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Insights into the biochemical and biophysical mechanisms mediating the longevity of the transparent optics of the eye lens

Open AccessPublished:September 26, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102537
      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.

      Keywords

      Abbreviations:

      ARC (age-related cataract), AQP0 (aquaporin 0), BFSP1 (beaded filament structural protein 1), BFSP2 (beaded filament structural protein 2), CL (cataractogenic load), FGF (fibroblast growth factor), GAG (glycosaminoglycan), GRN (gene regulatory network), HMW (high molecular weight), PDGF (platelet-derived growth factor), PG (proteoglycan), PTM (post-translational modification), SRO (short-range order), WIF (water-insoluble fraction)
      Image formation on the retina in the eye requires extraordinary biological specializations. Two tissues in particular are responsible for the optical refraction required to focus images onto the retina. These are the cornea and lens, and both are located in the front part (anterior segment) of the eye (Fig. 1A). The cornea (∼550 microns thick) and the lens (4.7–5.0 mm thick) are the two refractive transparent tissues (Fig. 1A), which project images onto the retina and its photoreceptors (
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      ). This requires the extracellular corneal stroma and the cytoplasm of lens fiber cells to conform to the same physical laws of transparency and light refraction (
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      ,
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      ). The importance of their combined optical contribution led to the proposal that the lens (Fig. 1B) and the cornea (Fig. 1A) should be considered as a single unit in the “refracton hypothesis” (
      • Piatigorsky J.
      Enigma of the abundant water-soluble cytoplasmic proteins of the cornea: the "refracton" hypothesis.
      ). With respect to the eye lens, the basis for the refracton hypothesis is the accumulation of diverse, water-soluble, and multifunctional proteins collectively called crystallins. The evolutionary selection of lens crystallins involves gene sharing, their dual function property (
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      Corneal crystallins and the development of cellular transparency.
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      ), and their sequence and conformational adaptions to deliver the required refractive index (
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      ). A logical extension of the refracton hypothesis is that the lens and cornea share common biochemical and biophysical properties required for the development and maintenance of a high index of refraction, “n,” via protein short-range order (SRO; Fig. 2) and water regulation necessary for transparency (
      • Piatigorsky J.
      Enigma of the abundant water-soluble cytoplasmic proteins of the cornea: the "refracton" hypothesis.
      ).
      Figure thumbnail gr1
      Figure 1Vertical section of the anterior chamber of a transparent adult eye and lens anatomy. A, the slit lamp image (
      • Clark J.I.
      Biology of the transparent lens and changes with age.
      ) provides an optical section through the anterior segment of the eye. The anterior cornea (bright curved band) is separated from the lens by a fluid-filled space (dark zone between the lens and cornea), known as the aqueous chamber. The different layers of the lens are visible. A single-layered lens epithelium sits on the inside of the lens capsule, the thickest basement membrane in the human body. The lens capsule and epithelium are seen as a curved bright line on the anterior surface of the lens. Immediately apposed to this surface layer is the cellular mass of the lens, comprising the outer cortical cell layers surrounding the central cell layers of the lens nucleus. The symmetry of the layers results from the coordinated differentiation of cells in the epithelium at the lens equator into lens fiber cells (described in B). The variations in the light scattered from the cortical layers are likely because of the different stages of differentiation of the lens fiber cells. Differentiation proceeds from the epithelial cells in the cortical periphery to the central (nuclear) core, so the oldest cells in the human body are in the center of the lens. These cells were produced before birth in the first trimester, so that the cells and proteins of the embryonic lens nucleus are older than the numerical age of the individual. Stability is key to the exceptional longevity of the proteins and cells of the deepest, and oldest, lens layers, where optical function must be maintained for the lifetime of an individual. Cytoplasmic protein concentrations are not only very high to provide help to the required refractive index, n, but transmittance is optimized by short-range order (SRO) and glass-like properties of the lens crystallins. In humans and nonhuman primates, the elasticity of the lens is important for accommodation. The complex gene regulatory networks (GRNs) that are responsible for symmetry, transparency, and longevity remain to be fully identified. B, the lens comprises concentric shells of fiber cells surrounding the embryonic nucleus. The oldest cells in the mammal are the lens fiber cells in the central core or embryonic lens nucleus. Lens fiber cells are the progeny of the epithelial monolayer, which generates all new cells in the developing and aging lens. The cortex comprises shells of differentiating lens fiber cells that connect to the anterior and posterior sutures and in cross-section have an iconic hexagonal profile. Differentiation in each growth shell is carefully coordinated, both spatially and temporally, so that the concentric layers are arranged symmetrically around the optical axis. The entire cellular mass is enclosed within the lens capsule, the thickest basement membrane in the human. The resulting optical symmetry is necessary for image formation. Adapted from Ref. (
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      ).
      Figure thumbnail gr2
      Figure 2Short-range order (SRO) and transparency. An electron micrograph of a cross section of collagen fibrils in the corneal stroma (
      • Gisselberg M.
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      A quantitative evaluation of Fourier components in transparent and opaque calf cornea.
      ) is compared with three simulations of fibril packing order. The concentration of collagen fibrils in the cornea accounts for the high refractive index (n ∼ 1.37), and the fibril packing order accounts for transparency. Random order of fibrils produces opacity. Long-range “crystalline” order results in transparency, but the arrangement of the fibrils has a periodic repeat. In the cornea, fibrils are arranged in SRO, where their spatial positions are variable but nonrandom. Proteoglycans (PGs) maintain the spatial organization of the collagen fibrils that allows transparency (see the text). Hydration of the cornea is carefully controlled to maintain SRO and corneal transmittance. Similarly, lens cell transparency results from SRO in the packing arrangement of the crystallins (bar represents 0.1 micron).
      The corneal stroma is approximately 90% of the thickness of the cornea and consists of 200 to 250 extracellular lamellae (sheets) of collagen fibrils embedded in a gel-like matrix of glycosaminoglycan (GAGs) and proteoglycans (PGs) with a small number of keratocytes dispersed throughout (
      • Hahnel C.
