Long-lived Proteins in Human Aging and Disease E-Book
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- Herausgeber: Wiley-VCH GmbH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
This authoritative overview on an emerging topic in the molecular life sciences covers all aspects of the aging of (long-lived) proteins. It describes the molecular mechanisms of aging on the protein level, in particular the most common side chain modifications and includes analytical methods to study protein half-life and the accumulation of modifications. Finally, the impact of protein aging on several age-related disases in humans is dissected, and their role in limiting human lifespan is discussed.
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Veröffentlichungsjahr: 2021
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Table of Contents
Cover
Title Page
Copyright
Introduction to the Book
Long‐Lived Proteins Are Ubiquitous
Aging
Autoimmunity
Age‐Related Diseases
Our Lenses in the Vanguard
Brain and Memory
1 Long‐Lived Cells and Long‐Lived Proteins in the Human Body
1.1 What Constitutes a Long‐Lived Cell and a Long‐Lived Protein?
1.2 Aim of the Chapter
1.3 Aging
1.4 Location of LLPs Within the Body
1.5 Extracellular LLPs
1.6 Intracellular LLPs and LLCs
1.7 Organs and Tissues that Contain LLCs or LLPs
1.8 Protein Changes and DNA Changes with Age
1.9 Processes Responsible for the Breakdown of LLPs
1.10 Oxidation: Methionine Sulfoxide Reductases and the Glutathione System
1.11 Consequences of LLP Decomposition
1.12 LLPs and Age‐Related Disorders
1.13 Neurological Diseases Where LLPs May be Implicated
1.14 Aging DNA and LLPs
1.15 How Can the Role of LLPs in Aging and Disease Be Investigated? What Can Be Done
1.16 We Will Not Live Forever
1.17 Conclusion
Acknowledgments
References
2 Imaging Mass Spectrometry of Long‐Lived Proteins
2.1 Introduction
2.2 Imaging Mass Spectrometry Methods
2.3 Protein Identification
2.4 LLPs in the Body
2.5 Long‐Lived Cells and Structures
2.6 Future Directions
References
3 Eye Lens Crystallins: Remarkable Long‐Lived Proteins
3.1 Introduction
3.2 Eye Lens and Its Transparency
3.3 Lens Crystallin Proteins
3.4 Congenital, Early Onset, and Age‐Related Cataract
3.5 Protein Aggregation and Disease, Particularly Cataract
3.6 Concluding Comments
References
4 Spontaneous Breakdown of Long‐Lived Proteins in Aging and Their Implications in Disease
4.1 Introduction
4.2 LLPs Are Found Throughout the Body
4.3 Spontaneous Modifications of Aging
4.4 LLPs and Onset of Disease: Is Correlation the Only Answer?
4.5 Spontaneous Modifications: Detrimental or Beneficial?
4.6 Protein Turnover Slows with Age
4.7 Potential Treatment of Diseases Initiated by LLPs
4.8 Future Outlook
Acknowledgments
References
5 Modifications of Long‐Lived Proteins that Affect Protein Solubility
5.1 Introduction
5.2 Insoluble Protein Definition
5.3 Insolubilization Due to Disulfide Bonding
5.4 Insolubilization Due to Nondisulfide Cross‐links
5.5 Insolublization Due to Protein Fragmentation
5.6 Insolublization Due to Deamidation, Isomerization, and Racemization
5.7 In vitro Studies of How PTMs Alter Protein Structure and Solubility
5.8 Proteomics Methods to Detect Post‐translation Modifications Contributing to Protein Insolublization
5.9 Future PTM Studies of Long‐Lived Proteins
5.10 Concluding Remarks
Acknowledgments
References
6 Degradation of Long‐Lived Proteins as a Cause of Autoimmune Diseases
6.1 Introduction
6.2 Long‐Lived Cells Are Widespread in the Body
6.3 Long‐Lived Proteins Are Present in Many Tissues
6.4 Long‐Lived Proteins Decompose Over Time
6.5 Defenses Against LLP Decomposition
6.6 Consequences of Long‐Lived Protein Decomposition
6.7 Individual Autoimmune Diseases
6.8 Person‐to‐Person Variability in Breakdown of LLPs: Multiple Sclerosis
6.9 Conclusions and Future Research
Acknowledgments
References
7 How Isomerization and Epimerization in Long‐Lived Proteins Affect Lysosomal Degradation and Proteostasis
7.1 Proteostasis
7.2 Invisible Modifications
7.3 Repair
7.4 Identification
7.5 Protein Turnover
7.6 Mechanistic Considerations
7.7 Prevention
7.8 Conclusion
Acknowledgments
References
Note
8 The Maillard Reaction: Protein Modification by Ascorbic Acid
8.1 Introduction
8.2 Ascorbic Acid Homeostasis in the Lens: A Dual Sword
8.3 Ascorbic Acid as a Source of Age‐Related Damage to the Lens
8.4 Chemical Pathways of Ascorbic Acid Degradation
In Vitro
and the Human Lens
8.5 Advanced Glycation End Products that have been Detected in the Human Lens
8.6 Glucose vs. Ascorbic Acid as a Source of Advanced Glycation End Products in the Lens
8.7 Ascorbic Acid as a Major Source of Oxoaldehydes in Lens and Brain
8.8 Significance of Advanced Glycation/Ascorbylation Products in the Lens and Brain
8.9 Conclusions
Acknowledgments
References
Index
End User License Agreement
List of Tables
Chapter 3
Table 3.1 Documented mutations in human α‐crystallins and their associated ca...
Table 3.2 Documented mutations in human β‐crystallins and their associated ca...
Table 3.3 Documented mutations in human γ‐crystallins and their associated ca...
Table 3.4 The major PTMs of human lens crystallins.
Chapter 4
Table 4.1 LLPs have been implicated in a number of diseases and ailments asso...
Chapter 6
Table 6.1 List of some common autoimmune diseases together with the relevant ...
List of Illustrations
Chapter 1
Figure 1.1. “Venus” by Titian is used to illustrate the many sites within th...
Figure 1.2.
Different cells within the pancreas
. An image illustrating the d...
