The Interaction of Proline-Rich Motifs in Signaling Proteins with Their Cognate Domains
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Metabolic Engineering of Escherichia Coli for Poly(3-Hydroxybutyrate)
Lin et al. Microb Cell Fact (2015) 14:185 DOI 10.1186/s12934-015-0369-3 RESEARCH Open Access Metabolic engineering of Escherichia coli for poly(3‑hydroxybutyrate) production via threonine bypass Zhenquan Lin1,2,3†, Yan Zhang1,2,3†, Qianqian Yuan1,2,3,4, Qiaojie Liu1,2,3, Yifan Li1,2,3, Zhiwen Wang1,2,3, Hongwu Ma4*, Tao Chen1,2,3,5* and Xueming Zhao1,2,3 Abstract Background: Poly(3-hydroxybutyrate) (PHB), have been considered to be good candidates for completely biode- gradable polymers due to their similar mechanical properties to petroleum-derived polymers and complete biodeg- radability. Escherichia coli has been used to simulate the distribution of metabolic fluxes in recombinant E. coli pro- ducing poly(3-hydroxybutyrate) (PHB). Genome-scale metabolic network analysis can reveal unexpected metabolic engineering strategies to improve the production of biochemicals and biofuels. Results: In this study, we reported the discovery of a new pathway called threonine bypass by flux balance analysis of the genome-scale metabolic model of E. coli. This pathway, mainly containing the reactions for threonine synthesis and degradation, can potentially increase the yield of PHB and other acetyl-CoA derived products by reutilizing the CO2 released at the pyruvate dehydrogenase step. To implement the threonine bypass for PHB production in E. coli, we deregulated the threonine and serine degradation pathway and enhanced the threonine synthesis, resulting in 2.23-fold improvement of PHB titer. Then, we overexpressed glyA to enhance the conversion of glycine to serine and activated transhydrogenase to generate NADPH required in the threonine bypass. -
The Role of Agmatine and Arginine Decarboxylase in Ischemic Tolerance After Transient Cerebral Ischemia in Rat Models
The role of agmatine and arginine decarboxylase in ischemic tolerance after transient cerebral ischemia in rat models Jin Young Jung Department of Medicine The Graduate School, Yonsei University The role of agmatine and arginine decarboxylase in ischemic tolerance after transient cerebral ischemia in rat models Directed by Professor Seung Kon Huh The Doctoral Dissertation submitted to the Department of Medicine, the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Jin Young Jung May 2007 This certifies that the Doctoral Dissertation of Jin Young Jung is approved. __________________________________ Thesis Supervisor: Seung Kon Huh __________________________________ Jong Eun Lee: Thesis Committee Member #1 __________________________________ Jin Woo Chang: Thesis Committee Member #2 __________________________________ Duck Sun Ahn: Thesis Committee Member #3 __________________________________ Ji Cheol Shin: Thesis Committee Member #4 The Graduate School Yonsei University May 2007 Acknowledgements Some may consider this short section of the thesis trivial but for me it is a chance to express my sincerest gratitude to those that I am truly thankful. First of all, I would like to express my deepest gratitude to my thesis supervisor and mentor Professor Seung Kon Huh. He has inspired me when I was troubled and always gave me a warm heart. I would also like to thank Professor Jong Eun Lee who shared her valuable time on the execution and interpretation of this study, Professor Jin Woo Chang who always inspiring me with passion and discerning insight. Professor Duck Sun Ahn whose insightful comments were essential in completing this thesis, Professor Ji Cheol Shin for the excellent suggestion for improvement in this thesis. -
Paxillin: a Focal Adhesion-Associated Adaptor Protein
