Old Proteins in Man: A Field in its Infancy

doi:10.1016/j.tibs.2016.06.004

Trends in Biochemical Sciences

Volume 41, Issue 8, August 2016, Pages 654-664

https://doi.org/10.1016/j.tibs.2016.06.004 Get rights and content

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LLPs are present throughout the body.

Over time, LLPs degrade as a result of spontaneous processes and modification by reactive metabolites.

When an LLP breaks down, it generates new structures that the body can recognize as foreign, and may therefore mount an immune response towards the modified as well as the parent protein.

These crucial aspects of human aging and age-related disease are very difficult to study using the traditional experimental approaches (e.g., animal models) and have been largely ignored.

It has only recently been appreciated that the human body contains many long-lived proteins (LLPs). Their gradual degradation over time contributes to human aging and probably also to a range of age-related disorders. Indeed, the role of progressive damage of proteins in aging may be indicated by the fact that many neurological diseases do not appear until after middle age. A major factor responsible for the deterioration of old proteins is the spontaneous breakdown of susceptible amino acid residues resulting in racemization, truncation, deamidation, and crosslinking. When proteins decompose in this way, their structures and functions may be altered and novel epitopes can be formed that can induce an autoimmune response.

Section snippets

Many Proteins Are Present in the Body for Decades

In the human body, old proteins are everywhere. Indeed the most-abundant protein in the body, collagen (which represents ∼30% of total protein), is one such old or long-lived protein (LLP) (see Glossary). Its half-life varies, depending on the anatomical site and type of collagen, but has been estimated to be 117 years in articular cartilage [1] and between 95–215 years in intervertebral discs [2].

As depicted in Figure 1, LLPs are present in the heart [3], arteries [4], lungs [5], skeleton [2],

Why Are Proteins Long-Lived?

It is likely that longevity is a consequence of several factors. First, it is wasteful energetically, from a resource standpoint, to continually renew proteins if this is unnecessary. Second, it may be difficult or impossible to achieve. For example, it is presumably challenging to rebuild a highly insoluble, crosslinked protein such as elastin, and to regenerate an internal lens cell, considering the architecture of the lens where cells are added continuously throughout life on top of a

What is an LLP?

For the purposes of this article, LLPs are considered to be those whose half-lives are longer than 48 h. This definition is somewhat arbitrary but is taken in the context of what is known about the typical lifetimes of proteins within cells. This ranges from <1 h to 22 h [18]. In fact, most proteins discussed in this review have lifetimes of months or years, with some showing no turnover at all. It is likely that many more proteins will be discovered to be long-lived.

How Do We Know that Proteins are Long-Lived?

There are three main ways in which the lifetime of a protein can be determined. In the case of humans, perhaps the most rigorous method is determination of ¹⁴C content. This assay takes advantage of the fact that atmospheric levels of ¹⁴C reached a maximum in the late 1950s as a result of above-ground testing of nuclear weapons. ¹⁴C levels in the atmosphere and food then declined once the 1963 nuclear test ban treaty was implemented. In a sense, all people born after this time are subjects in a

Why Are LLPs Important?

Unfortunately LLPs decompose over time. From the time that an LLP is formed (Figure 2), it comes under relentless attack by enzymes and reactive small molecules, resulting in degradation. Another source of degradation arises from spontaneous processes; that is, reactions that take place due to the intrinsic instability of some amino acids and that are the inevitable result of heat and time. Spontaneous degradation appears to be the most common cause of LLP decomposition [23].

What Happens to LLPs in the Body?