Figure 1.3.
Lifelong proteins breakdown at similar rates
. The two graphs sho...
Chapter 2
Figure 2.1 Workflow of MALDI‐IMS experiment showing the following steps: tis...
Figure 2.2 MALDI‐IMS of human lens αA‐crystallin products as a function of l...
Figure 2.3 MALDI‐IMS of human lens AQP0 as a function of age showing increas...
Figure 2.4 MALDI‐IMS of lipidated human lens AQP0 as a function of lens age ...
Figure 2.5 MALDI‐IMS of deamidated human lens AQP0 as a function of age. Aft...
Figure 2.6 MALDI‐IMS of a human optic nerve section showing various protein ...
Chapter 3
Figure 3.1
Eye lens structure
and the development of
cataract
. (a) A cross sec...
Figure 3.2
The crystallins of the eye lens
. (a) An atomic model of the αB‐cr...
Figure 3.3
Various morphologies of cataract
and their locations within the ey
...
Figure 3.4
Protein folding, unfolding, and aggregation
. (a) Schematic of the...
Figure 3.5
The structure of the amyloid‐β (Aβ)
amyloid fibril
Figure 3.6
The formation, structure, and visualization of β
2
‐microglobu
...
Figure 3.7
Schematic of the mechanism of sHsp, e.g. αB‐crystallin, chaperone
...
Chapter 4
Scheme 4.1 Spontaneous modification of LLPs as we age can alter (a) charge, ...
Scheme 4.2 Spontaneous breakdown of LLPs can generate reactive intermediates...
Scheme 4.3 Cleavage of LLPs can occur via spontaneous processes at Asn, Asp,...
Scheme 4.4 Asparagine and aspartic acid readily break down in LLPs via two r...
Figure 4.1
γS crystallin
, a major LLP in the human lens, accumulates num
...
Chapter 5
Figure 5.1 The identification of deamidation in human γS‐crystallin peptide ...
Figure 5.2 Identification of isoasparate residues in peptide 106–117 (LTIFEQ...
Figure 5.3 Strategy to introduce a
D
‐isoaspartyl residue at position 14 in h...
Figure 5.4 A hypothetical model to explain the formation of light scattering...
Chapter 6
Figure 6.1
Aged, long‐lived proteins
are more heterogeneous than the or
...
Figure 6.2
Autoimmune diseases
affect many organs and tissues
. Just as the h...
Chapter 7
Figure 7.1 Illustration of balance and the major contributors active in prot...
Figure 7.2 Representative examples of peptide isomers (because of insertion ...
Scheme 7.1 Mechanism behind formation of isomerization and epimerization at ...
Scheme 7.2 Primary repair pathway for PIMT. The L‐succinimide can then reope...
Figure 7.3 Hypothesized connection between aging, lysosome function, and pro...
Chapter 8
Figure 8.1 Major degradation products of ascorbic acid and dehydroascorbic a...
Figure 8.2 Structure of selected advanced glycation end products present in ...
Figure 8.3 Mean levels of crystallin‐bound AGEs and oxidative modifications ...
Figure 8.4 The chemical relationship between advanced glycation end products...
Figure 8.5 Rapid
in vitro
browning of rabbit lenses incubated with each 10 m...
Guide
Cover Page
Title Page
Copyright
Table of Contents
Begin Reading
Index
WILEY END USER LICENSE AGREEMENT
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Long‐Lived Proteins in Human Aging and Disease
Edited by
Roger J.W. Truscott
Editor
Prof. Roger Truscott
University of Wollongong
Illawarra Health and Medical Research Institute
2522 Wollongong
Australia
Cover
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Cover Images: Engraving from de humani
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1543, is reproduced of the Het Allard Pierson.
Scanning Electron Micrograph
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fabrica libri septem by Andreas Vesalius, 1543.
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Print ISBN: 978‐3‐527‐34728‐5
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Introduction to the Book
Why is it important to understand long‐lived proteins (LLPs) and long‐lived cells (LLCs) and their breakdown? This question forms the theme of this book.
Long‐Lived Proteins Are Ubiquitous
A major finding in the past decade has been that LLPs and LLCs are found throughout the human body. Initially, this discovery was surprising because a common conception in biochemistry, reinforced by undergraduate teaching, was that proteins turnover. This is indeed true for the majority of cellular polypeptides.
Most proteins are created, perform their function, and then are destroyed. Indeed, this aspect of a protein's existence forms a plank of proteostasis, the realm of biochemistry that investigates the many regulatory processes that work together to maintain a relatively constant environment within the cell. Proteostasis also allows a cell to regulate the levels of certain proteins in response to external stresses. One example is the increased concentration of heat‐shock proteins in cells, which are synthesized in response to elevated temperatures. Without the ability to degrade old proteins and synthesize new proteins, proteostasis would not be possible.
Clearly, LLPs are a subset of proteins that do not fall within this proteostatic framework because they are, by definition, long lived. One of the aims of this book is to demonstrate that these long‐lived components are not simply inert bystanders but that they decompose and their breakdown in the body has a number of major outcomes.
Aging
One area where LLPs will assume much greater significance is in the field of human aging. At present, most research into the mechanism of aging uses short‐lived animals, e.g. fruit flies, nematodes, and rodents. It is obvious that LLP breakdown will have little, or no, significant role in the aging of species whose lifetime is measured in days or weeks. Therefore, to some extent, experiments that can be readily undertaken within the time frame of typical grants have shaped the direction of the overall field of aging research. A key question then is to what degree do short‐lived animal studies reproduce biochemical events that are responsible for human aging.
As will become clear after reading the chapters in this tome, there are many organs and tissues where cells are long lived and where proteins are LLPs. Furthermore, some of these LLPs are present for years, decades, or even, in some cases, our whole lives. All LLPs decompose. In many instances, this breakdown is due to spontaneous chemical processes that are mediated by the properties of the amino acid side chains. In other cases, reactive metabolites such as sugars and their metabolites bind to the LLPs or the proteins are modified enzymatically. In every instance, the structure of the LLP will be altered.
