Homocysteine in Protein Structure/Function and Human Disease : Chemical Biology of Homocysteine-containing Proteins

Excess of homocysteine, a product of the metabolism of the essential amino acid methionine, is associated with poor health, is linked to heart and brain diseases in general human populations, and accelerates mortality in heart disease patients. Neurological and cardiovascular abnormalities occur in patients with severe genetic hyperhomocysteinemia and lead to premature death due to vascular complications. Although it is considered a non-protein amino acid, studies over the past dozen years have discovered mechanisms by which homocysteine becomes a component of proteins. Homocysteine-containing proteins lose their normal biological function and become auto-immunogenic and pro-thrombotic. In this book, the author, a pioneer and a leading contributor to the field, describes up-to date studies of the biological chemistry of homocysteine-containing proteins, as well as pathological consequences and clinical implications of their formation. This is a comprehensive account of the broad range of basic science and medical implications of homocysteine-containing proteins for health and disease. ​ Preface 8 Contents 12 Chapter 1: Introduction 15 Chapter 2: An Overview of Homocysteine Metabolism 20 2.1 Homocysteine Metabolite Levels 21 2.1.1 ``Total Homocysteine ́ ́ 22 2.1.2 S-Homocysteinyl-Protein 22 2.1.3 Homocysteine-Thiolactone 23 2.1.4 N-Homocysteinyl-Protein 23 2.1.5 Nepsi-Homocysteinyl-Lysine 24 2.1.6 S-Adenosylhomocysteine 24 2.1.7 Cystathionine 24 2.1.8 Homocysteic Acid and Homocysteine Sulfinic Acid 24 2.2 Toxicity of Homocysteine Metabolites 25 2.2.1 Free Reduced Homocysteine 26 2.2.2 S-Homocysteinyl-Protein 26 2.2.3 Homocysteine-Thiolactone 27 2.2.4 N-Homocysteinyl-Protein 29 2.2.5 Nepsi-Homocysteinyl-Lysine 30 2.2.6 S-Adenosylhomocysteine 30 2.2.7 Cystathionine 31 2.2.8 Homocysteic Acid 31 Chapter 3: Homocysteine-Thiolactone 32 3.1 Chemical Synthesis 32 3.1.1 Demethylation of Methionine 32 3.1.2 Acid-Dependent Cyclization of Homocysteine 33 3.2 Physicochemical Properties 33 3.2.1 UV Spectrum 33 3.2.2 pKa value 36 3.2.3 Stability 36 3.2.4 Reactivity Toward Amino Groups 38 3.2.5 Reactivity Toward Carbonyl Compounds 40 3.3 Quantification Methods 42 3.3.1 Assays Based on Radiolabeling 42 3.3.2 Assays Based on Direct UV Monitoring 42 3.3.3 High-Performance Liquid Chromatography-Based Assays 42 3.3.3.1 High-Performance Liquid Chromatography with UV Detection 43 3.3.3.2 High-Performance Liquid Chromatography with Fluorescence Detection 43 3.3.4 Gas Chromatography/Mass Spectrometry Assay 45 3.3.5 Gold Nanoparticle Homocysteine-Thiolactone Sensor 45 3.4 Biosynthesis 46 3.4.1 The Involvement of Methionyl-tRNA Synthetase 46 3.4.2 Molecular Mechanism 50 3.4.2.1 The Synthetic/Editing Active Site of Methionyl-tRNA Synthetase 50 3.4.2.2 The Thiol-Binding Subsite of Methionyl-tRNA Synthetase 54 3.4.2.3 S-NO-Hcy Is Transferred to tRNA and Participates in Protein Biosynthesis 55 3.5 Turnover 58 3.5.1 Enzymatic Turnover 58 3.5.1.1 Paraoxonase 1 58 3.5.1.2 Bleomycin Hydrolase 61 3.5.2 Urinary Excretion 63 3.6 Clinical Significance 64 Chapter 4: Discoveries of Protein S- and N-Homocysteinylation 67 Chapter 5: N-Homocysteinyl-Proteins 70 5.1 Synthesis In Vitro 70 5.2 Biological Formation 73 5.2.1 N-Hcy-Protein Levels In Vivo 