The most commonly used clinical method for assessing diabetic compliance (the overall control of blood sugar levels) employs an assay for glycosylated hemoglobin. If glucose levels are raised in the blood then the α-amino group of hemoglobin becomes more highly modified by the aldehyde moiety of glucose. This simple example illustrates that proteins can be significantly modified by molecules that exist throughout the body and that modification can take place over a period of days given that the

Spontaneous Decomposition of Amino Acids within LLPs Is a Major Source of Breakdown

The lens of the eye is composed of proteins that do not turn over [12]. It is an ideal tissue for investigating the sources of LLP breakdown because of its peculiar manner of growth, its simple composition, and the availability of samples across the age-range. The center of the lens was present when you were born, the mature fiber cells lack cellular organelles, and the ability to synthesize macromolecules is therefore absent. Over time, the enzymes that were present at birth in the lens centre

Racemization, Cleavage, and Crosslinking

If indeed spontaneous reactions are a major factor in alterations to LLPs over time, what are they? Four main types of reactions occur, most often involving Asp, Asn, and Ser. These are amino acid racemization, deamidation, cleavage, and covalent crosslinking. These are briefly summarized in this section.

Consequences of Protein Decomposition for Aging

While it seems clear that spontaneous processes within LLPs are a major contributor to age-related conditions of the human lens, such as presbyopia and cataract [23], the situation for other tissues is more difficult to determine. Correlations must suffice.

It is well known that the physical properties and functionality of blood vessels, lungs, and teeth change over time. For example, forced expiratory volume declines by 1–2% per year after the age of 25. The capacity of the heart is diminished

The Increase in D-Asp with Age Is Not Always Linear

In collagen extracted from cartilage [42] and bone [43] there is also a roughly linear increase in the content of D-Asp with age, although the levels are about half those seen in aorta and lung. Interestingly, in the human lens the age-related increase in modification of LLPs is typically not linear; however, the reasons for non-linearity are unclear. The increase in D-Asp content with age in elastin isolated from arteries [4] mirrors more closely the age-dependent profile for total D-Asp from

Consequences of Protein Decomposition for Disease

Once the scale of age-related changes in human LLPs is appreciated, it becomes more apparent how these alterations could translate into the inevitable decline of body parts with age, as well as disease outcomes. This is a new area of medicine and data are, at present, scarce; however, there are some intriguing observations. It is well known that the incidence of diseases such as cataract, Alzheimer's disease, Parkinson's disease, and a host of others, is markedly age-dependent. The LLP changes

LLPs and Autoimmunity

It is apparent that significant changes to an LLP will markedly affect its structure. This has implications for autoimmune diseases because the modified LLP may now be seen by the body as being foreign. This is an area that deserves much greater attention. Mamula and colleagues have shown that changing an L-Asp residue to an isoAsp in a peptide can render it a potent antigen 55, 56. Supporting this idea, isomerization of Asp25 in histone H2B is implicated in lupus erythematosis [57].

Concluding Remarks and Future Perspectives

A great deal of research needs to be undertaken before we understand clearly the impact of LLP deterioration on human health. To emphasize this it is not yet widely appreciated that LLPs exist in the human body, or that they probably play an important role in aging, age-related diseases, and even lifespan [60]. The field of old proteins is still in its infancy. To illustrate this point, novel spontaneous PTMs are still being characterized 38, 50, 61. It will be vital in future research to

Glossary

Amino acid racemization: conversion of L-amino acids to D-amino acids.
Autoimmune disease: an inappropriate immune response initiated towards the body's own cells or structures.
Crosslink: covalent bonds that are formed between previously separate protein chains.
Deamidation: introduction of a negative charge by conversion of a neutral amide side chain into a carboxylic acid.
Long-lived protein (LLP): a protein whose half-life is measured typically in months or years.
Post-translational modification (PTM)