Although it is possible to analyze the deterioration of these LLPs over time, it is much harder to measure the physiological impact of the gradual deterioration of such LLPs. This is particularly so because aging is accompanied by several factors that may each contribute to the observed loss of function. One major difficulty is that such studies must be performed in long‐lived species: ideally, in humans or in other primates. In the case of humans, the number of experiments that are possible is restricted, and it is very difficult to have replicates. Researchers must also accept a diverse genetic background. Despite these restrictions, it will become increasingly important to examine aging in long‐lived animals with particular reference to the age‐dependent degradation of LLPs. This in no way diminishes the need to complement such studies with parallel experiments on DNA degradation. These two pillars are not separate. One can demonstrate their interrelationship using two examples: the longevity of some histones and the deterioration of some LLP components of the nuclear pore. If long‐lived histones deteriorate, it may affect gene transcription. Breakdown of the structure of the nuclear pore in postmitotic cells could influence the transport of molecules into the realm of the chromosome as well as the transport of molecules, such as mRNA, out of the nucleus.
Absent from this book is a discussion of another macromolecular group: lipids. This field is of interest because lens membrane phospholipids change significantly in humans as a function of age. Polysaccharides are also not covered, and data on the age‐dependent alterations to glycoproteins is scant.
Autoimmunity
Another area of LLPs that, at the time of writing, is almost an empty page is that of LLP breakdown and autoimmunity. In Chapter 6, it is hypothesized that the age‐related decomposition of human LLPs is responsible for many, if not most, cases of autoimmune disease. The fact that LLPs and LLCs are ubiquitous and that they decompose over time to form new structures that were not present at the time of birth effectively means that the body of an adult human contains many “foreign” antigens. In this case, “foreign” does not refer to extraneous antigens but simply to a normal human LLP that has, over time, become significantly altered. Very little is known about the immunogenicity of the suite of novel post‐translational modifications that accompany aging of LLPs. One exception is where it has been shown that replacement of one L‐Asp by an isoAsp residue increases the immune response greatly, which suggests that the introduction of other novel structures into LLPs is likely to act as a trigger for an autoimmune response. This is amplified by the recognition that most LLPs will accumulate a number of different modifications that may act synergistically as immune stimulants. In some cases, this response to an altered self may lead to the development of an autoimmune disease.
Age‐Related Diseases
Sadly, little headway has been made in efforts to combat major age‐related diseases, particularly neurological ones, such as Parkinson's, multiple sclerosis, motor neurone disease (Amyotrophic lateral sclerosis) and Alzheimer's disease. Their incidence increases as we live longer lives, with the result that there is already a huge impact on the health care budgets of all nations, particularly those of developed countries.
It is noteworthy that many of the age‐related diseases listed above involve proteinopathies, where protein aggregates accumulate inside the cells. The reason for this accumulation is poorly understood. It is my contention that the age‐related decomposition of proteins that is documented in this book will be found to play a role in the formation of such protein aggregates. Once this factor is recognized, the focus of research should necessarily alter.
One important implication of this perspective is that the thrust of age‐related disease research will necessarily change to humans because short‐lived animals cannot recapitulate the sorts of changes that are observed as a result of long‐lived macromolecular breakdown.
To illustrate this point, a number of age‐related PTMs, including racemization and covalent cross‐linking, cannot be readily processed by the protein recycling machinery of the cell. An inevitable consequence is that such modified LLPs accumulate in the lysozyme and cytoplasm. This aspect is covered in Chapter 7.
Once the eyes of scientists are opened to the ubiquitous phenomenon of age‐related protein degradation, greater progress should be made in understanding these neurological diseases.
Our Lenses in the Vanguard
It will become apparent from the chapters in this book that much of our current understanding of the consequences of LLPs on the cells and proteins contained within them has been derived from investigations of the human lens. Although this is a well‐studied tissue, there are still fundamental aspects that remain to be understood. It is likely that some of these will have relevance for other tissues of the body. As just one example, it appears that once the levels of chaperones, such as α‐crystallin, within fiber cells drop significantly with age, that protein aggregates bind tightly to the interior of cell membranes. This attachment impedes cell‐to‐cell transport within the lens with dire consequences for the concentration of glutathione in the lens nucleus because this antioxidant is synthesized and reduced exclusively in the outer part of the lens. Is this observation lens specific or could a similar phenomenon occur in other aged postmitotic cells such as neurons? Neurons are dependent on a supply of cysteine from astrocytes for glutathione synthesis because they lack the capacity to import glutathione directly. It is not known if aging affects this requisite transport process. It will be important to determine the extent to which lens data can be applied more generally.
It is my hope, and I am sure that of the other authors in this book, that by illustrating the many and varied aspects of LLPs and LLCs, that readers will come to appreciate the importance of this newly recognized family of cells and macromolecules. At the moment, this is a nascent field, but I am confident that in the near future, it will blossom exponentially.
Brain and Memory
I will finish with a musing on memory, the molecular basis of which still remains largely a scientific enigma. I have long thought that LLPs could play a part in, or could even be responsible for, memory. In support of this notion, recent findings have demonstrated the existence of LLPs in brain synapses and reinforce a potential role for them in learning and memory. A key issue is the degree of turnover of the macromolecules within neuronal connections that are thought to be responsible for memory storage.
In the future, scientists may confirm that memories are, at least in part, due to LLPs. If this is validated, the field of LLPs will unquestionably assume a role far greater than it does today. As a corollary, if this statement is borne out, your remembrance of reading this statement, as well as all others, would depend in no small part on the maintenance of LLPs within your brain.
Roger J.W. Truscott
February, 2020
1Long‐Lived Cells and Long‐Lived Proteins in the Human Body
Roger J.W. Truscott
University of Wollongong, Illawarra Health and Medical Research Institute, Wollongong, NSW, 2522, Australia
1.1 What Constitutes a Long‐Lived Cell and a Long‐Lived Protein?
It is sometimes stated in textbooks that all proteins in the body are being continuously degraded and resynthesized. It will become clear after reading this chapter that this statement is untrue.
