaling requires an orchestrated coupling0020between lipid metabolism, lipid organization, and the action of protein machines that execute appropriate downstream reactions. Using membrane trafficking control as primary context, we explore the idea that the Sec14-protein superfamily defines a set of modules engineered for the sensing of specific aspects of lipid metabolism and subsequent transduction of ‘sensing’ information to a phosphoinositide-driven ‘execution phase’. In this manner, the Sec14–superfamily connects diverse territories of the lipid metabolome with phosphoinositide signaling in a productive ‘crosstalk’ between these two systems. Mechanisms of crosstalk, where non-enzymatic proteins integrate metabolic cues with the action of interfacial enzymes, represent unappreciated regulatory themes in lipid signaling. Lipids and pathways for membrane trafficking The identities of proteins that regulate the membrane deformations required for biogenesis and fusion of transport vesicles were discovered by the pioneering studies of Rothman and Schekman some 25 years ago [reviewed in refs 1,2].
    [Show full text]
  • Biochemical Approaches for the Diagnosis and Treatment of Lafora Disease
    University of Kentucky UKnowledge Theses and Dissertations--Molecular and Cellular Biochemistry Molecular and Cellular Biochemistry 2019 BIOCHEMICAL APPROACHES FOR THE DIAGNOSIS AND TREATMENT OF LAFORA DISEASE Mary Kathryn Brewer University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0001-5089-3570 Digital Object Identifier: https://doi.org/10.13023/etd.2019.120 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Brewer, Mary Kathryn, "BIOCHEMICAL APPROACHES FOR THE DIAGNOSIS AND TREATMENT OF LAFORA DISEASE" (2019). Theses and Dissertations--Molecular and Cellular Biochemistry. 41. https://uknowledge.uky.edu/biochem_etds/41 This Doctoral Dissertation is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Molecular and Cellular Biochemistry by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known.
    [Show full text]
  • Regulation of Amyloid Processing in Neurons by Astrocyte-Derived Cholesterol
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.18.159632; this version posted June 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Regulation of amyloid processing in neurons by astrocyte-derived cholesterol Hao Wang1,2,3,6, Joshua A. Kulas4,5,6, Heather A. Ferris4,5*, Scott B. Hansen1,2* 1Department of Molecular Medicine, 2Department of Neuroscience, 3Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Jupiter, FL 33458, USA 4Division of Endocrinology and Metabolism, 5Department of Neuroscience, University of Virginia, Charlottesville, VA 22908, USA; 6these authors contributed eQually *Correspondence: [email protected], [email protected] ABSTRACT Alzheimer’s Disease (AD), a common and burdensome neurodegenerative disorder, is characterized by the presence of β-Amyloid (Aβ) plaques, inflammation, and loss of cognitive function. A cholesterol- dependent process sorts Aβ-producing enzymes into nanoscale lipid compartments (also called lipid rafts). Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic marker for sporadic AD. Evidence suggests apoE links to Aβ production through lipid rafts, but so far there has been little scientific validation of this link in vivo. Here we use super-resolution imaging to show apoE utilizes astrocyte-derived cholesterol to specifically traffic amyloid precursor protein (APP) into lipid rafts where it interacts with β- and g-secretases to generate Aβ-peptide. We find that targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid burden in a mouse model of AD.
