<p>Introduction to the Book 1</p> <p>Long-Lived Proteins Are Ubiquitous 1</p> <p>Aging 1</p> <p>Autoimmunity 2</p> <p>Age-Related Diseases 3</p> <p>Our Lenses in the Vanguard 3</p> <p>Brain and Memory 4</p> <p><b>1 Long-Lived Cells and Long-Lived Proteins in the Human Body 5<br /></b><i>Roger J.W. Truscott</i></p> <p>1.1 What Constitutes a Long-Lived Cell and a Long-Lived Protein? 5</p> <p>1.2 Aim of the Chapter 6</p> <p>1.3 Aging 6</p> <p>1.4 Location of LLPs Within the Body 7</p> <p>1.4.1 ECM and Tissue Function 7</p> <p>1.5 Extracellular LLPs 7</p> <p>1.5.1 Several ECM Components Are Long Lived 7</p> <p>1.5.1.1 Elastin 7</p> <p>1.5.1.2 Structural Glycoproteins and Proteoglycans 8</p> <p>1.5.1.3 Collagens 8</p> <p>1.6 Intracellular LLPs and LLCs 10</p> <p>1.6.1 LLCs and LLPs in the Organs of the Body 10</p> <p>1.7 Organs and Tissues that Contain LLCs or LLPs 11</p> <p>1.7.1 Long-Lived Cells 11</p> <p>1.7.1.1 Eye 11</p> <p>1.7.1.2 Oocytes 14</p> <p>1.7.1.3 Kidneys 15</p> <p>1.7.1.4 Adipose Tissue 15</p> <p>1.7.1.5 Brain 15</p> <p>1.7.1.6 Heart 17</p> <p>1.7.1.7 Lung 17</p> <p>1.7.1.8 Skeleton 18</p> <p>1.7.1.9 Teeth 18</p> <p>1.7.1.10 Hair 18</p> <p>1.7.1.11 Joints 19</p> <p>1.7.1.12 Pancreas 19</p> <p>1.7.1.13 Liver 20</p> <p>1.7.1.14 Intestine 20</p> <p>1.7.1.15 Dividing Cells and LLPs 21</p> <p>1.7.2 Sensory Tissues 21</p> <p>1.7.2.1 Hearing 21</p> <p>1.7.2.2 Smell 21</p> <p>1.8 Protein Changes and DNA Changes with Age 21</p> <p>1.9 Processes Responsible for the Breakdown of LLPs 22</p> <p>1.10 Oxidation: Methionine Sulfoxide Reductases and the Glutathione System 23</p> <p>1.11 Consequences of LLP Decomposition 24</p> <p>1.11.1 Protein Modification and Cellular Processing 24</p> <p>1.11.2 Lifelong Proteins and the Consequences 24</p> <p>1.12 LLPs and Age-Related Disorders 25</p> <p>1.12.1 Modified LLPs Acting as Novel Antigens: Autoimmune Diseases 25</p> <p>1.12.2 Defects in Cytosol/Nuclear Communication 25</p> <p>1.12.3 Defects in Nuclear Transcription 26</p> <p>1.12.4 Breakdown of Abundant Macromolecules 26</p> <p>1.12.5 Elastin 26</p> <p>1.12.6 Collagen 26</p> <p>1.13 Neurological Diseases Where LLPs May be Implicated 27</p> <p>1.13.1 Multiple Sclerosis 27</p> <p>1.13.2 Motor Neuron Disease (MND)/Amyotrophic Lateral Sclerosis (ALS) 27</p> <p>1.13.3 Alzheimer Disease (AD) 27</p> <p>1.14 Aging DNA and LLPs 28</p> <p>1.15 How Can the Role of LLPs in Aging and Disease Be Investigated? What Can Be Done 28</p> <p>1.15.1 Heterogeneity of Aged LLPs: A Large Hurdle to Overcome 29</p> <p>1.16 We Will Not Live Forever 29</p> <p>1.16.1 LLP Degradation and Tissue Function: Is There a Threshold for Decay? 30</p> <p>1.16.2 Lifelong Proteins May Degrade at Similar Rates 30</p> <p>1.16.3 Decay in Tissue Function with Age and Its Effect on Fitness, Health, and Mortality 32</p> <p>1.16.4 LLPs and Life Span 32</p> <p>1.16.5 Heart 32</p> <p>1.16.6 Lung 33</p> <p>1.16.7 Nerves and Brain 33</p> <p>1.17 Conclusion 33</p> <p>Acknowledgments 33</p> <p>References 33</p> <p><b>2 Imaging Mass Spectrometry of Long-Lived Proteins 43<br /></b><i>Kevin L. Schey</i></p> <p>2.1 Introduction 43</p> <p>2.2 Imaging Mass Spectrometry Methods 44</p> <p>2.2.1 General Considerations 44</p> <p>2.2.2 MALDI-IMS 44</p> <p>2.2.3 Desorption Electrospray Ionization (DESI)-IMS 46</p> <p>2.2.4 Secondary Ion Mass Spectrometry (SIMS)-IMS 46</p> <p>2.2.5 Other IMS Methods 46</p> <p>2.3 Protein Identification 47</p> <p>2.4 LLPs in the Body 48</p> <p>2.4.1 Lens 48</p> <p>2.4.2 Optic Nerve 51</p> <p>2.4.3 Retina 52</p> <p>2.4.4 Brain and CNS 52</p> <p>2.4.5 Cartilage 53</p> <p>2.5 Long-Lived Cells and Structures 