Although one subject of this book is long‐lived proteins (LLPs), the definition of what constitutes an LLP is somewhat arbitrary. Clearly, it does not apply to a polypeptide whose half‐life is measured in minutes or a few hours; however beyond that, the importance of half‐life in terms of its effect on biology is intimately linked to the susceptibility of a particular protein to modification.
In some cases, LLPs have been subdivided into categories, for example, by creating a subgroup of extremely long‐lived proteins [1]. For reasons of simplicity in this book, LLPs will be regarded as one category. As a guide, a half‐life of greater than 48 hours can be regarded as a yard stick for the classification of an LLP. In most cases, half‐lives will be considerably longer than this, and in some cases, there is no turnover during a lifetime. The members of this latter category will be referred to as lifelong proteins.
In this context, one year of life, which we typically consider to be a very short period of time within a normal life span, corresponds to incubation of a lifelong protein at 37 °C for 8760 hours. We can infer that in many cases, a period of several weeks may be sufficient to lead to significant LLP breakdown and that this may have major consequences. Predominantly, this conclusion arises from studies in which the main enzyme involved in protection from protein deterioration, protein isoaspartate methyl transferase (PIMT), was deleted. In the absence of PIMT, mice underwent seizures and died 42 days after birth [2]. Over this time period, progressive damage to cytosolic proteins was detected in the brain, heart, liver, and blood cells. Because this protein damage is due to spontaneous reactions, the extent of damage is likely to be very similar in mice and men. Information on the sites of LLPs in the body has been outlined previously [3] and will be summarized in this chapter.
Similar considerations with regard to definition apply to long‐lived cells (LLCs). Typically, LLCs are present in the body for a period of many weeks and often may be present for years. Long‐lived plasma cells within the bone marrow are responsible for the fact that serum antibodies arising from childhood vaccinations against smallpox are still present in adults, despite the disease being eradicated more than 30 years ago [4]. It should be noted that the focus of this chapter will be human studies.
1.2 Aim of the Chapter
The purpose of this chapter is not to provide a comprehensive review of particular sections that are highlighted but rather, within the confines of word limits, to provide an overview of the field of LLPs and LLCs with a particular reference to their locations within the body.
Where information is available, an attempt will be made to link age‐related decline in function of the tissue to the presence of LLPs or LLCs. This is a nascent field and it will evolve rapidly.
1.3 Aging
Although aging is obviously intimately related to the degradation of LLPs, and this is encapsulated in the title of this book, it is not the purpose of this chapter to review aging. Aging is a large, complex subject with a number of theories. Broadly, it can be stated that there are two main camps: researchers who believe that aging is genetically regulated and the other group who believe that aging is primarily the result of molecular degradation. In the latter group, DNA deterioration, particularly as it concerns telomeres, has been a prime focus.
There are numerous phenotypic changes that accompany human aging, among which are wrinkling of skin, increase in abdominal fat, decrease in bone density, loss of cartilage and muscle mass, and, in late stages, a compilation of adverse features that can be termed frailty.
Partridge et al. [5] subdivided the “hallmarks of aging” into nine categories: genomic instability, telomere attrition, epigenetic alterations, stem cell exhaustion, loss of proteostasis, deregulated nutrient sensing, altered intercellular communication, mitochondrial dysfunction, and cellular senescence.
It may seem surprising that LLPs and their age‐dependent deterioration were not incorporated as one of the hallmarks of aging in this article [5]. This omission probably reflects the current lack of awareness of the existence of LLPs and will no doubt be rectified in subsequent reviews on the mechanisms of aging. The author predicts that it will rise to become recognized as one of the most important factors responsible for aging of humans and the associated age‐related decline in organ, tissue, and overall bodily function. The detailed role of LLP breakdown in determining human life span may remain more elusive; however, a framework for this was published a decade ago [6]. As the name implies, aging is the major risk factor for many age‐related diseases including cataract and neurological diseases such as dementia.
1.4 Location of LLPs Within the Body
Many LLPs serve a structural function. LLPs will be considered in two classes: those that lie within the cell and those that are extracellular. It is important to recognize that these are not two independent classes.
It is now known that the extracellular matrix (ECM) is a complex structure composed of many different macromolecules whose structural integrity is crucial for maintaining tissue function. Abnormalities in ECM biosynthesis and catabolism are responsible for a number of inherited and acquired diseases, but it is unknown whether insidious age‐related modifications to LLPs within the ECM affect the functions of tissues. For a more detailed discussion of ECM, the reader should refer to Ref. [7].
1.4.1 ECM and Tissue Function
The ECM has a profound effect on cellular function. Four broad types of interactions regulate the growth, development, and function of cells: growth factors/cytokines, cell‐to‐cell contact, hormones/vitamins, and ECM. Each of these operates via specific cell surface or sometimes via cytoplasmic receptors. This is further complicated by the knowledge that many growth factors and cytokines are bound specifically by matrix components and also that ECM can modulate the expression of cellular receptors for growth factors. Thus, there is an intimate reciprocal relationship between cellular function and the ECM. These complex interactions should be borne in mind when evaluating the aging of individual components within the ECM.
1.5 Extracellular LLPs
1.5.1 Several ECM Components Are Long Lived
The LLPs of the ECM can be subdivided into four groups: elastin, structural glycoproteins, proteoglycans, and collagens. These will be considered individually with particular attention to data that pertain to longevity. In a recent study in mice using stable isotope labeling, ECM proteins were among the longest lived of any of the ~3500 proteins analyzed [1].
1.5.1.1 Elastin
Elastic fibers and sheets confer strength, distensibility, and flexibility to tissues. Unlike other ECM family members, elastin is present in DNA as a single‐copy gene. This codes for the soluble precursor tropoelastin. Elastic fibers are insoluble, which is a consequence of the high proportion of hydrophobic amino acids and the presence of unique intermolecular covalent cross‐links: desmosine and isodesmosine [8]. Elastin has a similar amino acid composition to collagen, in that approximately one‐third is composed of glycine. Alanine and valine are also prominent with substantial amounts of proline. The sulfur‐containing amino acids, cysteine, and methionine are absent.