    [Show full text]
  • 2006 Summer Research Program Office of Educational Programs Medical School
    2006 Summer Research Program Student Abstracts ii Contents Preface Acknowledgments Lab Research Ownership Index Medical Students ……………..……….……. 1 International Medical Students ……………... 3 Undergraduate Students …………..….……. 5 Abstracts – Medical Students ………….….... 7 Abstracts – International Medical Students… 43 Abstracts – Undergraduates ……….….…..... 51 Faculty Mentors ……………...……….……... 101 Departments ….………………….……..……. 105 Medical Student Class Photo ……...….…..… 109 Undergraduate Class Photo ……….….…..… 111 Student Abstracts, Volume XXI, Summer 2006 iii iv Preface The University of Texas at Houston Medical School (UTHMS) Summer Research Program provides intensive, hands-on laboratory research training for MS-1 medical students and undergraduate college students under the direct supervision of experienced faculty researchers and teachers. These faculty members’ enthusiasm for scientific discovery and commitment to teaching is vital for a successful training program. It is these dedicated scientists who organize the research projects to be conducted by the students. The trainee’s role in the laboratory is to participate to the fullest extent of her/his ability in the research project being performed. This involves carrying out the technical aspects of experimental analyses, interpreting data and summarizing results. The results are presented as an abstract and are written in the trainees’ own words that convey an impressive degree of understanding of the complex projects in which they were involved. To date, more than 1,500 students have gained research experience through the UTHMS Summer Research Program. Past trainees have advanced to pursue research careers in the biomedical sciences, as well as gain an appreciation of the relationship between basic and clinical research and clinical practice. This the second year of a new program which was initiated for international medical students from schools with whom we have cooperative agreements.
    [Show full text]
  • Phospholipase D Transduces Force to TREK-1 Channels in a Biological Membrane E
    bioRxiv preprint doi: https://doi.org/10.1101/758896; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Phospholipase D Transduces Force to TREK-1 Channels in a Biological Membrane E. Nicholas Petersen1,4, Manasa Gudheti5, Mahmud Arif Pavel1, Keith R. Murphy2,3, William W. Ja2,3, Erik M. Jorgensen5, Scott B. Hansen1* 1Departments of Molecular Medicine, 2Department of Neuroscience, 3Center on Aging, 4Scripps Research Skaggs Graduate School of Chemical and Biological Sciences, The Scripps Research Institute, Scripps, Florida 33458, USA 5Department of Biology, Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA *Correspondence: [email protected] ABSTRACT ordered proteins and lipids is a non-canonical sensor of mechanical force in biological The transduction of force into a biological signal is membranes4,5. Force was shown to disassemble critical to all living organisms. Recently, disruption and flatten caveolae4 and force disrupts ordered of ordered lipids has emerged as an ‘atypical’ force lipids and lipid clusters5. The ordered phase, sensor in biological membranes; however, sometimes called a lipid raft, is enriched in disruption has yet to link with canonical channel saturated lipids and palmitoylated proteins6,7 and is mechanosensation. Here we show that force- subject to disruption (see Supplemental Fig. 1a-b). induced disruption and lipid mixing activates TWIK- + In C2C12 muscle cells, chemical disruption related K channel (TREK-1), and that this releases a palmitoylated protein phospholipase D activation is dependent on phospholipase D2 (PLD2) from lipid rafts activating a mechano- (PLD2).
    [Show full text]
  • Studies on the Mechanism of Membrane Mediated General Anesthesia
    bioRxiv preprint doi: https://doi.org/10.1101/313973; this version posted June 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Studies on the mechanism of membrane mediated general anesthesia. 2 3 Mahmud Arif Pavel1,2, E. Nicholas Petersen1,2, Hao Wang1,2, Richard A. Lerner3, and Scott B. 4 Hansen1,2,* 5 6 1Department of Molecular Medicine, 2Department of Neuroscience, The Scripps Research 7 Institute, Jupiter, Florida 33458, USA. 8 3Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, USA 9 10 *Correspondence: [email protected] 11 12 ABSTRACT 13 Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly 14 activate TWIK related K+ channels (TREK-1) and reversibly induce loss of consciousness. For a 15 hundred years anesthetics were speculated to target cellular membranes, yet no plausible 16 mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled 17 anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of palmitate- 18 mediated localization of phospholipase D2 (PLD2) to lipid rafts and subsequent production of 19 signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK- 20 1 currents in cell patch-clamp. Localization of PLD2 renders the anesthetic-insensitive TRAAK 21 channel sensitive. General anesthetics chloroform, isoflurane, diethyl ether, xenon, and propofol 22 disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and 23 PLDnull flies resist anesthesia. Our results establish a membrane mediated target of inhaled 24 anesthesia and suggest PA helps set anesthetic sensitivity in vivo.