Oncogene (2001) 20, 6459 ± 6472 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Paxillin: a focal adhesion-associated adaptor protein Michael D Schaller*,1 1Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center and Comprehensive Center for In¯ammatory Disorders, University of North Carolina, Chapel Hill, North Carolina, NC 27599, USA Paxillin is a focal adhesion-associated, phosphotyrosine- The molecular cloning of paxillin revealed a number containing protein that may play a role in several of motifs that are now known to function in mediating signaling pathways. Paxillin contains a number of motifs protein ± protein interactions (see Figure 1) (Turner that mediate protein ± protein interactions, including LD and Miller, 1994; Salgia et al., 1995a). The N-terminal motifs, LIM domains, an SH3 domain-binding site and half of paxillin contains a proline-rich region that SH2 domain-binding sites. These motifs serve as docking could serve as an SH3 domain-binding site. Several sites for cytoskeletal proteins, tyrosine kinases, serine/ tyrosine residues conforming to SH2 domain binding threonine kinases, GTPase activating proteins and other sites were also noted. In addition, the N-terminal adaptor proteins that recruit additional enzymes into domain of paxillin contains ®ve copies of a peptide complex with paxillin. Thus paxillin itself serves as a sequence, called the LD motif, which are now known docking protein to recruit signaling molecules to a to function as binding sites for other proteins (see speci®c cellular compartment, the focal adhesions, and/ Table 1) (Brown et al., 1998a). The C-terminal half of or to recruit speci®c combinations of signaling molecules paxillin is comprised of four LIM domains, which are into a complex to coordinate downstream signaling. -
Amino Acid Chemistry
Handout 4 Amino Acid and Protein Chemistry ANSC 619 PHYSIOLOGICAL CHEMISTRY OF LIVESTOCK SPECIES Amino Acid Chemistry I. Chemistry of amino acids A. General amino acid structure + HN3- 1. All amino acids are carboxylic acids, i.e., they have a –COOH group at the #1 carbon. 2. All amino acids contain an amino group at the #2 carbon (may amino acids have a second amino group). 3. All amino acids are zwitterions – they contain both positive and negative charges at physiological pH. II. Essential and nonessential amino acids A. Nonessential amino acids: can make the carbon skeleton 1. From glycolysis. 2. From the TCA cycle. B. Nonessential if it can be made from an essential amino acid. 1. Amino acid "sparing". 2. May still be essential under some conditions. C. Essential amino acids 1. Branched chain amino acids (isoleucine, leucine and valine) 2. Lysine 3. Methionine 4. Phenyalanine 5. Threonine 6. Tryptophan 1 Handout 4 Amino Acid and Protein Chemistry D. Essential during rapid growth or for optimal health 1. Arginine 2. Histidine E. Nonessential amino acids 1. Alanine (from pyruvate) 2. Aspartate, asparagine (from oxaloacetate) 3. Cysteine (from serine and methionine) 4. Glutamate, glutamine (from α-ketoglutarate) 5. Glycine (from serine) 6. Proline (from glutamate) 7. Serine (from 3-phosphoglycerate) 8. Tyrosine (from phenylalanine) E. Nonessential and not required for protein synthesis 1. Hydroxyproline (made postranslationally from proline) 2. Hydroxylysine (made postranslationally from lysine) III. Acidic, basic, polar, and hydrophobic amino acids A. Acidic amino acids: amino acids that can donate a hydrogen ion (proton) and thereby decrease pH in an aqueous solution 1. -
Insights Into the Mn Binding Site in the Agmatinase-Like Protein (ALP): A
International Journal of Molecular Sciences Article Insights into the Mn2+ Binding Site in the Agmatinase-Like Protein (ALP): A Critical Enzyme for the Regulation of Agmatine Levels in Mammals María-Belen Reyes 1, José Martínez-Oyanedel 1,*, Camila Navarrete 1, Erika Mardones 1, Ignacio Martínez 1,Mónica Salas 2, Vasthi López 3, María García-Robles 4, Estefania Tarifeño-Saldivia 1, Maximiliano Figueroa 1, David García 1 and Elena Uribe 1,* 1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 4070386, Chile; [email protected] (M.-B.R.); [email protected] (C.N.); [email protected] (E.M.); [email protected] (I.M.); [email protected] (E.T.-S.); [email protected] (M.F.); [email protected] (D.G.) 2 Instituto de Bioquímica y Microbiología, Universidad Austral de Chile, Valdivia 5110566, Chile; [email protected] 3 Departamento de Ciencias Biomédicas, Universidad Católica del Norte, Coquimbo 1781421, Chile; [email protected] 4 Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción 3349001, Chile; [email protected] * Correspondence: [email protected] (J.M.-O.); [email protected] (E.U.) Received: 28 April 2020; Accepted: 5 June 2020; Published: 10 June 2020 Abstract: Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. -