73 5.2.1.1 Cellular Proteins 76 5.2.1.2 Blood Proteins 76 5.2.1.3 Collagen 78 5.2.1.4 Milk Protein 79 5.2.1.5 Hair Keratin 79 5.2.2 Site-Specific N-Homocysteinylation In Vivo 80 5.3 Turnover 82 5.3.1 Nepsi-Homocysteinyl-Lysine 82 5.3.1.1 Chemical Synthesis 82 5.3.1.2 Physicochemical Properties 84 5.3.1.3 Biological Formation 84 5.3.1.4 Quantification 85 5.3.1.5 Clinical Significance 86 5.4 Structural and Functional Consequences 87 5.4.1 N-Homocysteinylation and Redox Function 89 5.4.1.1 N-Hcy-Albumin 89 5.4.1.2 N-Hcy-Cytochrome c 93 5.4.2 N-Hcy-Fibrinogen and Fibrin Clot Properties 94 5.4.3 N-Homocysteinylation and LDL Function 95 5.4.4 N-Homocysteinylation and HDL Function 96 5.4.5 N-Homocysteinylation Induces Protein Aggregation and Amyloidal Conversion 97 5.4.5.1 N-Hcy-Albumin 97 5.4.5.2 N-Hcy-Insulin 101 5.4.5.3 N-Hcy-Prion 102 5.4.5.4 N-Hcy-Amyloid beta-Peptide 102 5.4.5.5 N-Hcy-Tau 103 5.4.5.6 N-Hcy-Caseins 103 5.4.5.7 N-Hcy-Crystallin 104 5.4.5.8 N-Hcy-Dynein 104 5.5 Quantification of N-Hcy-Protein 105 5.5.1 High-Performance Liquid Chromatography Assay with UV Detection 106 5.5.2 High-Performance Liquid Chromatography Assays with Fluorescence Detection 107 5.5.2.1 Derivatization with OPA 107 5.5.2.2 Derivatization with 4-Fluoro-7-sulfamoyl-benzofurazan 109 5.5.2.3 Derivatization with 7-Fluorobenzo-2-Oxa-1,3-Diazole-4-Sulfonate 109 5.5.3 Immunoassays with Rabbit Polyclonal Anti-N-Hcy-Protein Antibodies 110 5.5.4 Western Blot Immunoassay for N-Hcy-ApoAI 110 5.5.5 Detection of Protein N-Homocysteinylation by Selective Reactions with Aldehydes 111 5.5.6 Liquid Chromatography/Mass Spectrometry Analysis of the Site-Specific Protein N-Homocysteinylation In Vivo 113 5.5.6.1 N-Hcy-Albumin 113 5.5.6.2 N-Hcy-Fibrinogen 115 Chapter 6: Pathophysiological Consequences of Protein N-Homocysteinylation 117 6.1 The Hcy-Thiolactone Hypothesis 118 6.1.1 Hcy-Thiolactone Levels in Hyperhomocysteinemia 119 6.1.2 Protein N-Linked Hcy in Hyperhomocysteinemia 120 6.2 N-Hcy-Protein and Adaptive Autoimmune Responses 121 6.2.1 Atherosclerosis Is an Inflammatory Disease 121 6.2.2 N-Hcy-Protein Is Immunogenic in Rabbits 122 6.2.3 N-Hcy-Protein Is Autoimmunogenic in Humans 122 6.2.4 Anti-N-Hcy-Protein Autoantibodies in Atherosclerosis 124 6.3 N-Hcy-Protein and Innate Immune Responses 125 6.4 N-Hcy-Fibrinogen and Thrombosis 127 6.5 N-Hcy-Collagen and Connective Tissue Abnormalities 129 Chapter 7: S-Homocysteinylated Proteins 130 7.1 Plasma S-Homocysteinyl-Proteins 132 7.1.1 S-Hcy-Albumin 132 7.1.2 S-Hcy-Transthyretin 133 7.1.3 Apolipoprotein B and Lipoprotein[a] 135 7.2 Extracellular Matrix Proteins 136 7.2.1 Fibrillins 136 7.2.2 Fibronectin 138 7.2.3 Tropoelastin 138 7.3 Blood Homeostasis Proteins 139 7.3.1 Factor Va 139 7.3.2 Annexin A2 140 7.4 Intracellular Proteins 141 7.4.1 Heterogenous Nuclear Ribonucleoprotein E1 (hnRNP-E1) 141 7.4.2 Metallothionein 143 7.4.3 Dimethylarginine Dimethylaminohydrolase 143 References 145 Index 169 Front Matter....Pages i-xiii Introduction....Pages 1-5 An Overview of Homocysteine Metabolism....Pages 7-18 Homocysteine-Thiolactone....Pages 19-53 Discoveries of Protein S - and N -Homocysteinylation....Pages 55-57 N -Homocysteinyl-Proteins....Pages 59-105 Pathophysiological Consequences of Protein N -Homocysteinylation....Pages 107-119 S -Homocysteinylated Proteins....Pages 121-135 Back Matter....Pages 137-166