Thesis advisor: Eranthie Weerapana / A chemical-proteomic platform to monitor cysteine sensitivity to transnitrosation Yani Zhou Dissertation advisor: Dr. Eranthie Weerapana Abstract S-nitrosation has emerged as a ubiquitous endogenous protein posttranslational modification that significantly impacts cellular protein function through a variety of mechanisms. Despite the advent of chemical and proteomic methods to study S-nitrosation, the subset of cellular cysteine residues that show uniquely high reactivity to endogenous transnitrosation donors is poorly characterized. To further these existing global studies, a cysteine-reactivity profiling strategy was applied herein to rank ~600 cysteine residues by sensitivity to S-nitrosoglutathione. These proteomic studies revealed several previously uncharacterized sites of S-nitrosation, including Cys58 in HADH2. Further characterization revealed that HADH2 catalytic activity is allosterically regulated by S-nitrosation, and this modification occurs in cells at (patho)physiological levels of nitrosative stress. Functional role of Cys58 and its regulation by S-nitrosation facilitated the identification of RB-21-CA as a potential covalent Cys58 inhibitor. Global analysis of GSNO, S-nitroso-Coenzyme A and Thioredoxin-C73-SNO transnitrosation identified 756 cysteines with different sensitivity to each of three SNO donors. Systematic evaluation on transnitrosation selectivity revealed that specific interaction of transnitrosation donor with its protein target is a key component governing the selective transnitrosation of a specific cysteine residue. Together, these studies illustrated the potential of cysteine-reactivity profiling strategy for evaluating the substrate specificity of transnitrosation donors and enable the identification of previously uncharacterized, functionally relevant sites of S-nitrosation. Another cysteine oxoform, S-glutathionylation, is the disulfide formation of a protein cysteine residue with glutathione. Although glucose starvation is known to induce redox-disturbance, global and individual protein S-glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. By using a clickable glutathione approach, which forms clickable glutathione by the use of a mutant of glutathione synthetase, we found that protein S-glutathionylation is readily induced in response to glucose starvation when mitochondrial reactive oxygen species are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Application of a proteomic mass spectrometry platform identified over 1,300 S-glutathionylated. Confirmation of S-glutathionylation for selected proteins by in gel analysis further validated the mass spectrometry results, and highlights the dynamic change of S-glutathionylation on an individual protein level. In order to expand on the understanding of the functional role of the cysteine residues in biological systems, we evaluated a panel of 1,3,5-triazine- and 4-aminopiperidine-based cysteine-reactive small-molecules on two proteins, apoptosis signal-regulating kinase 1 (ASK1) and peroxiredoxin 1 (Prdx1), between which a intermolecular disulfide forms and results in the activation of mitogen-activated protein kinase pathway. In-gel fluorescence revealed that RB-11-CA and SMC-1, both of which contain an n-octyl group as a diversity element, showed greatest selectivity and potency to ASK1 and Prdx1, respectively. Further mass spectrometry analysis identified cysteine 225 of ASK1 and cysteine 173 of Prdx1 are the sites of covalent probe modification. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

by Nathan, Yiu-hung Yam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 139-157). / Acknowledgements --- p.i / Table of Contents --- p.ii / Abstract --- p.viii / List of Abbreviations --- p.x / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Aims of the present study --- p.2 / Chapter 1.3 --- Sequencing of an adult human heart cDNA library --- p.3 / Chapter 1.4 --- Rat/mouse CRIP --- p.5 / Chapter 1.5 --- LIM proteins --- p.13 / Chapter 1.6 --- Zinc-binding proteins --- p.17 / Chapter 1.7 --- Bacterial expression system using the pAED4 vector --- p.24 / Chapter Chapter 2 --- Identification and sequence analysis ofhCRHP --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and methods --- p.29 / Chapter 2.2.1 --- Bacterial strains and vectors --- p.29 / Chapter 2.2.2 --- "Mediums, buffers and solutions" --- p.31 / Chapter 2.2.3 --- Bacteriophage clones preparation --- p.34 / Chapter 2.2.4 --- Amplification of clones by PCR --- p.35 / Chapter 2.2.5 --- Cycle sequencing of PCR products --- p.36 / Chapter 2.2.6 --- DNA sequences analysis --- p.38 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Sequence analysis of hCRHP --- p.39 / Chapter 2.3.2 --- Comparison of hCRHP with CRIP --- p.52 / Chapter 2.3.3 --- Comparison of hCRHP with some LIM proteins --- p.56 / Chapter 2.4 --- Discussions --- p.61 / Chapter Chapter 3 --- Study of hCRHP at the nucleic acid level --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Materials and methods --- p.66 / Chapter 3.2.1 --- Animals --- p.66 / Chapter 3.2.2 --- "Mediums, buffers, enzymes and solutions" --- p.66 / Chapter 3.2.3 --- Preparation of total RNA --- p.70 / Chapter 3.2.3.1 --- Preparation of RNA by the CsCl method --- p.70 / Chapter 3.2.3.2 --- Preparation of RNA by the AGPC method --- p.71 / Chapter 3.2.4 --- Northern hybridization of hCRHP --- p.72 / Chapter 3.2.4.1 --- Formaldehyde agarose gel electrophoresis --- p.72 / Chapter 3.2.4.2 --- Preparation of radioactive probe --- p.73 / Chapter 3.2.4.3 --- RNA transfer and Northern hybridization --- p.74 / Chapter 3.2.5 --- Preparation of human genomic DNA --- p.77 / Chapter 3.2.6 --- Southern hybridization of hCRHP --- p.78 / Chapter 3.2.6.1 --- Restriction cutting and agarose gel electrophoresis of genomic DNA --- p.78 / Chapter 3.2.6.2 --- DNA transfer and Southern hybridization --- p.79 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Southern hybridization of hCRHP --- p.80 / Chapter 3.3.2 --- Identification of hCRHP in neonatal human heart --- p.83 / Chapter 3.3.3 --- Tissue distribution of CRIP mRNA in rat tissues --- p.85 / Chapter 3.3.4 --- Time course of CRIP expression in rat heart --- p.85 / Chapter 3.4 --- Discussions --- p.87 / Chapter Chapter 4 --- Subcloning and expression of hCRHP --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and methods --- p.90 / Chapter 4.2.1 --- Bacterial strains and vectors --- p.90 / Chapter 4.2.2 --- "Mediums, buffers, enzymes and solutions" --- p.92 / Chapter 4.2.3 --- Subcloning of hCRHP into pAED4 --- p.98 / Chapter 4.2.3.1 --- Primers design and PCR --- p.98 / Chapter 4.2.3.2 --- Purification of PCR products by Geneclean II´ёØ (BIO 101 Inc) --- p.99 / Chapter 4.2.3.3 --- Restriction digestion of purified PCR product and pAED4 --- p.100 / Chapter 4.2.3.4 --- Ligation and transformation of hCRHP --- p.101 / Chapter 4.2.3.5 --- Amplification and purification of pAED4-hCRHP --- p.103 / Chapter 4.2.4 --- Expression of hCRHP --- p.105 / Chapter 4.2.4.1 --- Induction of hCRHP expression --- p.105 / Chapter 4.2.4.2 --- SDS-PAGE and protein detection --- p.106 / Chapter 4.3 --- Results --- p.108 / Chapter 4.3.1 --- Subcloning of hCRHP into pAED4 --- p.108 / Chapter 4.3.2 --- Induction and optimization of hCRHP expression --- p.110 / Chapter 4.4 --- Discussions --- p.117 / Chapter Chapter 5 --- Partial purification and isoelectric focusing of hCRHP --- p.120 / Chapter 5.1 --- Introduction --- p.120 / Chapter 5.2 --- Materials and methods --- p.121 / Chapter 5.2.1 --- "Mediums, buffers and mediums" --- p.121 / Chapter 5.2.2 --- Purification of hCRHP by ammonium sulphate precipitation --- p.121 / Chapter 5.2.3 --- Purification of hCRHP by hydrochloric acid extraction --- p.122 / Chapter 5.2.4 --- Purification of hCRHP by ultrafiltration --- p.123 / Chapter 5.2.5 --- Isoelectric focusing of hCRHP --- p.127 / Chapter 5.3 --- Results --- p.128 / Chapter 5.3.1 --- Partial purification of hCRHP by ammonium sulphate precipitation --- p.128 / Chapter 5.3.2 --- Partial purification of hCRHP by hydrochloric acid extraction --- p.128 / Chapter 5.3.3 --- Partial purification of hCRHP by ultrafiltration --- p.131 / Chapter 5.3.4 --- Isoelectric focusing of hCRHP --- p.133 / Chapter 5.4 --- Discussions --- p.133 / Chapter Chapter 6 --- Discussions --- p.136 / Chapter 6.1 --- The possible role(s) of hCRHP/CRIP --- p.136 / Chapter 6.2 --- Future prospects --- p.137 / References --- p.139 / Appendix 1 --- p.158

