I. Understanding Membrane Interactions of Bacterial Exoproteins; II. Identification and Characterization of a Novel Mammalian cis-Aconitate Decarboxylase Author: Jiongjia Cheng Persistent link: http://hdl.handle.net/2345/bc-ir:104068 This work is posted on eScholarship@BC, Boston College University Libraries. Boston College Electronic Thesis or Dissertation, 2013 Copyright is held by the author, with all rights reserved, unless otherwise noted. Boston College The Graduate School of Arts and Sciences Department of Chemistry I. UNDERSTANDING MEMBRANE INTERACTIONS OF BACTERIAL EXOPROTEINS; II. IDENTIFICATION AND CHARACTERIZATION OF A NOVEL MAMMALIAN CIS- ACONITATE DECARBOXYLASE A Dissertation by JIONGJIA CHENG Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy December, 2013 © copyright by JIONGJIA CHENG 2013 I. Understanding Membrane Interactions of Bacterial Exoproteins; II. Identification and Characterization of a Novel Mammalian cis-Aconitate Decarboxylase Jiongjia Cheng Under the direction of Dr. Mary F. Roberts ABSTRACT Secreted phosphatidylinositol-specific phospholipase Cs (PI-PLCs) are often virulence factors in pathogenic bacteria. Understanding how these enzymes interact with target membranes may provide novel methods to control bacterial infections. In this work, two typical PI-PLC enzymes, from Bacillus thuringiensis (Bt) and Staphylococcus aureus (Sa), were studied and their membrane binding properties were examined and correlated with enzymatic activity. BtPI-PLC is kinetically activated by allosteric binding of a phosphatidylcholine (PC) molecule. MD simulations of the protein in solution suggested correlated loop and helix motions around the active site could regulate BtPI-PLC activity. Vesicle binding and enzymatic studies of variants of two proline residues, Pro245 and Pro254, that were associated with these motions showed that loss of the correlated motions between the two halves of PI-PLC were more critical for enzymatic activity than for vesicle binding. Furthermore, loss of enzyme activity could be rescued to a large extent with PC present in a vesicle. This suggests that binding to PC changes the enzyme conformation to keep the active site accessible. SaPI-PLC shows 41.3% sequence similarity with BtPI-PLC but has very different ways its activity is regulated. While it is kinetically activated by PC it does not in fact bind to that phospholipid. Enzymatic and membrane interaction assays showed that SaPI-PLC has evolved a complex, apparently unique way to control its access to PI or GPI-anchored substrate. (i) An intramolecular cation-π latch facilitates soluble product release under acidic conditions without dissociation from the membrane. (ii) There is a cationic pocket on the surface of enzyme that likely modulates the location of the protein. (iii) Dimerization of protein is enhanced in membranes containing phosphatidylcholine (PC), which acts not by specifically binding to the protein, but by reducing anionic lipid interactions with the cationic pocket that stabilizes monomeric protein. SaPI-PLC activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane depleted in anionic phospholipids. This protein also served as a way to test the hypothesis that a cation-π box provides for PC recognition site. This structural motif was engineered into SaPI-PLC by forming N254Y/H258Y. This variant selectively binds PC-enriched vesicles and the enzyme binding behavior mimics that of BtPI-PLC. Itaconic acid (ITA) is a metabolite synthesized in macrophages and related cell lines by a cis-aconitate decarboxylase (cADC). cADC activity is dramatically increased upon macrophage stimulation. In this work, the cell line RAW264.7 was used to show that cADC activity upon stimulation requires de novo protein synthesis. MS analyses of partially purified RAW264.7 protein extracts from stimulated cells show a large increase for immunoresponsive gene 1 protein (IRG1) and siRNA knockdown of the IRG1 reduces cADC activity upon stimulation. Suspected active site residues of IRG1 were identified by mutagenesis studies of the recombinant protein based on a homology structure model of fungal cADC. The cloning and overexpression of this enzyme should help clarify the cofactor-independent decarboxylation mechanism of this mammalian enzyme as well as open up future studies into the specific role of ITA in the mammalian immune system and cancers. Acknowledgements First and foremost, I would like to especially thank my adviser Prof. Mary F. Roberts for her guidance and support during my Ph.D. studies. She is always encouraging, giving me confidence and offering invaluable advice in my research. I am so proud to be her student and her extraordinary personality made my graduate