Other macromolecules such as lysyl oxidase, glycoproteins, and fibrillin occur together with elastin in the elastic fibers. Elastin plays a vital role in tissues such as the aorta where, as in other large arteries, it functions in pressure wave propagation and pulse dampening. It is also a major component of the lung, skin, ligaments, oesophagus, cartilage, and the bladder.
It is clear that there are major changes to tissues with aging. Cutaneous aging is the most obvious because its consequences are clearly visible. In the skin, most of the elastin is located in the dermis, which is the spongy middle layer [9]. Degeneration of the elastic fiber network coupled with loss/modification of collagen and a decrease in hydration are linked to visible changes in the dermis with age.
The expression of the tropoelastin gene mainly occurs in the first years of life when the cells of elastic tissues produce the elastin required for the body. After that time, gene expression is reduced significantly and less elastin is made, such that by middle age, only a trace of elastin is produced and we rely on the elastin that was deposited before and during childhood [10, 11]. Therefore, our elastic connective tissues depend on the persistence of elastin. To this end, elastin has been shown to have a half‐life of about 74 years [12], and aging of elastin has been proposed to limit human life expectancy [13]. During our life span, the elastin D‐Asp content increases to reach ~15% by the age of 60 years. Interestingly, the rate of increase in D‐Asp was most rapid in the years up to the age of 20 years and then became linear [10] (Figure 1.3). This mirrors the accumulation of D‐amino acids in the lens proteins; however, the reason for this biphasic graph is not known.
1.5.1.2 Structural Glycoproteins and Proteoglycans
It appears that both elastin and the non‐elastin components of the elastic fiber are long‐lived. This was the conclusion of a study by Shapiro et al. [12] who compared the nuclear weapon‐derived 14C content and the D‐Asp levels of elastin.
The content of D‐Asp in elastin from aged human lung tissues amounts to approximately 17% by the eighth decade [12]. Such a large extent of racemization would be expected to be accompanied by changes to the properties of the elastin fibres. This is especially so, given that racemization of Asp is but one of many age‐related post‐translational modifications (PTMs). Very few studies have examined the properties of isolated elastin itself, but there are a number of studies in which the properties of the tissues that depend on elasticity have been documented as a function of age. For example, in the case of lung elasticity, one study measured the age‐dependent change in the aerobic capacity of healthy adults [15]. Age‐related decline in peak oxygen consumption (VO2) was pronounced and the decline was not linear. The rate of decline was found to accelerate from 3% to 6% per decade in the 20s and 30s to more than 20% per decade in the 70s.
1.5.1.3 Collagens
Collagens are the most abundant animal proteins accounting for approximately one‐third of total body protein by dry weight. The term collagen encompasses more than 30 gene products with approximately 20 different types of collagens. For more information, the reader is referred to detailed reviews, e.g. Ref. [16].
Collagens are predominantly extracellular. The primary gene products form triple helices, and the polypeptide chains undergo extensive PTMs, in particular hydroxylations involving the formation of hydroxyproline (hydroxyPro) and hydroxylysine residues. A common repeating sequence in collagen is Gly‐X‐Y, with X and Y being Pro or hydroxyPro. The other unusual amino acid, hydroxylysine, acts as a site of covalent cross‐linking, as well as attachment of carbohydrate. Some collagens form sheets, others form fibrils, and yet others form filaments.
The presence of repeated amino acid sequences renders the collagens difficult to analyze using proteomics, although four collagen proteins were present in the list of LLPs identified by Toyama et al. [17]. Most data on the effect of aging on collagens have been derived from the measurement of D‐Asp.
A mean collagen half‐life of 197 years was calculated for the superficial digital flexor tendon (SDFT) in horses, which was significantly higher than that for the common digital extensor tendon (34 years). By comparison, the half‐life of noncollagenous proteins was two years in the SDFT [18]. In these tissues, the D‐Asp levels correlated with those of pentosidine. Pentosidine is an advanced glycation end (AGE) product and is a marker of carbohydrate modification. Evidence suggests that the turnover of collagen in patellar tendons may be similar to that from more metabolically active tissues such as skeletal muscle [19]. In biological terms, turnover appears minor and tendon collagen can be considered relatively inert.
Collagen half‐life has been calculated in other tissues with Sivan et al. [20] reporting a half‐life of 95–215 years in intervertebral disc collagen, whereas Verzijl et al. [21] reported 117 years in articular (knee) cartilage. By comparison, when a noncollagenous protein half‐life was calculated from the same tissues, values of 3–25 years were reported for aggrecan fractions in articular cartilage [21] and 6–26 years for intervertebral disc aggrecan [21]. Thus, collagen may be largely stable over our lifetimes in tendons and many other cartilage‐containing samples, whereas the other protein components of these same tissues are also LLPs, but with significantly shorter half‐lives.
In collagen isolated from both articular tissue and skin, AGE concentrations increased linearly in parallel with D‐Asp. The half‐life of human skin collagen was calculated to be approximately 15 years [22].
1.5.1.3.1 Aging Skin and Collagen Cutaneous aging occurs through two processes that can be categorized as intrinsic and extrinsic aging [23].
In the human dermis, intrinsic aging is characterized by atrophy because of loss of collagen, degeneration in the elastic fiber network, and loss of hydration. Extrinsic aging is due to environmental factors with a principal cause being ultraviolet (UV) exposure, although tobacco use can also contribute.
Intrinsic aging takes place over time, regardless of outside environmental influences. After the age of 20, a person produces about 1% less collagen in the skin each year, and the skin becomes thinner and more fragile. Less elastin and glycosaminoglycan (implicated in hydration) formation also contributes to skin aging, together with diminished functioning of the sweat and oil glands. Some of the age‐related changes to the mechanical properties of dermal connective tissue can be correlated with covalent cross‐linking [24]. Accumulation of advanced glycation cross‐links and other adducts is covered in Chapter 8. It should be noted that the processes underpinning collagen modification are still subject to revision. For example, it was long accepted that cross‐linking on the lysine aldehyde pathway, which is the major one in skin and cornea, involved histidine residues. It has recently been demonstrated that this cross‐link, histidinohydroxylysinonorleucine, is in fact a laboratory artifact [25].