The PX Domain Protein Interaction Network in Yeast
The PX domain protein interaction network in yeast Zur Erlangung des akademischen Grades eines DOKTORS DER NATURWISSENSCHAFTEN (Dr. rer. nat.) der Fakultät für Chemie und Biowissenschaften der Universität Karlsruhe (TH) vorgelegte DISSERTATION von Dipl. Biol. Carolina S. Müller aus Buenos Aires Dekan: Prof. Dr. Manfred Kappes Referent: Dr. Nils Johnsson Korreferent: HD. Dr. Adam Bertl Tag der mündlichen Prüfung: 17.02.2005 I dedicate this work to my Parents and Alex TABLE OF CONTENTS Table of contents Introduction 1 Yeast as a model organism in proteome analysis 1 Protein-protein interactions 2 Protein Domains in Yeast 3 Classification of protein interaction domains 3 Phosphoinositides 5 Function 5 Structure 5 Biochemistry 6 Localization 7 Lipid Binding Domains 8 The PX domain 10 Function of PX domain containing proteins 10 PX domain structure and PI binding affinities 10 Yeast PX domain containing proteins 13 PX domain and protein-protein interactions 13 Lipid binding domains and protein-protein interactions 14 The PX-only proteins Grd19p and Ypt35p and their phenotypes 15 Aim of my PhD work 16 Project outline 16 Searching for interacting partners 16 Confirmation of obtained interactions via a 16 second independent method Mapping the interacting region 16 The Two-Hybrid System 17 Definition 17 Basic Principle of the classical Yeast-Two Hybrid System 17 Peptide Synthesis 18 SPOT synthesis technique 18 Analysis of protein- peptide contact sites based on SPOT synthesis 19 TABLE OF CONTENTS Experimental procedures 21 Yeast two-hybrid assay -
The Role of Some of the Krebs Cycle Reactions in the Biosynthetic Functions of Thiobacillus Thioparus
AN ABSTRACT OF THE THESIS OF Gerald G. Still for the PhD in Chemistry (Name) (Degree) (Major) Date thesis is presented May 14, 1965 Title THE ROLE OF SOME OF THE KREBS CYCLE REACTIONS IN THE BIOSYNTHETIC FUNCTIONS OF THIOBACILLUS THIOPARUS Abstract approved Redacted for Privacy (Major professor) Aseptic radiorespirometry has been used to examine the utilization of external carbon sources by proliferat- ing Thiobacillus thioparus cells. These studies reveal that glucose, galactose, mannose, fructose, ribose, DL- glutamate, and L- aspartate were not utilized by this chemoautotrophic organism. However, it has been shown that trace amounts of acetate, glycine, DL- serine, DL- alanine, succinate and fumarate can be utilized by T. thioparus cells. To elucidate the nature of the biosynthetic pathways operative in this bacteria, proliferating cell cultures were allowed to metabolize specifically 14C labeled substrates. The resulting 14C labeled cells were sub- sequently hydrolyzed, their amino acids isolated and subjected to degradation experiments. Examination of the respective fates of the label in DL- alanine- 2 -14C, acetate- 1 -14C, or acetate -2 -14C in the cellular metabolism revealed that the Krebs Cycle path- way is not functioning as a respiratory mechanism in T. thioparus. However, most of the reactions of the Krebs Cycle pathway are involved in the biosynthesis of carbon skeletons for various amino acids. A CO2 fixa- tion pathway of the C3 +C1 type is instrumental in provid- ing C4 dicarboxylic acids and those amino acids derived therefrom. Acetate can be incorporated into a -keto- glutarate and those amino acids derived therefrom, but cannot be incorporated into the C4 dicarboxylic acids. -
Solutions to 7.012 Problem Set 1