53</p> <p>2.6 Future Directions 54</p> <p>References 54</p> <p><b>3 Eye Lens Crystallins: Remarkable Long-Lived Proteins 59<br /></b><i>Aidan B. Grosas and John A. Carver</i></p> <p>3.1 Introduction 59</p> <p>3.2 Eye Lens and Its Transparency 59</p> <p>3.3 Lens Crystallin Proteins 61</p> <p>3.3.1 α-Crystallins 61</p> <p>3.3.2 β- and γ-Crystallins 63</p> <p>3.4 Congenital, Early Onset, and Age-Related Cataract 65</p> <p>3.5 Protein Aggregation and Disease, Particularly Cataract 71</p> <p>3.5.1 Protein Unfolding and Aggregation and Molecular Chaperones 71</p> <p>3.5.2 Amyloid Fibril and Amorphous Protein Aggregates 73</p> <p>3.5.3 Diseases Associated with Protein Aggregation 74</p> <p>3.5.4 Crystallin Aggregation and Cataract 75</p> <p>3.6 Concluding Comments 77</p> <p>References 78</p> <p><b>4 Spontaneous Breakdown of Long-Lived Proteins in Aging and Their Implications in Disease 97<br /></b><i>Michael G. Friedrich</i></p> <p>4.1 Introduction 97</p> <p>4.2 LLPs Are Found Throughout the Body 98</p> <p>4.3 Spontaneous Modifications of Aging 99</p> <p>4.3.1 Deamidation, Racemization, and Isomerization 99</p> <p>4.3.2 Cross-linking 101</p> <p>4.3.3 Truncation 102</p> <p>4.3.4 Age, Disease, and Spontaneous PTMs: General Considerations 103</p> <p>4.4 LLPs and Onset of Disease: Is Correlation the Only Answer? 105</p> <p>4.4.1 Eye 106</p> <p>4.4.1.1 Lens and Age-Related Nuclear Cataract 106</p> <p>4.4.1.2 Retina, Vitreous Humor, and Sclera 108</p> <p>4.4.2 Central Nervous System 108</p> <p>4.4.2.1 Multiple Sclerosis 109</p> <p>4.4.2.2 Alzheimer’s Disease 109</p> <p>4.4.2.3 Parkinson’s Disease 110</p> <p>4.4.2.4 Amyotrophic Lateral Sclerosis/Motor Neuron Disease 110</p> <p>4.4.2.5 Systemic Lupus Erythematosus 111</p> <p>4.4.3 Extracellular Matrix Proteins 111</p> <p>4.4.3.1 Articular Cartilage, Intervertebral Disc, and Osteoarthritis 112</p> <p>4.4.3.2 Circulatory System 112</p> <p>4.4.3.3 Respiratory System 112</p> <p>4.4.4 Digestive System 112</p> <p>4.4.4.1 Diabetes 113</p> <p>4.5 Spontaneous Modifications: Detrimental or Beneficial? 113</p> <p>4.5.1 NGR Motifs 113</p> <p>4.5.2 Bcl-xL 113</p> <p>4.6 Protein Turnover Slows with Age 113</p> <p>4.7 Potential Treatment of Diseases Initiated by LLPs 114</p> <p>4.8 Future Outlook 114</p> <p>Acknowledgments 115</p> <p>References 115</p> <p><b>5 Modifications of Long-Lived Proteins that Affect Protein Solubility 127<br /></b><i>Larry L. David</i></p> <p>5.1 Introduction 127</p> <p>5.2 Insoluble Protein Definition 128</p> <p>5.3 Insolubilization Due to Disulfide Bonding 128</p> <p>5.3.1 Disulfide Bonding Is Strongly Correlated with Age-Related Cataracts 128</p> <p>5.3.2 Levels of Disulfide Bonding at Individual Cysteines in Cataractous Lenses 129</p> <p>5.3.3 Identity of Individual Disulfide Cross-links in Crystallins of Aged Lenses 129</p> <p>5.4 Insolubilization Due to Nondisulfide Cross-links 130</p> <p>5.4.1 Cross-links Due to Dehydroalanine Formation 130</p> <p>5.4.2 Cross-links Due to C-Terminal Anhydrides 130</p> <p>5.5 Insolublization Due to Protein Fragmentation 131</p> <p>5.5.1 Introduction: Protein Hydrolysis and Insolubilization 131</p> <p>5.5.2 Proteolysis as a Driver of Protein Insolublization in Animal Lenses 131</p> <p>5.5.3 Nonenzymatic Hydrolysis as a Driver of Protein Insolublization in Human Lenses 131</p> <p>5.6 Insolublization Due to Deamidation, Isomerization, and Racemization 132</p> <p>5.7 In vitro Studies of How PTMs Alter Protein Structure and Solubility 133</p> <p>5.7.1 In vitro Studies of Disulfide Bonding 133</p> <p>5.7.2 In Vitro Studies of Deamidation 135</p> <p>5.8 Proteomics Methods to