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Citation Excerpt :
To categorize PTMs with protective or damaging effects on lens function, datasets that quantify differences in deamidation rates within crystallins need to be characterized by age and transparency. This also applies to levels of racemization and isomerization, which potentially perturb protein structure greater than deamidation alone (131–139). In long-lived biological cells and tissues, racemization and deamidation can be an accurate measure of age, comparable to radiocarbon dating (140).
In the human eye, a transparent cornea and lens combine to form the “refracton” to focus images on the retina. This requires the refracton to have a high refractive index “n,” mediated largely by extracellular collagen fibrils in the corneal stroma and the highly concentrated crystallin proteins in the cytoplasm of the lens fiber cells. Transparency is a result of short-range order in the spatial arrangement of corneal collagen fibrils and lens crystallins, generated in part by post-translational modifications (PTMs). However, while corneal collagen is remodeled continuously and replaced, lens crystallins are very long-lived and are not replaced and so accumulate PTMs over a lifetime. Eventually, a tipping point is reached when protein aggregation results in increased light scatter, inevitably leading to the iconic protein condensation–based disease, age-related cataract (ARC). Cataracts account for 50% of vision impairment worldwide, affecting far more people than other well-known protein aggregation–based diseases. However, because accumulation of crystallin PTMs begins before birth and long before ARC presents, we postulate that the lens protein PTMs contribute to a “cataractogenic load” that not only increases with age but also has protective effects on optical function by stabilizing lens crystallins until a tipping point is reached. In this review, we highlight decades of experimental findings that support the potential for PTMs to be protective during normal development. We hypothesize that ARC is preventable by protecting the biochemical and biophysical properties of lens proteins needed to maintain transparency, refraction, and optical function.

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Trends in Biochemical Sciences

OpinionOld Proteins in Man: A Field in its Infancy

Trends

Section snippets

Many Proteins Are Present in the Body for Decades

Why Are Proteins Long-Lived?

What is an LLP?

How Do We Know that Proteins are Long-Lived?

Why Are LLPs Important?

What Happens to LLPs in the Body?

Spontaneous Decomposition of Amino Acids within LLPs Is a Major Source of Breakdown

Racemization, Cleavage, and Crosslinking

Consequences of Protein Decomposition for Aging

The Increase in D-Asp with Age Is Not Always Linear

Consequences of Protein Decomposition for Disease

LLPs and Autoimmunity

Concluding Remarks and Future Perspectives

Glossary

J. Biol. Chem.

J. Biol. Chem.

Atherosclerosis

Cell

Cell

Biochim. Biophys. Acta Gen. Subjects

J. Biol. Chem.

J. Biol. Chem.

Ageing Res. Rev.

Micron

Neuron

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Ageing Res. Rev.

Exp. Eye Res.

J. Biol. Chem.

Arch. Oral Biol.

J. Biol. Chem.

J. Biol. Chem.

Neurochem. Int.

Forensic Sci. Int.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Aspartic acid racemisation in purified elastin from arteries as basis for age estimation

Int. J. Legal Med.

Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon

J. Clin. Invest.

Collagen, cross-linking, and advanced glycation end products in aging human skeletal muscle

J. Appl. Physiol.

Amino acid racemization reveals differential protein turnover in osteoarthritic articular and meniscal cartilages

Arthritis Res. Ther.

Aspartic acid racemization: evidence for marked longevity of elastin in human skin

Br. J. Dermatol.

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

Nature

Aspartic acid racemisation in dentine as a measure of ageing

Nature

Dynamics of fat cell turnover in humans

Nature

Radiocarbon dating of the human eye lens crystallins reveal proteins without carbon turnover throughout life

PLoS One

Age estimation based on aspartic acid racemization in human sclera

Int. J. Legal Med.

Localization of D-β-aspartic acid-containing proteins in human eyes

Invest. Ophthalmol. Vis. Sci.

Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis

Int. J. Legal Med.

Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes

Genes Dev.

Proteome half-life dynamics in living human cells

Science

In vivo kinetic approach reveals slow SOD1 turnover in the CNS

The Journal of Clinical Investigation

Simultaneous determination of post-translational racemization and isomerization of N-terminal amyloid-β in Alzheimer's brain tissues by covalent chiral derivatized ultraperformance liquid chromatography tandem mass spectrometry

Anal. Chem.

Opinion
Old Proteins in Man: A Field in its Infancy