MIT Biology Department 7.012: Introductory Biology - Fall 2004 Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel Solutions to 7.012 Problem Set 1 Question 1 Bob, a student taking 7.012, looks at a long-standing puddle outside his dorm window. Curious as to what was growing in the cloudy water, he takes a sample to his TA, Brad Student. He wanted to know whether the organisms in the sample were prokaryotic or eukaryotic. a) Give an example of a prokaryotic and a eukaryotic organism. Prokaryotic: Eukaryotic: All bacteria Yeast, fungi, any animial or plant b) Using a light microscope, how could he tell the difference between a prokaryotic organism and a eukaryotic one? The resolution of the light microscope would allow you to see if the cell had a true nucleus or organelles. A cell with a true nucleus and organelles would be eukaryotic. You could also determine size, but that may not be sufficient to establish whether a cell is prokaryotic or eukaryotic. c) What additional differences exist between prokaryotic and eukaryotic organisms? Any answer from above also fine here. In addition, prokaryotic and eukaryotic organisms differ at the DNA level. Eukaryotes have more complex genomes than prokaryotes do. Question 2 A new startup company hires you to help with their product development. Your task is to find a protein that interacts with a polysaccharide. a) You find a large protein that has a single binding site for the polysaccharide cellulose. Which amino acids might you expect to find in the binding pocket of the protein? What is the strongest type of interaction possible between these amino acids and the cellulose? Cellulose is a polymer of glucose and as such has many free hydroxyl groups. -
Biomoleculesbiomolecules
1414Unit Objectives BiomoleculesBiomolecules After studying this Unit, you will be able to • explain the characteristics of “It is the harmonious and synchronous progress of chemical biomolecules like carbohydrates, reactions in body which leads to life”. proteins and nucleic acids and hormones; • classify carbohydrates, proteins, A living system grows, sustains and reproduces itself. nucleic acids and vitamins on the The most amazing thing about a living system is that it basis of their structures; is composed of non-living atoms and molecules. The • explain the difference between pursuit of knowledge of what goes on chemically within DNA and RNA; a living system falls in the domain of biochemistry. Living • describe the role of biomolecules systems are made up of various complex biomolecules in biosystem. like carbohydrates, proteins, nucleic acids, lipids, etc. Proteins and carbohydrates are essential constituents of our food. These biomolecules interact with each other and constitute the molecular logic of life processes. In addition, some simple molecules like vitamins and mineral salts also play an important role in the functions of organisms. Structures and functions of some of these biomolecules are discussed in this Unit. 14.114.114.1 Carbohydrates Carbohydrates are primarily produced by plants and form a very large group of naturally occurring organic compounds. Some common examples of carbohydrates are cane sugar, glucose, starch, etc. Most of them have a general formula, Cx(H2O)y, and were considered as hydrates of carbon from where the name carbohydrate was derived. For example, the molecular formula of glucose (C6H12O6) fits into this general formula, C6(H2O)6. But all the compounds which fit into this formula may not be classified as carbohydrates. -
Insulin Receptor Tyrosine Kinase Substrate Links the E. Coli O157:H7
Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspFU during pedestal formation Didier Vingadassaloma, Arunas Kazlauskasb, Brian Skehana, Hui-Chun Chengc, Loranne Magouna, Douglas Robbinsa, Michael K. Rosenc, Kalle Sakselab, and John M. Leonga,1 aDepartment of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655; bDepartment of Virology, Haartman Institute, University of Helsinki and HUSLAB, Helsinki University Central Hospital, FIN-00014, Helsinki, Finland; and cDepartment of Biochemistry and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390 Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved March 2, 2009 (received for review September 12, 2008) Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effec- host cell plasma membrane with N- and C-terminal intracellular tors to trigger localized actin assembly in mammalian cells, result- domains and a central extracellular domain that binds to the ing in filamentous actin ‘‘pedestals.’’ One effector, the translocated bacterial outer membrane protein intimin. Clustering of Tir in the intimin receptor (Tir), is localized in the plasma membrane and host cell membrane upon intimin binding initiates a signaling clustered upon binding the bacterial outer membrane protein cascade, ultimately leading to actin pedestal formation. intimin. The second, the proline-rich effector EspFU (aka TccP) For the canonical EPEC strain, serotype O127:H6, Tir is the activates the actin nucleation-promoting factor WASP/N-WASP, only translocated effector required for pedestal formation, and and is recruited to sites of bacterial attachment by a mechanism after becoming phosphorylated on tyrosine residue 474 (Y474) dependent on an Asn-Pro-Tyr (NPY458) sequence in the Tir C- by mammalian kinases, recruits the SH2 domain-containing terminal cytoplasmic domain. -
The Hippo Pathway Target, YAP, Promotes Metastasis Through Its TEAD-Interaction Domain
The Hippo pathway target, YAP, promotes metastasis PNAS PLUS through its TEAD-interaction domain John M. Lamara, Patrick Sterna,1, Hui Liua,b,2, Jeffrey W. Schindlera,b, Zhi-Gang Jianga,c, and Richard O. Hynesa,b,c,3 cHoward Hughes Medical Institute, aKoch Institute for Integrative Cancer Research, and bDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 Contributed by Richard O. Hynes, July 23, 2012 (sent for review February 28, 2012) The transcriptional coactivator Yes-associated protein (YAP) is 14-3-3 proteins (1, 9) and α-catenin (10, 11). LATS-mediated a major regulator of organ size and proliferation in vertebrates. phosphorylation of YAP also can promote YAP ubiquitination As such, YAP can act as an oncogene in several tissue types if its and subsequent proteasomal degradation (12). Several addi- activity is increased aberrantly. Although no activating mutations tional proteins are involved in Hippo pathway-dependent and in the yap1 gene have been identified in human cancer, yap1 is -independent regulation of YAP and TAZ, including the FERM located on the 11q22 amplicon, which is amplified in several hu- domain proteins Merlin/NF2 and FRMD6, the junctional pro- man tumors. In addition, mutations or epigenetic silencing of teins ZO-2 and AJUB, the polarity complex proteins Crumbs, members of the Hippo pathway, which represses YAP function, Angiomotin, Scribble, and KIBRA, and the protein phosphatases have been identified in human cancers. Here we demonstrate that, PP2A and ASPP1 (6–8). Thus, YAP protein levels and activity are in addition to increasing tumor growth, increased YAP activity is regulated tightly at multiple levels. -
Importance of Acidic, Proline/Serine/Threonine-Rich, And
Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2501–2506, March 1997 Immunology Importance of acidic, prolineyserineythreonine-rich, and GTP- binding regions in the major histocompatibility complex class II transactivator: Generation of transdominant- negative mutants KEH-CHUANG CHIN*†,GEORGE G.-X. LI†‡, AND JENNY P.-Y. TING†‡ *Department of Biochemistry and Biophysics, †Lineberger Comprehensive Cancer Center, and ‡Department of Microbiology–Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295 Communicated by George Stark, Cleveland Clinic Foundation, Cleveland, OH, December 31, 1996 (received for review September 3, 1996) ABSTRACT The class II transactivator (CIITA) is a (11). CIITA was cloned by its ability to complement RJ2.2.5, master transcription regulator of gene products involved in an in vitro-generated MHC class II negative cell derived from the exogenous antigen presentation pathway, including major Raji (11, 18). Several groups, including our own, have shown histocompatibility complex (MHC) class II, invariant chain, that CIITA is induced by IFN-g and that transfection of CIITA and DM. An extensive analysis of the putative functional alone into cells is sufficient to activate MHC class II (19–21), domains of CIITA is undertaken here to explore the action of invariant chain (19, 22), and HLA-DM genes (22). CIITA. Antibodies to CIITA protein were produced to verify A major issue in the field concerns the mode of action of that these mutant proteins are expressed. Both acidic and CIITA. Although CIITA is a strong transactivator, it does not prolineyserineythreonine-rich domains are essential for class bind MHC class II promoter elements, nor does it appear to II MHC promoter activation.