Some wrinkle formation as a result of intrinsic aging is inevitable. In purified elastin from skin, the increase in D‐Asp was highly correlated with age. Racemization rates were found to be higher in elastin from skin than from lung parenchyma and from aorta [26].
Because of the multiple events taking place in collagens, it is difficult to highlight one degradation or cross‐linking process as being responsible for the changes in physical properties associated with aging. It is very likely that multiple chemical and biochemical events are responsible. Aging of elastic fibers and its influence on the elasticity of tissues has been reviewed [27].
1.6 Intracellular LLPs and LLCs
Several questions need to be addressed before the role of LLPs and LLCs can be properly evaluated in terms of the impact of the longevity of macromolecules and their deterioration on overall human health and age‐related diseases.
Firstly, how many tissues/organs in the body contain components that are long‐lived? Once such sites have been examined, which of the cells within them are old, and which of the macromolecules within these LLCs are also old? Lastly, is there evidence of LLP breakdown and what is known about the consequences?
Before discussing these aspects, it should be recognized that, at the moment, little is known about many issues, partly because LLPs/LLCs is a newly recognized field. It is also worth noting in terms of background that in mice the abundance of the vast majority of proteins in organs and tissues remains unchanged with age [28]; however, some alterations in protein abundance between the livers and brains of young and old rats has been detected [29].
1.6.1 LLCs and LLPs in the Organs of the Body
Individual organs and tissues will be discussed where information about LLPs or LLCs is known. At the end of the discussion of each tissue, a section on aging and possible age‐related consequences will be discussed.
A very brief summation of the main function(s) of the particular organ is provided at the beginning of each section. Under each heading, some of the main conditions or diseases associated with aging of that particular organ will be described. Within the scope of this book, it is not possible to cover these comprehensively.
Following this, evidence will be provided for the existence of LLCs or LLPs within the organ.
Before this general description of cells and organs within the body, a recent comprehensive program to examine the lifetimes of brain proteins has revealed some remarkable results [1]. Neurofilament proteins and transmembrane proteins were found to be stable. The cytoskeleton was composed of protein components with varied lifetimes. For example, neurofilaments, tubulin, and intermediate filament proteins were found to be LLPs, whereas the lifetimes of actins approximated that of the average proteome. A picture is emerging in which the actin cytoskeletal network within a cell may be dynamic, whereas the microtubule‐based cytoskeleton is more stable. Generally, individual protein lifetimes were conserved across the organs examined. Mitochondrial proteins tend to be long‐lived, although there was some heterogeneity [1]. It is important to recognize that proteomic investigations despite being very elegant cannot provide data on all proteins in a sample because of issues of solubility, amino acid sequence repeats, PTM, and low copy numbers.
1.7 Organs and Tissues that Contain LLCs or LLPs
1.7.1 Long‐Lived Cells
The following organs and tissues will be discussed in relation to their content of LLCs and LLPs: the eye, oocytes, kidney, adipose tissue, brain, heart, lung, skeleton, teeth, hair, joints, liver, pancreas, and intestine (see Figure 1.1).
Figure 1.1. “Venus” by Titian is used to illustrate the many sites within the human body where long‐lived proteins and long‐lived cells are present. Each site is described in greater detail in this chapter. Mark Twain in “A Tramp Abroad” described this masterpiece as the “the foulest, the vilest, the obscenest picture the world possesses.”
Source: Adam eastland/Alamy Stock Photo.
1.7.1.1 Eye
The eye is composed of the lens, cornea, aqueous humor, vitreous humor, and the retina. Tissues will be discussed separately.
1.7.1.1.1 Lens The lens focuses and filters external light onto the retina. The lens is known to be made up of many LLCs. This may be a direct consequence of the growth pattern of the lens. During our lives, epithelial cells are continuously added on the outside of a lens that was present at birth. These epithelial cells differentiate into very long fiber cells that are packed with crystallin proteins but lack nuclei, mitochondria, and other cellular organelles. Lens proteins are known to be LLPs [30].
The human lens has proven to be an ideal tissue for studying the age‐related decomposition of LLPs (see also Chapters 3–5). Partly, this is a consequence of there being only a few major proteins, so proteomic investigations are made easier. Another factor is the absence of active enzymes in the center of the adult lens, i.e. the lens that was present at the time of birth. This is due to the fact that all proteins in this region have been exposed to an elevated temperature (~35 °C) for 175 000 hours by age 20. Thermal denaturation of the enzymes has taken place.
The lack of enzyme activity indicates that any changes to LLPs in the lens have come about as a result of spontaneous processes. The interior of the adult lens is thus the realm of chemistry and not biochemistry.
This fact has been enormously useful for researchers because it indicates that events such as protein truncation, if found, can be attributed not to enzyme activity but to other reactions such as spontaneous peptide bond cleavage [31, 32]. Similarly, when protein methylation was detected in the nuclear (central part of the lens) researchers recognized that this could arise only from non‐enzymatic reactions. This lead to the discovery that exposure to S‐adenosyl methionine alone was sufficient to cause methylation of cysteine residues [33]. As an aside, it should be noted that methylation of DNA bases (as well as of histone Lys and Arg residues) is one of the primary means by which epigenetic processes are mediated. It is not known if non‐enzymatic methylation might also be involved in the nuclei of cells or if this might increase with age.
Indeed, the human lens has proven to be a gold mine for elucidating the processes responsible for age‐related degradation of LLPs. These various modifications, including racemization, deamidation, truncation, and cross‐linking, will be documented elsewhere. The degree of modification is great and increases with age. To illustrate this point, by the age of 60 years, every single lens polypeptide contains, on average, two to three D‐amino acids.