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      The keratocyte network of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.
      ). The stroma is ∼80% water and ∼15% densely packed collagen fibrils, which increase the refractive index to “n” ∼1.37 (
      • Dai E.
      • Boulton M.
      Basic science of the lens.
      ,
      • Hassell J.R.
      • Birk D.E.
      The molecular basis of corneal transparency.
      ,
      • Meek K.M.
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      ). In addition to high refractive index, collagen fibrils provide stability to the cornea, preserving its smooth curvature needed for optical quality (
      • Cheng X.
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      Mechanisms of self-organization for the collagen fibril lattice in the human cornea.
      ,
      • Meek K.M.
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      ,
      • Meek K.M.
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      ,
      • McCally R.L.
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      ). Theoretically, the concentrated collagen fibrils are consistent with an opaque cornea (Fig. 1A; (
      • Farrell R.A.
      • McCally R.L.
      Corneal Transparency.
      ,
      • Benedek G.B.
      Theory of transparency of the eye.
      )). In the cornea, the spatial arrangement of the fibrils is coordinated by the PGs to limit light scatter. The research on the cornea is extensive, and its transparency is explained by the SRO of the collagen fibrils that optimizes transmittance (Fig. 2; (
      • Meek K.M.
      • Knupp C.
      Corneal structure and transparency.
      • Matsuura T.
      • Gorti S.
      • Tanaka T.
      • Hara Y.
      • Saishin M.
      Determination of corneal gel dynamics.
      ,
      • Lewis P.N.
      • Pinali C.
      • Young R.D.
      • Meek K.M.
      • Quantock A.J.
      • Knupp C.
      Structural interactions between collagen and proteoglycans are elucidated by three-dimensional electron tomography of bovine cornea.
      )). Similarly, SRO in the lens is thought to account for lens cell transparency (see later). An endothelial cell layer on the posterior surface of the cornea maintains the osmotic balance and hydration necessary for maintenance of uniform optical refraction and image formation required for corneal visual function (Fig. 1A; (
      • Cheng X.
      • Pinsky P.M.
      Mechanisms of self-organization for the collagen fibril lattice in the human cornea.
      ,
      • Meek K.M.
      • Dennis S.
      • Khan S.
      Changes in the refractive index of the stroma and its extrafibrillar matrix when the cornea swells.
      ,
      • Meek K.M.
      • Leonard D.W.
      • Connon C.J.
      • Dennis S.
      • Khan S.
      Transparency, swelling and scarring in the corneal stroma.
      ,
      • McCally R.L.
      • Farrell R.A.
      Light scattering from cornea and corneal transparency.
      )).
      In the cornea, PGs and GAGs coordinate the spatial position of the collagen fibrils, largely through hydrogen bonds. The spacing can be altered by increased intraocular pressure or when hydration is not controlled (
      • Hassell J.R.
      • Birk D.E.
      The molecular basis of corneal transparency.
      ,
      • Meek K.M.
      • Knupp C.
      Corneal structure and transparency.
      ,
      • Lewis P.N.
      • Pinali C.
      • Young R.D.
      • Meek K.M.
      • Quantock A.J.
      • Knupp C.
      Structural interactions between collagen and proteoglycans are elucidated by three-dimensional electron tomography of bovine cornea.
      ). Disruption of the noncovalent hydrogen bonds results in voids or “lakes” that produce inhomogeneities large enough to scatter light ((
      • Gisselberg M.
      • Clark J.I.
      • Vaezy S.
      • Osgood T.B.
      A quantitative evaluation of Fourier components in transparent and opaque calf cornea.
      ); discussed later). Under normal homeostasis, the GAGs and PGs form a resilient scaffold to order the collagen fibril spacing that together resembles a gel-like matrix (
      • Meek K.M.
      • Knupp C.
      Corneal structure and transparency.
      ,
      • Matsuura T.
      • Gorti S.
      • Tanaka T.
      • Hara Y.
      • Saishin M.
      Determination of corneal gel dynamics.
      ,
      • Lewis P.N.
      • Pinali C.
      • Young R.D.
      • Meek K.M.
      • Quantock A.J.
      • Knupp C.
      Structural interactions between collagen and proteoglycans are elucidated by three-dimensional electron tomography of bovine cornea.
      ). Just as in the lens, hydration is vital to the development and maintenance of the optical function of the cornea.

      Embryology and SRO in the eye lens

      Very early in human embryonic development, a few ectodermal cells, adjacent to the neural plate, swell, thicken, and compress the intercellular space to form a small placode. The placode invaginates and forms a fluid-filled lens vesicle that separates from the surface epithelium that will help form the cornea (
      • Clark J.I.
      Biology of the transparent lens and changes with age.
      ,
      • Cvekl A.
      • McGreal R.
      • Liu W.
      Lens development and crystallin gene expression.
      ,
      • Gunhaga L.
      The lens: a classical model of embryonic induction providing new insights into cell determination in early development.
      ,
      • Robinson M.L.
      • Lovicu F.J.
      Early Lens Development.
      ,
      • Cvekl A.
      • Ashery-Padan R.
      The cellular and molecular mechanisms of vertebrate lens development.
      ,
      • Graw J.
      Eye development.
      ,
      • Feneck E.M.
      • Lewis P.N.
      • Meek K.M.
      Identification of a primary stroma and novel endothelial cell projections in the developing human cornea.
      ). Migration of neural crest–derived mesodermal fibroblasts behind the remaining ectoderm generates the matrix of the corneal stroma (with its corneal epithelium) directly anterior to the aqueous chamber (Fig. 1A).
      Adjacent to the aqueous chamber is the lens vesicle, where the posterior epithelial cells exit the cell cycle and elongate to fill the vesicle and form the primary embryonic nucleus of the lens. Figure 1A shows the location of the aqueous chamber in the adult eye, and this is the same as in the embryo. A monolayer of epithelial cells remains beneath the thickening anterior capsule ((
      • Cvekl A.
      • McGreal R.
      • Liu W.
      Lens development and crystallin gene expression.
      ,
      • Griep A.E.