    [Show full text]
  • Symposium 1: Functional Genomics, Proteomics and Bioinformatics 1.1
    Symposium Abstracts SYMPOSIUM 1: FUNCTIONAL GENOMICS, PROTEOMICS AND BIOINFORMATICS 1.1. Epigenetics: DNA Methylation and Far Beyond IL 1.1–1 where through epigenetic means nonequivalent sister chromatids The role of MeCP2 in the brain are generated by chromosome replication. Second, epigenetic states controlling gene repression are inherited in mitosis and A. Bird, P. Skene, R. Illingworth and J. Guy Wellcome Trust Centre for Cell Biology, University of Edinburgh, meiosis as remarkably stable conventional Mendelian markers (1). Edinburgh, UK We propose that likewise asymmetric cell divisions in higher eukaryotes might result by further postulating biased segregation The DNA of every cell in the body carries a pattern of chemical of differentiated sister chromatids of both copies of a specific modifications due to the methylation of cytosine in the dinucleo- chromosome to daughter cells (2,3). Can we explain hitherto tide sequence 5¢CG. It is thought that these chemical marks help unexplained developmental traits/disorders in humans and verte- to define the pattern of gene expression that is appropriate for brates by invoking such principles? The causes of schizophrenia each cell type. The nuclear protein MeCP2 was originally discov- and bipolar human psychiatric disorders are unknown. A novel ered because of its ability to specifically bind to methylated CG somatic cell genetics, SSIS (Somatic Strand-specific Imprinting sites, but not to CGs lacking the methyl moiety. Because of its and Selective strand segregation) model, postulated biased segre- DNA binding preference, it was hypothesised that MeCP2 inter- gation of differentiated older ‘Watson’ versus ‘Crick’ DNA chains prets the DNA methylation signal.
    [Show full text]
  • Molecular Mechanism of General Anesthesia Genel Anesteziklerin Moleküler Mekanizması
    DERLEME Experimed 2020; 10(3): 140-3 REVIEW DOI: 10.26650/experimed.2020.824840 Molecular Mechanism of General Anesthesia Genel Anesteziklerin Moleküler Mekanizması Özge Köner1 , Sibel Temür1 , Turgay İsbir2 1Department of Anesthesiology and Reanimation, Yeditepe University, Istanbul, Turkey 2Department of Molecular Medicine, Yeditepe University, Istanbul, Turkey ORCID ID: Ö.K. 0000-0002-5618-2216; S.T. 0000-0002-4494-2265; T.İ. 0000-0002-7350-6032 Cite this article as: Köner Ö, Temür S, İsbir T. Molecular Mechanism of General Anesthesia. Experimed 2020; 10(3): 140-3. ABSTRACT ÖZ Despite the widespread changes induced by general anesthetic Genel anestezik ajanlar, nörotransmitterleri modüle ederek santral agents, their exact effect sites are not clearly defined in the central sinir sisteminde yaygın nöronal değişime neden olmaktadır. Yeni nervous system (CNS). Recent molecular studies have pointed out yapılan moleküler araştırmalarda anestezik ajanların etki ettiği specific sites in CNS on which anesthetic drugs show their effects. spesifik alanlar üzerinde durulmaktadır. Hipnoz, amnezi, sedasyon Hypnosis, amnesia, sedation are mediated by different receptors, santral sinir sisteminde farklı reseptör, nörotransmitter ve nöronal neurotransmitters and neuronal pathways in the CNS. Protein base yolaklar aracılığıyla sağlanır. 1980'lerin başında yapılan çalışmalar theory of anesthesia, which focuses on ion channels, took the sonrasında, iyon kanallarına odaklanan protein temelli anestezi te- place of lipid-based theory in the 1980’s. There are two types of orisi, lipid temelli anestezi teorisinin yerini almıştır. Protein temelli receptors, which are known to be responsible for the general anes- teoriye göre, genel anestezik etkiden sorumlu iki tip reseptör mev- thetic action: neurotransmitter receptors and ion channels.
    [Show full text]