Age‐Related Changes to the Lens: Presbyopia and Cataract Presbyopia, the inability to focus on nearby objects, affects almost all individuals in the fifth decade. This results primarily from a massive increase in the stiffness of the lens [34]. By this stage in life, all free α‐crystallin has been consumed by binding to other lens proteins as they denatured [35]. At this time, an internal barrier to diffusion arises within the lens at a position that corresponds approximately to the lens at birth. The result is that essential antioxidants such as glutathione, which are synthesized or re‐reduced only in the outer region of the lens, cannot readily access the central part of the lens. Ultimately, this increasingly oxidative environment leads to the widespread oxidation of lens crystallin proteins in the lens center (nucleus). This oxidation is accompanied by insolubility and coloration [36]. Age‐related nuclear cataract is the outcome.
1.7.1.1.2 Cornea Major changes in the cornea with age include thickening of both the epithelial and the endothelial basement membranes. One change in the cornea with age is the decrease in the density of the single layer of corneal endothelial cells, which line the posterior surface of the cornea. Corneal endothelial cells are not known to proliferate [37], so may be LLCs.
1.7.1.1.3 Vitreous Humor The vitreous humor is a transparent gel situated between the lens and the retina, occupying most of the eye's volume. It is composed of hyaluronic acid interspersed in an aqueous network of collagen type II and collagen type IX fibrils [38]. The properties of the vitreous humor change with age, becoming more like a two‐phase system. More than half of the vitreous humor becomes liquified in 25% of individuals aged 40–49 years, and this increases to 62% of individuals aged 80–89 years [39]. Although the volume of liquid vitreous increases with age due to phase separation, the gel component becomes stiffer [40].
Age‐Related Changes to the Vitreous Humor: Retinal Detachment The most common age‐related pathology of the vitreous humor is posterior vitreous detachment (PVD). This involves separation of the posterior vitreous cortex from the lamina of the retina. The incidence of PVD rises from 53% in people older than 50 years to approximately 65% over the age of 65 years.
Aggregation of collagen fibrils may cause vitreous liquefaction in older people, which predisposes them to PVD. The total amount of collagen in the vitreous humor does not change throughout life, so the concentration decreases as the eye grows. It is thought that there is no, or very little, postnatal synthesis of vitreous collagen [41]. Thus, vitreous collagen is probably an LLP that undergoes typical age‐related degradation; however, these have not been documented. It is not known if this possible change in the structure and properties of collagen in aged eyes is a factor that predisposes the elderly to PVD; however, this proposition is worthy of investigation.
The most common reason for retinal detachment is age‐related shrinkage of the vitreous gel, described above, which can lead to tearing at a weak point in the retina. Once such a tear, or a hole, develops, fluid can collect beneath it and reduce the adhesion of the retina to the choroid, resulting in retinal detachment.
1.7.1.1.4 Retina The retina is the innermost, light‐sensitive part of the eye. Photons from the outside world are converted into a neural signal that is transported to the brain via the optic nerve. The structure of the vertebrate retina is complex, consisting of 10 layers. In addition to three types of glial cells [42], five types of neurons are present in the retina: amacrine cells, ganglion cells, photoreceptors, horizontal cells, and bipolar cells. Within retinal ganglion cell neurons, and other neurons, a stable neurofilament network is present, which is composed of proteins that self‐assemble, and such structures tend to remain intact [43]. While these may be LLPs, the vast majority of labeled actin is cleared within seven months of its synthesis [44].
Age‐Related Changes to the Retina Like other neurons in the central nervous system (CNS), there may be little or no turnover of nerve cells after childhood years. This includes the optic nerve. Photoreceptors are renewed continuously in a process termed disc shedding. Equatorial cones and retinal pigment epithelial (RPE) cells decrease at uniform rates from the second to the ninth decade [45]. Interestingly, the rates of rod and ganglion cell loss were faster between the second and fourth decades.
The RPE is a single layer of cells located in the outermost part of the retina, which nurtures adjacent photoreceptor cells and helps in the renewal of photoreceptor outer segment membranes. The age‐related pigment, lipofuscin, accumulates in the human RPE and by the age of 40, approximately 8% of the cytoplasmic volume of macular RPE cells is occupied by lipofuscin granules, whereas by the eighth decade of life, lipofuscin content reaches an extraordinary 19% of the cytoplasmic volume [46, 47]. Such an observation implies, but does not necessarily mean, that the RPE cells are LLCs because in this case, the accumulation of lipofuscin may be influenced by exposure to light [48].
Age‐Related Conditions Affecting the Retina A number of diseases affect the retina, and there is insufficient space to describe them all.
Age‐related macular degeneration (AMD) is one of the most common. In AMD, the macula (the central part of the retina responsible for high‐resolution color vision) is degraded, which means that vision is distorted. Thus, reading, for example, becomes difficult. The cause of AMD is not known.
Retinitis pigmentosa is characterized by photoreceptor degeneration and progressive blindness. The molecular mechanisms involved in photoreceptor death are also not understood.
Glaucoma is caused by damage to the optic nerve. In glaucoma, the ganglion cells, as well as other cells within the optic nerve, die. One reason is excessive fluid pressure within the eye. Because regeneration of the nerve does not take place, damage is irreversible.
Lamin B1 and lamin B2 are known LLPs that have distinct functions in retinal homeostasis and are present in both rod and cone photoreceptors [49]. In the absence of Lamin B1, cone photoreceptor survival is decreased and synaptogenesis is impaired; however, it is not known if the age‐related modification of lamin B1 and lamin B2 affects cell function.
1.7.1.2 Oocytes
Every woman is born with all the eggs already inside her ovaries, and human egg cells are huge (~100 μm diameter). A female will release one of these eggs during every menstrual cycle throughout her fertile lifetime. This remarkable sequence of events means that a mother carries egg cells, one of which may one day be fertilized and grow into her own grandchild. The consequence of this process is that eggs are decades old and are thus LLCs. Despite this longevity, once ovulation occurs, the egg cell deteriorates very quickly and dies after 12–24 hours.
1.7.1.2.1 Age‐Related Changes to Oocytes Fertilization and pregnancy rates of unfertilized oocytes were measured in three groups of women based on age (group 1, ≤34 years; group 2, between 35 and 39 years; and group 3, ≥40 years). [50] Under the same conditions, fertilization rates in the three groups were indistinguishable; however, pregnancy rates dropped by one‐third (group 1, 43.2%; and group 3, 14.3%). The authors concluded that the age‐related decline in fertility was due to degeneration in the oocytes. On this basis, “women in the older age group have a higher chance of achieving pregnancy from ovum‐donation programs than by persisting in using their own aged oocytes.”