The type Iα cGMP-dependent protein kinase (PKG Iα) is an essential regulator of vascular tone and systemic blood pressure. Located in the smooth muscle of resistance vessels, PKG Iα stimulates vasodilation through the phosphorylation of multiple intracellular substrates. Its primary regulator is the small molecule, 3',5'-cyclic guanosine monophosphate (cGMP); however, the Iα isoform can also be activated by oxidation. Despite the established physiological importance of PKG Iα, the structural underpinnings of these two activation mechanisms are largely unknown. The work presented in this dissertation demonstrates the importance of the cGMP-binding domain A (CBD-A) in regulating both of these mechanisms of PKG Iα activation. Using a monomeric, N-terminally truncated form of PKG Iα (Δ53), Chapter 2 investigates the mechanism of inhibition through the autoinhibitory domain and the influence of dimerization on cooperative cGMP-dependent activation and cyclic nucleotide selectivity. We observed that autoinhibition occurs in cis, whereas cooperativity requires interprotomer contacts facilitated by the N-terminal dimerization domain. Furthermore, the loss of selectivity for cGMP over cAMP of this construct suggests the dimerization domain plays a critical role in preventing cross-reactivity with cAMP-dependent signaling. These observations culminate into an overarching model wherein binding of cGMP to CBD-A is necessary and sufficient for activation and cooperativity is driven by the dimerization domain. Chapter 3 investigates the cysteine residues that mediate oxidation-dependent activation of PKG Iα. Using PKG Iα constructs with point mutations at specific cysteine residues, it was found that oxidation-dependent activation is driven by C117 in CBD-A. Furthermore, the interprotomer disulfide bond that forms in the dimerization domain at C42 does not contribute to this mechanism. Finally, we propose a model wherein the disulfide bond that forms between C117 and the adjacent cysteine at position 195 acts as a protective mechanism to prevent activation and higher oxidation states form contacts with nearby residues in the linker region of PKG Iα to disrupt binding of the adjacent autoinhibitory domain to the catalytic domain. Finally, Chapter 4 provides a discussion of the results presented herein in context with previous studies and suggests future directions for the PKG field.

Neuronal nicotinic acetylcholine receptors can have their function modulated by zinc. Depending on concentration and subunit composition, zinc either inhibits or potentiates receptor function. The zinc ion potentiates the alpha4beta4 receptor at non-agonist binding interfaces or "pseudo sites." Zinc potentiation is reduced if certain residues are mutated or spatially interfered with. The residue contributing most to this potentiation reduction effect is histidine 162 on the alpha4 subunit. The anthelmintic compound levamisole potentiates acetylcholine response of certain neuronal nicotinic receptors. Levamisole and its functional analogues morantel, oxantel, and pyrantel all were found to potentiate alpha4beta4 receptors at low (µM) concentrations and inhibit them at high (mM) concentrations. Oxantel showed the greatest degree of potentiation, about a third of the maximal zinc potentiation measured. Oxantel was screened using the substituted cysteine accessibility method (SCAM) against the residue histidine 162 as well as nearby alpha4 residues histidine 61 and glutamate 59 and the beta4 residue aspartate 195. Screening was carried out by mutating said residues into cysteine, followed by covalent linkage with a disulfide bridge of that residue with a methanethiosulfonate compound. SCAM experiments allowed testing of the effects of a single residue and the area immediately adjacent to it. Receptors that lost zinc potentiation capacity from site-directed mutagenesis at the his 162 residue and subsequent methanethiosulfonate reaction still showed regular potentiation following oxantel treatment. Although these compounds exhibit similar biphasic potentiation dose-response curves as zinc, their mechanism for potentiation is not through the same mechanism.