Detect Post-translation Modifications Contributing to Protein Insolublization 135</p> <p>5.8.1 Crystallins as Ideal Proteins to Detect Age-Related PTMs 135</p> <p>5.8.2 Two-Dimensional Liquid Chromatography/Mass Spectrometry to Detect PTMs 136</p> <p>5.8.3 Searches for Known PTMs 136</p> <p>5.8.4 Searches for Unknown PTMs 137</p> <p>5.8.5 Identifying Disulfide Cross-links 138</p> <p>5.8.6 Identifying Deamidation Sites 139</p> <p>5.8.7 Identifying Isomerization Sites 142</p> <p>5.8.8 Identifying Racemization Sites 143</p> <p>5.8.9 Peptide Standards to Study Deamidation, Isomerization, and Racemization 145</p> <p>5.9 Future PTM Studies of Long-Lived Proteins 145</p> <p>5.10 Concluding Remarks 148</p> <p>Acknowledgments 150</p> <p>References 150</p> <p><b>6 Degradation of Long-Lived Proteins as a Cause of Autoimmune Diseases 159<br /></b><i>Roger J.W. Truscott</i></p> <p>6.1 Introduction 159</p> <p>6.1.1 Background 159</p> <p>6.1.2 Autoimmunity: Long-Lived Proteins and Long-Lived Cells 159</p> <p>6.1.3 Focus of this Chapter 159</p> <p>6.2 Long-Lived Cells Are Widespread in the Body 160</p> <p>6.3 Long-Lived Proteins Are Present in Many Tissues 160</p> <p>6.4 Long-Lived Proteins Decompose Over Time 161</p> <p>6.5 Defenses Against LLP Decomposition 162</p> <p>6.5.1 Rebuilding Degraded Asp and Asn Sites Within a Protein 162</p> <p>6.5.2 Oxidation-Related Modification Repair Enzymes and Antioxidants 163</p> <p>6.6 Consequences of Long-Lived Protein Decomposition 163</p> <p>6.7 Individual Autoimmune Diseases 165</p> <p>6.7.1 Pancreas 165</p> <p>6.7.2 Nerves 165</p> <p>6.7.3 Stomach 166</p> <p>6.7.4 Blood Vessels 166</p> <p>6.7.5 Gastrointestinal Tract 166</p> <p>6.7.6 Liver 166</p> <p>6.7.7 Thyroid Gland 166</p> <p>6.7.8 Adrenal Gland 166</p> <p>6.7.9 Joints 167</p> <p>6.7.10 Multiple Sites 167</p> <p>6.7.11 Skin 167</p> <p>6.7.12 Moisture-Secreting Glands 167</p> <p>6.7.13 Blood 167</p> <p>6.7.14 Muscles 168</p> <p>6.7.15 Heart 168</p> <p>6.8 Person-to-Person Variability in Breakdown of LLPs: Multiple Sclerosis 168</p> <p>6.8.1 Why Do Not All Adults Develop Autoimmune Disorders? 168</p> <p>6.8.2 Widespread LLPs and Modulation of an Immune Response 169</p> <p>6.9 Conclusions and Future Research 169</p> <p>Acknowledgments 170</p> <p>References 170</p> <p><b>7 How Isomerization and Epimerization in Long-Lived Proteins Affect Lysosomal Degradation and Proteostasis<br /></b><i>Ryan R. Julian 175</i></p> <p>7.1 Proteostasis 175</p> <p>7.2 Invisible Modifications 176</p> <p>7.3 Repair 179</p> <p>7.4 Identification 180</p> <p>7.5 Protein Turnover 180</p> <p>7.6 Mechanistic Considerations 181</p> <p>7.7 Prevention 182</p> <p>7.8 Conclusion 184</p> <p>Acknowledgments 184</p> <p>References 184</p> <p><b>8 The Maillard Reaction: Protein Modification by Ascorbic Acid 189<br /></b><i>Vincent M. Monnier, David R. Sell, Grant Hom, Shiyuan Dong, Benlian Wang and Xingjun Fan</i></p> <p>8.1 Introduction 189</p> <p>8.2 Ascorbic Acid Homeostasis in the Lens: A Dual Sword 190</p> <p>8.3 Ascorbic Acid as a Source of Age-Related Damage to the Lens 190</p> <p>8.4 Chemical Pathways of Ascorbic Acid Degradation In Vitro and the Human Lens 191</p> <p>8.5 Advanced Glycation End Products that have been Detected in the Human Lens 192</p> <p>8.6 Glucose vs. Ascorbic Acid as a Source of Advanced Glycation End Products in the Lens 193</p> <p>8.7 Ascorbic Acid as a Major Source of Oxoaldehydes in Lens and Brain 195</p> <p>8.8 Significance of Advanced Glycation/Ascorbylation Products in the Lens and Brain 196</p> <p>8.9 Conclusions 197</p> <p>Acknowledgments 197</p> <p>References 197</p> <p>Index 203</p>