Less is known about the biochemical changes that take place within the body in the oocyte at any stage. A protein involved in chromosome separation in eggs may be implicated in the age‐related decline in fertility. The many functional changes associated with oocyte aging have been reviewed [51].
Because of the importance of oocyte health for assisted reproduction, much has been published on the influence of culture conditions and a variety of additives, e.g. [51]. As illustrated by the substantial increase in the rate of trisomy (2–3% for women in their 20s to ~30% for women in their 40s), it is clear that chromosomal errors, particularly relating to segregation, are a feature of oocytes from older women [52]. It is not easy to tease out the relative impact of DNA degradation and LLP deterioration in these various age‐related phenomena. Expression of BRCA1 and other DNA repair proteins decreases with age, but it is not known if such tumor suppressor proteins are LLPs.
Acetylation of lysine 14 on histone H3 and lysines 8 and 12 on histone H4 in mouse oocytes gradually increased during aging, and it is known that acetylation of nuclear histones can play an important role in various cellular functions [53]. As noted elsewhere, histones H4 and H3 are LLPs, and if they are modified significantly as a result of aging, this may contribute to the alterations in the functional properties of the oocytes.
1.7.1.3 Kidneys
The kidneys maintain fluid balance and excrete waste products produced by metabolism. The functional unit of the kidney is the nephron, which filters the blood supplied to it. The end result is the reuptake of fluid and ions and production of urine.
1.7.1.3.1 Age‐Related Changes to the Kidney There is a linear decrease in renal function with age [54], and this is associated with a decrease in the mass of the organ. A number of other physiological functions correlate with age, for example, glomerular size increases. There are approximately 1 million functional nephrons per kidney; however, this number progressively decreases over time [55]. All these changes lead to an overall decrease in glomerular filtration rate.
The kidney is widely thought of as being unable to repair itself once damaged, and it would appear that this organ is composed largely of LLCs [56]. More specifically, the adult kidney possesses some ability to repair the existing nephrons but cannot replace nephrons lost with age with new ones [57].
1.7.1.4 Adipose Tissue
Adipose tissue is present in two types. One type exists primarily for the storage of lipid, whereas brown adipose tissue produces heat by thermogenesis and functions in thermoregulation. Brown adipocytes contain numerous small lipid droplets and many mitochondria, which gives the tissue its color. For a review, see Ref. [58].
1.7.1.4.1 Age‐Related Changes to Adipose Tissue Brown adipose tissue is abundant in newborns (~5% body weight), where it has an important role in providing resistance to hypothermia; however, the amount decreases with age. Graja and Schulz evaluated a number of mechanisms for the age‐related loss of brown adipocytes. A decline in brown adipogenic stem/progenitor cell function was one [59].
The number of adipocytes is set during childhood. Using bomb‐pulse data to calculate cell turnover, it was revealed that neither adipocyte death nor generation rate was altered in early onset obesity and that only 10% of fat cells were renewed annually [60].
1.7.1.5 Brain
It is well known that neurons are LLCs [61]. With regard to proteins within the brain, the vast majority of total proteins have lifetimes between 3 and 13 days [1], although some are much longer. Within this latter group, the proteins of myelin have been the most thoroughly studied by proteomics, with the evidence strongly supporting the case that the proteins in myelin are LLPs [17] and are probably lifelong. Myelin proteins such as MBP deteriorate with age and to a greater degree in multiple sclerosis patients [62]. The degradation pattern of MBP closely resembles that of lens crystallins, supporting the fact that MBP and presumably the other proteins that compose myelin are LLPs.
Other intracellular proteins are also long‐lived. The axons of peripheral nerves contain a large population of very stable microtubules [63, 64] composed of tubulin. Recent pulse‐labeling studies have also confirmed the long lifetime of neurofilament proteins [1].
It is also clear that the regions of the brain may differ in terms of protein stability. The lifetimes of some histones, septins, cell adhesion molecules, and exo‐ and endocytosis cofactors differed significantly in cortex compared with cerebellum [1]. Comparison of whole mouse 13‐C Lys labeling with cell culture studies [65, 66] uncovered a greater range of protein lifetimes [1] that more likely reflects the true nature of these within the brain.
1.7.1.5.1 Age‐Related Changes to the Brain There are many diseases associated with aging of the brain and CNS. Alzheimer's disease (AD), Parkinson's, motor neurone disease (ALS) are some of the more common diseases. Due to an increase in the longevity of the population, these age‐related neurological diseases are becoming more prevalent and are already consuming a huge proportion of the health budget. Despite an enormous research effort, it could be argued that there has been little progress made in understanding the fundamental basis of this group of diseases. At the time of writing, major pharmaceutical companies have scaled down, or exited AD research, because of the failure of many clinical trials. To some degree, this author believes that the lack of progress is due to the widespread use of animal models, which do not adequately replicate the human afflictions, as well as an overall lack of appreciation of the role of LLPs and LLCs in these diseases.
With regard to the cell types other than neurons, it appears that the oligodendrocytes that are responsible for myelination of axons in the CNS are also LLCs [67]. In mice, most oligodendrocytes are formed in the first six weeks of life, and in the corpus callosum, more than 90% of labeled cells survived for more than 1.5 years and as the authors stated “probably outlive the mouse.” Despite the vast majority being LLCs, a very small number may be made after one year [68].
With regard to nerves and other cells outside of the CNS, less is known. Myelination is complete in the peripheral nervous system by 20–40 months. In one study, Schwann cells showed very little turnover in the adult nerve, with not a single instance of myelinating Schwann cell division observed over a 70‐day period in mice. Despite this quiescence, cells could proliferate within days of an injury [69].
Elastin within cerebral arteries from older people loses its functionality [70], with large differences apparent by confocal microscopy, and this contributes to stiffening of the arterial walls with implications outlined elsewhere in this chapter.
1.7.1.5.2