X-ray crystallography provides detailed information of the atomic structure of macromolecules that aides in the understanding of their molecular function. In this study, the three-dimensional structures of the Toluene-4-monooxygenase catalytic effector protein (T4moD), NAD(P)H oxidase and choline oxidase were determined. The structures of wild-type and two mutant isoforms of T4moD were solved up to 1.7 resolution. Results from the crystallographic studies indicate that there are significant differences between the X-ray structure and the structure previously solved by NMR. The high-resolution structures have helped to define the potential differences in electrostatic surfaces that may govern the feasibility of protein-protein interactions and also reveal a single, well-defined cavity suitable for toluene binding that has substantial different electrostatic properties among the effector protein family members. The structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis was determined to 1.8 resolution. The flavoenzyme is of considerable interest as it catalyzes the oxidation of two equivalents of NAD(P)H and reduces one equivalent of oxygen to yield two equivalents of water without releasing hydrogen peroxide from the active site. The structure reveals the presence of a redox active cysteine residue that exists as a sulfenic acid and plays an important mechanistic role by reducing hydrogen peroxide to water. Additionally, a tightly bound ADP molecule was discovered in the enzyme which is hypothesized to play an important role in influencing the dual substrate specificity exhibited by the enzyme. The structure of choline oxidase from Arthrobacter globiformis was solved to 1.86 resolution. Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine via two sequential FAD-dependent reactions. The structure reveals a cavity within the active site, which is suitable for choline binding. This allows for the identification of the putative binding site for choline and residues involved in substrate-binding and catalysis. Additionally, the structure reveals a highly distorted FAD cofactor that contains a C4a-adduct that is proposed to be either an FAD-C4a-OH or FAD-C4a-O2- complex.

Hemoglobin plays an important role in transporting oxygen in human beings and other mammals. Hemoglobin is a tetrameric protein composed of two alpha and two beta subunits. The £\ and £] subunits are both necessary and the stoichiometric ratio of the two dislike subunits is critical for hemoglobin to perform its oxygen-carrying function properly. To better understand the coupling between the £\ and £] subunits and the subunit disassembly pathway, p-hydroxymercuri-benzoate (PMB) has been used to react with the cysteine residues in hemoglobin. The hemoglobin tetramer becomes unstable and disassembles into £\ and £] subunits when the cysteine sites are perturbed upon reacting with PMB. There are three kinds of cysteine residues, £]93, £\104 and £]112, in human hemoglobin. The reactivity of different cysteine residues with PMB and their reaction sequence have been studied via the Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The resonance Raman spectroscopy has been used to investigate the structural changes of hemoglobin accompanying the PMB-modification under the oxygenated and deoxygenated conditions. At last, a hemoglobin subunit disassembly mechanism is proposed and the site-specific roles of cysteine residues in human hemoglobin are discussed in detail.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 72-81).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 98-106).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Parenteral nutrition (PN) is a method of feeding those incapable of absorbing nutrients from the gastrointestinal tract. All required nutrients are combined in one "big bag". Consequently, many chemical interactions are possible between components. Ascorbic acid (AA) is ubiquitous to both animal and plant kingdoms. Although its biochemistry is not fully understood, dietary deficiency is detrimental to well being, with the most extreme condition being scurvy. AA is water-soluble and frequent intake is therefore required to maintain nutritional status. AA is possibly the most reactive additive in PN mixtures, readily reacting with dissolved oxygen, initially producing dehydroascorbic acid (OHAA). OHAA retains the biological activity of AA. It was the purpose of this study to further knowledge regarding stability of AA and OHAA in PN mixtures, informing pharmaceutical practice to improve safety and efficacy of PN. A stability-indicating HPLC method was optimised for the study of AA and OHAA in PN mixtures. A study of the kinetics of OHAA degradation was undertaken to provide data that could be used to predict OHAA stability. Results obtained indicated a first order reaction. In direct contrast to AA degradation, trace elements did not catalyse OHAA degradation. A further product of AA degradation is oxalic acid (OA) which is potentially toxic. A HPLC method for the determination of OA in PN mixtures was developed and validated, although minimum quantification limits were relatively high (~10J.Lg/ml).The method was used to assess OA appearance in stored PN mixtures, with results indicating that concentrations remained below 10J.Lg/ml even after 35 days storage. The final aspect of this research was to investigate the most likely components of a PN mixture which may "protect" AA from oxidation. a-tocopherol photo-oxidises and therefore may compete with AA for oxygen. As light catalyses the reaction it is possible oxygen reacts more rapidly with a-tocopherol compared with AA. Results indicated 0.- Tocopherol did not oxidise in preference to AA and therefore offered no "protection". Cysteine is a reducing agent included in some amino acid preparations. The average dissolved oxygen content of standard adult PN mixtures was determined, from which the amount of cysteine required to react with dissolved oxygen was calculated. AA instability in PN mixtures was compared with and without cysteine. Results indicated that adding cysteine to PN mixtures 24 hours before addition of AA, resulted in retention of >95% AA. Results obtained from this study have furthered knowledge of the AA degradation profile, its kinetics and the potential influence of other components in PN mixtures. In particular potential strategies for minimising AA degradation are identified therefore ensuring patients receive quantities approaching those prescribed.