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      ); Fig. 1B). These anterior epithelial cells are the source of all secondary fiber cells forming symmetric layers that surround the primary lens nucleus (
      • Wang K.
      • Venetsanos D.
      • Wang J.
      • Pierscionek B.K.
      Gradient moduli lens models: how material properties and application of forces can affect deformation and distributions of stress.
      ).
      Beginning at the lens equator, the extensive elongation and migration of the secondary fiber cells give rise to mature, millimeter-long, and transparent lens fibers that are never replaced (Fig. 1B). The proliferation, migration, and elongation of lens cells occur in the absence of direct contact with surrounding eye tissues, vasculature, innervation, and the immune system. This is due to a thick lens capsule, which defines the physical boundary of the lens. While the differentiation process of the epithelial cells proceeds, water-soluble crystallin proteins are expressed at very high levels, plasma membrane is added as lens fiber cells elongate, all major cell organelles are removed, and an extensive cytoskeletal network forms. The cytoskeleton becomes a scaffold for the condensed crystallins (
      • Maisel H.
      The Ocular Lens.
      ,
      • Li Y.
      • Liu X.
      • Xia C.H.
      • FitzGerald P.G.
      • Li R.
      • Wang J.
      • et al.
      CP49 and filensin intermediate filaments are essential for formation of cold cataract.
      ) as SRO of the lens cytoplasmic proteins is established in lens fiber cells to optimize lifelong transparency. The massive accumulation of crystallins increases the refractive index, “n,” inside each fiber cell. The ordered close-packed membranes form the iconic hexagonal profile typical of these newly differentiated lens fiber cells (Fig. 1B). The embryology of the lens is coordinated with the differentiation of the transparent cornea, as both become integrated into the refracton. These optics adapt continuously to the growing eye (
      • Piatigorsky J.
      Enigma of the abundant water-soluble cytoplasmic proteins of the cornea: the "refracton" hypothesis.
      ) over dimensions from 1.0 to 100 mm and over ages that can exceed 300 years without loss of function or cell replacement. Understanding the early embryological basis for the continuous development of the growth shells in the transparent lens is important for two reasons. It shows how the preplacodal ectoderm associated with sensory placodes is modified to generate the refractive properties needed to transform the primordial photoreceptors into a fully functional visual system. It also explains the processes that continue to assemble transparent refractile layers as the lens grows and ages. There is much to learn about the genetics and the gene regulatory networks (GRNs; (
      • Xie Q.
      • Cvekl A.
      The orchestration of mammalian tissue morphogenesis through a series of coherent feed-forward loops.
      )) of refracton embryology, one of the most remarkable processes in evolution (
      • Cvekl A.
      • Ashery-Padan R.
      The cellular and molecular mechanisms of vertebrate lens development.
      ,
      • Cvekl A.
      • Zhang X.
      Signaling and gene regulatory networks in mammalian lens development.
      ).
      To summarize, among the important factors needed for the molecular and cellular embryology of the lens are:
      • (1)
        Embryologically, the lens begins as a placode of a few elongated and swollen cells at the periphery of the neural ectoderm in the trilaminar embryo. It is unsurprising that many important molecular components are common to both neurons and elongated lens fiber cells (
        • Graw J.
        From eyeless to neurological diseases.
        ,
        • Frederikse P.
        • Kasinathan C.
        Lens biology is a dimension of neurobiology.
        ,
        • Giegling I.
        • Hartmann A.M.
        • Genius J.
        • Konte B.
        • Maul S.
        • Straube A.
        • et al.
        Polymorphisms in CRYBB2 encoding βB2-crystallin are associated with antisaccade performance and memory function.
        ).
      • (2)
        Lens development begins at approximately 50 days embryonic age, and all cells are retained for its lifetime. The cytoskeleton is intimately involved in every developmental stage; in most individuals, this is many decades.
      • (3)
        Longevity of optical function is a unique characteristic of the transparent aging lens.
      • (4)
        Loss of transparency as manifested by the appearance of cataracts is the leading protein condensation disease associated with aging, far more prevalent than either Alzheimer’s or Lewy body or other aging-associated condensation diseases of the nervous system.
      Detailed analyses of the lens epithelium and differentiating fiber cells reveal a more complex cellular organization particularly for the fiber cells ((
      • Hogan M.
      • Alvarado J.
      • Weddell J.
      Histology of the Human Eye; an Atlas and Textbook.
      ,
      • Kuszak J.R.
      • Costello M.J.
      Structure of the Vertebrate Lens.
      ,
      • Kuszak J.R.
      • Zoltoski R.K.
      • Sivertson C.
      Fibre cell organization in crystalline lenses.
      ); Fig. 1B). There are two types of elongating lens fiber cells. A few “straight” fiber cells originate at one pole of the lens but stop short of the opposite pole. Instead, they establish longitudinal meridians known as the lens sutures. Most fiber cells adjacent and parallel to the meridians must curve slightly, with their posterior and anterior ends facing matching parallel fiber cells from the opposing side of each meridional suture line to fill-in each layer or shell. These are known as “s-shaped” lens fiber cells (
      • Kuszak J.R.
      • Clark J.I.
      • Cooper K.
      • Rae J.L.
      Biology of the Lens: Lens Transparency as a Function of Embryology, Anatomy, and Physiology.
      ,
      • Kuszak J.R.
      • Zoltoski R.K.
      • Tiedemann C.E.
      Development of lens sutures.
      ). The result is a slight spiral, yet symmetric, organization of lens fiber cells within each cortical layer that could contribute to its accommodative mechanism (
      • Kuszak J.R.
      • Mazurkiewicz M.
      • Zoltoski R.
      Computer modeling of secondary fiber development and growth: I. Nonprimate lenses.
      ). Whether there is mechanistic and genetic significance to the synchronous and coordinated organization of straight and “s-shaped” fiber cells remains to be determined. The suture lines, however, resemble seams oriented symmetrically about the optical axis at the anterior and posterior poles but arranged to minimize the impact upon the optical efficiency of primate lenses (
      • Kuszak J.R.
      • Peterson K.L.