studies enjoyable and will influence me for the rest of my life. I would like to thank all the collaborators for their contributions in this work: Prof. Anne Gershenson for her guidance and help in the PI-PLC projects, especially for the use of FCS instrument; Prof. Patrick L. Wintrode and Prof. Nathalie Reuter for their MD simulations of BtPI-PLC; Dr. Boguslaw Stec for his help in protein crystallization and structural modeling; Prof. Thomas Chiles for his guidance in mammalian cell culture; and Prof. Eranthie Weerapana for her help in the MS proteomics study. Special thanks are expressed to Dr. Fay Dufort and Shannon Heyse, who gave me helpful advice on cell biology techniques. These collaborative experiences were especially valuable to me. I would like to thank all the members of Roberts group, the present and the past, for their friendship and helpful discussions. I would like to give special thanks to Dr. Rebecca Goldstein for her work in the crystallization of SaPI-PLC. I would also like to thank Dr. Mingming Pu and Dr. Xiaomeng Shi for their guidance in my research during my first year; Dr. Cheryl L. Strelko for her advice in cADC identification, and Delilah Jewel for her help in RAW cell growth and cADC mutagenesis. I shared a lot of fun time with Dr. Wei Chen, Dr. Su Guo, Dr. Yang Wei, Dr. Jingfei Cai and Tao He, and they all provided me with useful suggestions. Lastly and most importantly, I want to thank my parents for their love and care during my whole life. I also want to thank my husband, Qian, for his love, support and encouragement, and for the happiness he brings to my life. My daughter, Emily, although only 6 months, does bring me a lot of happiness, and I would like to thank her too. Abbreviations A. terreus Aspergillus terreus ActD actinomycin D AF488-Cys Alexa Fluor 488 C5 maleimide AF488-N-term Alexa Fluor 488 carboxylic acid, succinimidyl ester APS ammonium persulfate B. cereus Bacillus cereus B. thuringiensis Bacillus thuringiensis BSA bovine serum albumin BtPI-PLC Bacillus thuringiensis phosphatidylinositol-specific phospholipase C C2 protein kinase C conserved region 2 cADC cis-aconitate decarboxylase CBD chitin binding domain CD circular dichroism cDNA complementary deoxyribonucleic acid cIP inositol 1,2-cyclic phosphate CMC critical micelle concentration CNS central nervous system CoA coenzyme A CSA chemical shift anisotropy i D2O deuterium oxide DAG diacylglycerol DC detergent compatible diC6PC dihexanoyl-phosphatidylcholine diC7PC diheptanoyl-phosphatidylcholine diC8PE dioctanoyl-phosphatidylethanolamine diC8PG dioctanoyl-phosphatidyglycerol diC8PS dioctanoyl-phosphatidylseine DLS dynamic light scattering DMEM Dulbecco’s minimal essential medium DMPC 1,2-dimyristoyl-phosphocholine DMSO dimethyl sulfoxide DOPA 1,2-dioleoyl-phosphatidic acid DOPG 1,2-dioleoyl-phosphatidyglycerol DOPMe 1,2-dioleoyl-phosphatidylmethanol DOPS 1,2-dioleoyl-phosphatidylserine dsRNA double-stranded RNA DTNB 5,5'-dithiobis-(2-nitrobenzoic acid) DTT dithiothreitol E. coli Escherichia coli EDTA ethylenediaminetetraacetic acid EGF epidermal growth factor ii FBS fetal bovine serum fc-P-NMR high resolution field cycling 31P NMR FCS fluorescence correlation spectroscopy fmax apparent maximum fraction bound FRET fluorescence resonance energy transfer GPI glycosylphosphatidylinositol GST glutathione S-transferase HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIC hydrophobic interaction chromatography His-tag poly histidine tag I-1-P inositol 1-phosphate IDS iminodisuccinate IFN-γ interferon γ IMPDH inosine monophosphate dehydrogenase IPTG isopropyl β-D-1-thiogalactopyranoside IRG1 immunoresponsive gene 1 ITA itaconic acid Kd apparent dissociation constant Km Michaelis constant L. monocytogenes Listeria monocytogenes LAB lactic acid bacteria LP lipopeptide or lipoprotein iii LPS lipopolysaccharide MBP maltose-binding protein MD molecular dynamics MES 2-(N-morpholino) ethane-sulfonic acid MS mass spectrometry MSA multiple sequence alignment MTSL 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl-methanethio- sulfonate MyD88 myeloid differentiation factor-88 NAD+ β-nicotinamide adenine dinucleotide hydrate NF-κB nuclear factor κ-light-chain-enhancer of activated B cells Ni-NTA nickel-nitrilotriacetic acid NMR nuclear magnetic resonance NOS nitric oxide O.D. optical density ORF open reading frame PA phosphatidic acid PBS phosphate buffered saline PC phosphatidylcholine PCA principal component analyses PCR polymerase chain reaction PE phosphatidylethanolamine iv PG phosphatidylglycerol PH pleckstrin homology pI isoelectric point PI phosphatidylinositol PI3K phosphatidylinositol 3-kinase PIA phosphatidylinositol analog PI-PLC phosphatidylinositol-specific