      • Sivak J.G.
      • Herbert K.L.
      The interrelationship of lens anatomy and optical quality. II. Primate lenses.
      ). The most important role for the sutures is to facilitate entry of water, ions, small molecules, and nutrients via the two lens poles. This is integral to the coordinated internal circulation system within the lens (see later). In the absence of a tissue vasculature, the lens “microcirculation” proves vital to symmetrical lens development and growth (
      • Berthoud V.M.
      • Gao J.
      • Minogue P.J.
      • Jara O.
      • Mathias R.T.
      • Beyer E.C.
      Connexin mutants compromise the lens circulation and cause cataracts through biomineralization.
      ,
      • Jara O.
      • Minogue P.J.
      • Berthoud V.M.
      • Beyer E.C.
      Do connexin mutants cause cataracts by perturbing glutathione levels and redox metabolism in the lens?.
      ,
      • Valiunas V.
      • Brink P.R.
      • White T.W.
      Lens connexin channels have differential permeability to the second messenger cAMP.
      ,
      • Vaghefi E.
      • Donaldson P.J.
      The lens internal microcirculation system delivers solutes to the lens core faster than would be predicted by passive diffusion.
      ,
      • Gupta A.
      • Ruminski D.
      • Jimenez Villar A.
      • Duarte Toledo R.
      • Manzanera S.
      • Panezai S.
      • et al.
      In vivo SS-OCT imaging of crystalline lens sutures.
      ).

      Transparency and SRO

      Transparency in both the lens and the cornea is facilitated by structural constraints. Instead of ordered collagen fibrils, lens cells contain concentrated solutions of crystallins. In the cornea, weak noncovalent bonds maintain SRO between collagen fibers. In the lens, weak protein–protein interactions between crystallins account for SRO and transparency. In both, destructive interference accounts for light transmission (Fig. 2; (
      • Clark J.I.
      Biology of the transparent lens and changes with age.
      ,
      • Benedek G.B.
      Theory of transparency of the eye.
      ,
      • Meek K.M.
      • Knupp C.
      Corneal structure and transparency.
      ,
      • Matsuura T.
      • Gorti S.
      • Tanaka T.
      • Hara Y.
      • Saishin M.
      Determination of corneal gel dynamics.
      )). The lens fiber cell cytoplasm is carefully regulated to control the pH, ionic strength, and hydration of the proteins. This happens at the same time as specific post-translational modifications (PTMs) of the surface-exposed side chains of the amino acid residues occur to favor transparency at body temperature. The range of PTMs include racemization, deamidation, oxidation, and phosphorylation. Transparency depends on maintaining the dimensions of protein scatterers below half the wavelength of visible light (400–700 nm). Slight alterations in protein–protein interactions can therefore affect transparency. The difference between SRO in transparent and opaque (cataractous) lenses can be very small as evidenced by data from electron microscopy and small-angle X-ray scattering analyses (Fig. 3, A and B, respectively; (
      • Gulik-Krzywicki T.
      • Tardieu A.
      • Delaye M.
      Spatial reorganization of low-molecular-weight proteins during cold cataract opacification.
      ,
      • Metlapally S.
      • Costello M.J.
      • Gilliland K.O.
      • Ramamurthy B.
      • Krishna P.V.
      • Balasubramanian D.
      • et al.
      Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye-Bueche theory.
      ,
      • Clark J.I.
      • Mengel L.
      • Benedek G.B.
      Scanning electron microscopy of opaque and transparent states in reversible calf lens cataracts.
      )). The data reported in the literature suggest that endogenous mechanisms involving PTMs can help maintain protein solubility to protect and regulate SRO, transparency, and optical function as the lens ages.
      Figure thumbnail gr3
      Figure 3Comparison of transparent and opaque human lenses by electron microscopy and SAXS. A, electron micrographs of cells in a transparent and opaque human lens demonstrate that very small changes in SRO can reduce transparency. Very small differences in SRO between the opaque and transparent lens cell cytoplasm may not be obvious. Quantitative analysis is necessary to determine which cells contain transparent SRO, where destructive interference of scattered light permits transparency, despite the highly concentrated proteins (
      • Metlapally S.
      • Costello M.J.
      • Gilliland K.O.
      • Ramamurthy B.
      • Krishna P.V.
      • Balasubramanian D.
      • et al.
      Analysis of nuclear fiber cell cytoplasmic texture in advanced cataractous lenses from Indian subjects using Debye-Bueche theory.
      ). B, SAXS from fresh samples of opaque and transparent lens cytoplasm confirms the observations by electron microscopy (A). The plots are nearly identical except for a small increase in large scattering components in opaque cytoplasm (dashed line). The result demonstrates the impact that very small differences in cytoplasmic SRO can have on lens transparency and can be a model for the earliest reversible stages of formation of pathological cataract. “s” is size; “A” is angstroms; and “I” is intensity (
      • Gulik-Krzywicki T.
      • Tardieu A.
      • Delaye M.
      Spatial reorganization of low-molecular-weight proteins during cold cataract opacification.
      ). SAXS, small-angle X-ray scattering; SRO, short-range order.

      Protein solubility and transparency

      Even before the transparent lens was shown to be cellular, early investigations of the lens protein composition recognized the significance of soluble and insoluble fractions in the lens. The insoluble fraction was originally termed albuminoid by Mörner, and the term was adopted by others (
      • Mörner C.
      Untersuchung der Proteinsubstanzen in den leichtbrechenden Medien des Auges I.
      ,
      • Pirie A.
      Color and solubility of the proteins of human cataracts.
      ,
      • Waley S.G.
      The problem of albuminoid.
      ,
      • Dische Z.
      • Borenfreund E.
      • Zelmenis G.
      Changes in lens proteins of rats during aging.
      ). By the 1970s, opaque water-insoluble fractions (WIFs), often known as the high molecular weight (HMW) fraction (
      • Jedziniak J.A.
      • Kinoshita J.H.
      • Yates E.M.
      • Hocker L.O.
      • Benedek G.B.
      On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses.
      ), were predicted to account for the opacity observed in cataracts (
      • Benedek G.B.
      Theory of transparency of the eye.
      ). These studies determined that the WIF is enriched in cross-linked crystallins (
      • Dische Z.
      • Borenfreund E.
      • Zelmenis G.
      Changes in lens proteins of rats during aging.
      ,
      • Dische Z.
      • Zil H.
      Studies on the oxidation of cysteine to cystine in lens proteins during cataract formation.
      ,
      • Lasser A.
      • Balazs E.A.
      Biochemical and fine structure studies on the water-insoluble components of the calf lens.
      ). The relative proportions of these different fractions were found to be age dependent, and this led to the tacit assumption that cross-linked products in these fractions were the cause of aging cataracts (reviewed in Ref. (
      • Harding J.J.
      • Dilley K.J.
      Structural proteins of the mammalian lens: a review with emphasis on changes in development, aging and cataract.
      )). The analysis of light scattered directly from intact human lenses made it possible to measure diffusivity of cytoplasmic proteins and calculate the approximate dimensions of scatterers. The calculated dimension of HMW protein scatterers in intact lenses was roughly the same as the dimension of HMW proteins isolated from lens extracts (
      • Jedziniak J.A.
      • Kinoshita J.H.
      • Yates E.M.
      • Hocker L.O.
      • Benedek G.B.
      On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses.
      ,
      • Jedziniak J.A.
      • Nicoli D.F.
      • Baram H.
      • Benedek G.B.
      Quantitative verification of the existence of high molecular weight protein aggregates in the intact normal human lens by light-scattering spectroscopy.
      ).
      Historically, it was incorrectly thought that lens transparency results from the precise spacing of cytoplasmic crystallins arranged in long-range crystalline order. SRO is accounted for by crystallin solubility in the absence of protein crystallization or aggregation at very high (up to 600 mg/ml) protein concentrations that are very rarely found in nonlens cells (
      • Clark J.I.
      Order and disorder in the transparent media of the eye.
      ,
      • Benedek G.B.
      Theory of transparency of the eye.
      ,
      • Delaye M.
      • Clark J.I.
      • Benedek G.B.
      Identification of the scattering elements responsible for lens opacification in cold cataracts.
      ,
      • Delaye M.
      • Tardieu A.
      Short-range order of crystallin proteins accounts for eye lens transparency.
      ,
      • Bloemendal H.
      • de Jong W.
      • Jaenicke R.
      • Lubsen N.H.
      • Slingsby C.
      • Tardieu A.
      Ageing and vision: structure, stability and function of lens crystallins.
      ,
      • Bettelheim F.A.
      • Paunovic M.
      Light scattering of normal human lens I. Application of random density and orientation fluctuation theory.
      ). Interestingly though, some lens crystallins retain the enzymatic activities (e.g., lactate dehydrogenase) of the cytoplasmic proteins from which they were derived as a result of “gene sharing” mechanisms (
      • Piatigorsky J.
      • Wistow G.
      The recruitment of crystallins: new functions precede gene duplication.
      ,
      • Piatigorsky J.
      Multifunctional lens crystallins and corneal enzymes. More than meets the eye.
      ). Even though the protein concentrations in lens fiber cells can exceed those found in transparent protein crystals used for X-ray crystallography, these multifunctional crystallins remain uncrystallized and in solution inside lens cells.
      Numerous publications allude to a cytoplasmic protein network or gel consistent with the original description of a water insoluble albuminoid fraction in the lens (
      • Waley S.G.
      The problem of albuminoid.
      ,
      • Lasser A.
      • Balazs E.A.
      Biochemical and fine structure studies on the water-insoluble components of the calf lens.
      ,
      • Spector A.
      The search for a solution to senile cataracts. Proctor lecture.
      ,
      • Benedek G.
      • Clark J.
      • Serrallach E.
      • Young C.
      • Mengel L.
      • Sauke T.
      • et al.
      Light scattering & reversible cataracts in calf and human lenses.
      ,
      • Latina M.
      • Chylack Jr., L.T.
      • Fagerholm P.
      • Nishio I.
      • Tanaka T.
      • Palmquist B.M.
      Dynamic light scattering in the intact rabbit lens. Its relation to protein concentration.
      ,
      • Morgan C.F.
      • Schleich T.
      • Caines G.H.
      • Farnsworth P.N.
      Elucidation of intermediate (mobile) and slow (solidlike) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy.
      ,
      • Taylor V.L.
      • al-Ghoul K.J.
      • Lane C.W.
      • Davis V.A.
      • Kuszak J.R.
      • Costello M.J.
      Morphology of the normal human lens.
      ,
      • Millar A.
      • Hooper A.
      • Copeland L.
      • Cummings F.
      • Prescott A.
      Reorganisation of the microtubule cytoskeleton and centrosomal loss during lens fibre cell differentiation.
      ,
      • Bettelheim F.A.
      Syneresis and its possible role in cataractogenesis.
      ,
      • Bettelheim F.A.
      • Siew E.L.
      Effect of change in concentration upon lens turbidity as predicted by the random fluctuation theory.
      ,
      • Logan C.M.
      • Menko A.S.
      Microtubules: evolving roles and critical cellular interactions.
      ,
      • Bassnett S.
      • Costello M.J.
      The cause and consequence of fiber cell compaction in the vertebrate lens.
      ). Biochemical and biophysical analyses indicate that the lens cytoplasm resembles a gel, in vitro and in vivo, and this can account for its glass-like transparency (
      • Lasser A.
      • Balazs E.A.
      Biochemical and fine structure studies on the water-insoluble components of the calf lens.
      ,
      • Bloemendal H.
      • de Jong W.
      • Jaenicke R.
      • Lubsen N.H.
      • Slingsby C.
      • Tardieu A.
      Ageing and vision: structure, stability and function of lens crystallins.
      ,
      • Latina M.
      • Chylack Jr., L.T.
      • Fagerholm P.
      • Nishio I.
      • Tanaka T.
      • Palmquist B.M.
      Dynamic light scattering in the intact rabbit lens. Its relation to protein concentration.
      ,
      • Bassnett S.
      • Costello M.J.
      The cause and consequence of fiber cell compaction in the vertebrate lens.
      ,
      • Bassnett S.
      • Shi Y.
      • Vrensen G.F.
      Biological glass: structural determinants of eye lens transparency.
      ,
      • Tanaka T.
      • Ishimoto C.
      In vivo observation of protein diffusivity in rabbit lenses.
      ). A gel has properties of both a liquid and a solid (
      • Flory P.J.
      Introductory lecture.
      ,
      • Tanaka T.
      Gels.
      ), can stabilize a transparent protein matrix, and is isolated as the WIF. At high protein concentrations, the SRO of the lens crystallin stabilized in a gel can decrease light scattering in comparison to that predicted for proteins acting as independent scatterers (
      • Trokel S.
      The physical basis for transparency of the crystalline lens.
      ,
      • Benedek G.B.
      Theory of transparency of the eye.
      ,
      • Benedek G.
      • Clark J.
      • Serrallach E.
      • Young C.
      • Mengel L.
      • Sauke T.
      • et al.
      Light scattering & reversible cataracts in calf and human lenses.
      ,
      • Bettelheim F.A.
      • Siew E.L.
      Effect of change in concentration upon lens turbidity as predicted by the random fluctuation theory.
      ). Whilst close packing of lens cytoplasmic proteins can decrease light scattering, protein crowding is often associated with the formation of unstable oligomers, fibrils, aggregates, or gels (
      • Ellis R.J.
      • Minton A.P.
      Protein aggregation in crowded environments.
      ,
      • Boob M.
      • Wang Y.
      • Gruebele M.
      Proteins: "Boil 'Em, mash 'Em, stick 'Em in a stew".
      ,
      • Minton A.P.
      Influence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations.
      ,
      • Grosas A.B.
      • Rekas A.
      • Mata J.P.
      • Thorn D.C.
      • Carver J.A.
      The aggregation of αb-crystallin under crowding conditions is prevented by αA-crystallin: implications for α-crystallin stability and lens transparency.
      ,
      • Feig M.
      Virtual issue on protein crowding and stability.
      ). Lens fiber cells are exposed to a variety of stresses, including hypoxia, high ionic strength, changes in osmotic pressure, decreasing pH, and high protein concentration (
      • Greiner J.V.
      • Kopp S.J.
      • Sanders D.R.
      • Glonek T.
      Organophosphates of the crystalline lens: a nuclear magnetic resonance spectroscopic study.
      ,
      • Willis J.A.
      • Schleich T.
      The effect of prolonged elevated glucose levels on the phosphate metabolism of the rabbit lens in perfused organ culture.
      ,
      • Beaulieu C.F.
      • Clark J.I.
      31P nuclear magnetic resonance and laser spectroscopic analyses of lens transparency during calcium-induced opacification.
      ), as the HMW and WIF (Fig. 4A), and PTMs (Fig. 4B) increase progressively with age. Under these crowded conditions, SRO and transparency are retained even as proteins slowly become insoluble (
      • Bloemendal H.
      • de Jong W.
      • Jaenicke R.
      • Lubsen N.H.
      • Slingsby C.
      • Tardieu A.
      Ageing and vision: structure, stability and function of lens crystallins.
      ,
      • Slingsby C.
      • Wistow G.J.
      • Clark A.R.
      Evolution of crystallins for a role in the vertebrate eye lens.
      ,
      • Ellis R.J.
      Macromolecular crowding: an important but neglected aspect of the intracellular environment.
      ).
      Figure thumbnail gr4
      Figure 4Progressive insolubility and accumulation of post-translational modifications (PTMs) in cytoplasmic protein with lens age. A, total dry weight (%) and water soluble and water insoluble (water insoluble fraction [WIF] = H2O insoluble) protein both increase linearly with age in normal human lenses (solid lines). These lenses were without cataracts. The WIF increases from approximately 1 to 2% of the total cytoplasmic protein in very young lenses to nearly 40% in very old lenses. Circles plot the dry weights for soluble (open circles) and WIF protein (closed circles) in extracts from cataractous (opaque) lenses. The selected cataract samples had extensive opacity in both the cortical and nuclear lens regions. The total dry weights were comparable for both cataract and age-matched noncataractous human lenses. From ∼55 years of age, the WIF of cataractous lenses start to increase at a faster rate (dashed line) than in normal age-matched control lenses (solid line). As some older transparent lenses have a greater amount of water insoluble protein than samples with severe cataracts obtained from younger individuals, this suggests that the age-dependent insolubilization of lens proteins may not be a measure of cataractogenesis (
      • Spector A.
      The search for a solution to senile cataracts. Proctor lecture.
      ). B, progressive accumulation of aspartic acid racemization with age (
      • Masters P.M.
      • Bada J.L.
      • Zigler Jr., J.S.
      Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.
      ). Aspartic acid racemization in the central nuclei of 13 group I and II cataracts plotted versus age. Fitting the data to a first-order rate equation using least squares (solid line) gives kasp = 1.29 × 10−3/year (r = 0.875) or about 0.14%/year. This is identical to the rate observed in the lens during normal aging, although D/L ratios in Asp are observed to be higher in the WIF than the water-soluble fraction (
      • Masters P.M.
      • Bada J.L.
      • Zigler Jr., J.S.
      Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.
      ,
      • Masters P.M.
      • Bada J.L.
      • Zigler Jr., J.S.
      Aspartic acid racemisation in the human lens during ageing and in cataract formation.
      ). This result, among many others, raises the possibility that racemization may be important for the normal development of lens transparency.

      Cataractogenic load hypothesis and its implication for lens transparency

      The cataractogenic load (CL) hypothesis acknowledges the collective effect of the progressive modification of lens cell constituents (proteins, DNA, and lipid membranes) that occur with age and with environmental, nutritional, metabolic, and genetic stresses upon the lens. The primary risk factor for reduced optical function and loss in lens transparency is age (
      • Clark J.I.
      Biology of the transparent lens and changes with age.
      • Benedek G.B.
      • Pande J.
      • Thurston G.M.
      • Clark J.I.
      Theoretical and experimental basis for the inhibition of cataract.
      ,
      • Harding J.
      Cataract: Biochemistry, Epidemiology and Pharmacology.
      ,
      • Wishart T.F.L.
      • Flokis M.
      • Shu D.Y.
      • Das S.J.
      • Lovicu F.J.
      Hallmarks of lens aging and cataractogenesis.
      ,
      • Regnault F.
      • Hockwin O.
      • Courtois I.
      Ageing of the Lens: Proceedings of the Symposium on Ageing of the Lens.
      ,
      • Young R.W.
      Age Related Cataract.
      ,
      • Asbell P.A.
      • Dualan I.
      • Mindel J.
      • Brocks D.
      • Ahmad M.
      • Epstein S.
      Age-related cataract.
      ,
      • Taylor H.R.
      Epidemiology of age-related cataract.
      ,
      • Truscott R.J.
      Age-related nuclear cataract: a lens transport problem.
      ,
      • Sharma K.K.
      • Santhoshkumar P.
      Lens aging: effects of crystallins.
      ). Some of the oldest cells in the body, and therefore the oldest proteins (
      • Stewart D.N.
      • Lango J.
      • Nambiar K.P.
      • Falso M.J.
      • FitzGerald P.G.
      • Rocke D.M.
      • et al.
      Carbon turnover in the water-soluble protein of the adult human lens.
      ,
      • Nielsen J.
      • Hedeholm R.B.
      • Heinemeier J.
      • Bushnell P.G.
      • Christiansen J.S.
      • Olsen J.
      • et al.
      Eye lens radiocarbon reveals centuries of longevity in the Greenland shark (Somniosus microcephalus).
      ,
      • Liu P.
      • Edassery S.L.
      • Ali L.
      • Thomson B.R.
      • Savas J.N.
      • Jin J.
      Long-lived metabolic enzymes in the crystalline lens identified by pulse-labeling of mice and mass spectrometry.
      ), are in the eye lens, making the lens an excellent model for research on molecular and cellular aging. In systematic studies of aging lenses, proteins, DNA, and lipid membranes are modified with age (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      ,
      • Ahmadi M.
      • Barnard S.
      • Ainsbury E.
      • Kadhim M.
      Early responses to low-dose ionizing radiation in cellular lens epithelial models.
      ,
      • Chitra P.S.
      • Chaki D.
      • Boiroju N.K.
      • Mokalla T.R.
      • Gadde A.K.
      • Agraharam S.G.
      • et al.
      Status of oxidative stress markers, advanced glycation index, and polyol pathway in age-related cataract subjects with and without diabetes.
      ,
      • Pendergrass W.R.
      • Penn P.E.
      • Li J.
      • Wolf N.S.
      Age-related telomere shortening occurs in lens epithelium from old rats and is slowed by caloric restriction.
      ). The progressive effect on transparency can be characterized as the CL, eventually leading to aging cataracts (Fig. 5; (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      )). The CL hypothesis implies that the progressive modification of lens constituents may be protective during development and lens aging, prior to any appearance of insult-induced or aging cataracts.
      Figure thumbnail gr5
      Figure 5Cataractogenic load (CL) hypothesis of lens aging. During the development of transparency, the post-translational modification (PTM) rate (ΔCLN) is slow and progressive. Late in life, at the intersection of CL and the initial stage of opacification (horizontal red line), a tipping point (red dot) occurs. This is often observed in a slit lamp examination of the eye lens in an individual. When cataractogenic conditions develop, the ΔCLR (dashed line) increases and the tipping point (red diamond) occurs at an early age for any single eye. A corollary to the CL hypothesis states that a decrease in ΔCL can protect transparency and extend the longevity of the lens function. The CL hypothesis (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      ) is based on concepts discussed by C. Kupfer, the first director of the National Eye Institute (
      • Kupfer C.
      Bowman lecture. The conquest of cataract: a global challenge.
      ) and by G.R. Merriam, A. Szechter, and B.V. Worgul (
      • Benedek G.B.
      • Pande J.
      • Thurston G.M.
      • Clark J.I.
      Theoretical and experimental basis for the inhibition of cataract.
      ,
      • Merriam Jr., G.R.
      • Worgul B.V.
      Experimental radiation cataract--its clinical relevance.
      ,
      • Merriam G.R.
      • Szechter A.
      The effect of age on the radiosensitivity of rat lenses.
      ,
      • Merriam G.R.
      • Worgul B.V.
      Experimental radiation cataract--its clinical relevance.
      ).
      Based on the multiple effects of ionizing radiation on lens cells and their constituents, a model is proposed to recognize the potential protective impact of PTMs accumulated with development and aging (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      ). Ionizing radiation is considered one of the most effective ways to accelerate lens aging in vivo and in vitro (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      ,
      • Ainsbury E.A.
      • Barnard S.
      • Bright S.
      • Dalke C.
      • Jarrin M.
      • Kunze S.
      • et al.
      Ionizing radiation induced cataracts: recent biological and mechanistic developments and perspectives for future research.
      ,
      • Merriam Jr., G.R.
      • Worgul B.V.
      Experimental radiation cataract--its clinical relevance.
      ,
      • Kleiman N.J.
      Radiation cataract.
      ,
      • Richardson R.B.
      Ionizing radiation and aging: rejuvenating an old idea.
      ). Few cataract models have been studied more than the lens opacities resulting from ionizing radiation exposure, in part not only because of their similarities to opacification with age but also because of the exposure of individuals involved in air travel, space exploration, medical imaging, and intervention therapies (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      ,
      • Blakely E.A.
      • Kleiman N.J.
      • Neriishi K.
      • Chodick G.
      • Chylack L.T.
      • Cucinotta F.A.
      • et al.
      Radiation cataractogenesis: epidemiology and biology.
      ,
      • Pendergrass W.
      • Zitnik G.
      • Tsai R.
      • Wolf N.
      X-ray induced cataract is preceded by LEC loss, and coincident with accumulation of cortical DNA, and ROS; similarities with age-related cataracts.
      ,
      • Clark J.I.
      • Giblin F.J.
      • Reddy V.N.
      • Benedek G.B.
      Phase separation of X-irradiated lenses of rabbit.
      ,
      • Horwitz M.
      • Auquier P.
      • Barlogis V.
      • Contet A.
      • Poiree M.
      • Kanold J.
      • et al.
      Incidence and risk factors for cataract after haematopoietic stem cell transplantation for childhood leukaemia: an LEA study.
      ). Irradiated lenses show altered metabolism, PTMs, oxidative and osmotic stress, increased insoluble protein and protein aggregation, decreased pH and ion imbalance, and light scattering (
      • Ainsbury E.A.
      • Barnard S.G.R.
      Sensitivity and latency of ionising radiation-induced cataract.
      ,
      • Ainsbury E.A.
      • Dalke C.
      • Mancuso M.
      • Kadhim M.
      • Quinlan R.A.
      • Azizova T.
      • et al.
      Introduction to the special LDLensRad focus issue.
      ,
      • Barnard S.
      • Uwineza A.
      • Kalligeraki A.
      • McCarron R.
      • Kruse F.
      • Ainsbury E.A.
      • et al.
      Lens epithelial cell proliferation in response to ionizing radiation.
      ,
      • Richardson R.B.
      • Ainsbury E.A.
      • Prescott C.R.
      • Lovicu F.J.
      Etiology of posterior subcapsular cataracts based on a review of risk factors including aging, diabetes, and ionizing radiation.
      ). These published data provide insight into the basis for molecular and cellular longevity in the aging lens (
      • Blakely E.A.
      • Kleiman N.J.
      • Neriishi K.
      • Chodick G.
      • Chylack L.T.
      • Cucinotta F.A.
      • et al.
      Radiation cataractogenesis: epidemiology and biology.
      ) and the mechanisms of aging on the sequence of molecular changes that occur in aging cells and tissues (
      • Harding J.
      Cataract: Biochemistry, Epidemiology and Pharmacology.
      ,
      • Regnault F.
      • Hockwin O.
      • Courtois I.
      Ageing of the Lens: Proceedings of the Symposium on Ageing of the Lens.
      ,
      • Young R.W.
      Age Related Cataract.
      ,
      • Truscott R.J.
      Age-related nuclear cataract: a lens transport problem.
      ). Extensive research on radiation-induced cataracts led to a novel interpretation (the CL hypothesis) for molecular and cellular longevity in the lens (Fig. 5) and identifies the potential role of PTMs in protecting lens transparency and optical function (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      ,
      • Berry V.
      • Georgiou M.
      • Fujinami K.
      • Quinlan R.
      • Moore A.
      • Michaelides M.
      Inherited cataracts: molecular genetics, clinical features, disease mechanisms and novel therapeutic approaches.
      ).

      PTMs in lens proteins and transparency

      Even before birth (
      • Hains P.G.
      • Truscott R.J.
      Proteome analysis of human foetal, aged and advanced nuclear cataract lenses.
      ,
      • Hains P.G.
      • Truscott R.J.
      Age-dependent deamidation of lifelong proteins in the human lens.
      ,
      • Hanson S.R.
      • Smith D.L.
      • Smith J.B.
      Deamidation and disulfide bonding in human lens gamma-crystallins.
      ), lens proteins start to accumulate PTMs (
      • Song S.
      • Landsbury A.
      • Dahm R.
      • Liu Y.
      • Zhang Q.
      • Quinlan R.A.
      Functions of the intermediate filament cytoskeleton in the eye lens.
      ,
      • Kalligeraki A.
      • Quinlan R.
      Structural proteins | crystallins of the mammalian eye lens.
      ,
      • Ma Z.
      • Hanson S.R.
      • Lampi K.J.
      • David L.L.
      • Smith D.L.
      • Smith J.B.
      Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.
      ). In 1942, G.L. Walls observed that “The lens is unique among the organs of the body in that its development never ceases, while senescence commences even before birth” (
      • Van Heyningen R.
      The lens.
      ,
      • Walls G.L.
      The Vertebrate Eye and its Adaptive Radiation.
      ). The tacit assumption has been that the PTMs associated with lens development are eventually responsible for cataracts (
      • Hanson S.R.
      • Smith D.L.
      • Smith J.B.
      Deamidation and disulfide bonding in human lens gamma-crystallins.
      ,
      • Ma Z.
      • Hanson S.R.
      • Lampi K.J.
      • David L.L.
      • Smith D.L.
      • Smith J.B.
      Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.
      ,
      • Hanson S.R.
      • Hasan A.
      • Smith D.L.
      • Smith J.B.
      The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage.
      ,
      • Lampi K.J.
      • Ma Z.
      • Hanson S.R.
      • Azuma M.
      • Shih M.
      • Shearer T.R.
      • et al.
      Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry.
      ). A different interpretation is that these PTMs may protect and maintain SRO, transparency, and symmetry as the lens ages (see later). The most abundant PTM by an order of magnitude in aged transparent human lenses is racemization, particularly of Asp/Asn and Ser (l- to d-isomer; (
      • Masters P.M.
      • Bada J.L.
      • Zigler Jr., J.S.
      Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.
      )) followed then by deamidation (
      • Hooi M.Y.
      • Truscott R.J.
      Racemisation and human cataract. D-Ser, D-Asp/Asn and D-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses.
      ,
      • Norton-Baker B.
      • Mehrabi P.
      • Kwok A.O.
      • Roskamp K.W.
      • Sprague-Piercy M.A.
      • Stetten D.v.
      • et al.
      Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging.
      ,
      • Wilmarth P.A.
      • Tanner S.
      • Dasari S.
      • Nagalla S.R.
      • Riviere M.A.
      • Bafna V.
      • et al.
      Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?.
      ,
      • Vetter C.J.
      • Thorn D.C.
      • Wheeler S.G.
      • Mundorff C.
      • Halverson K.
      • Wales T.E.
      • et al.
      Cumulative deamidations of the major lens protein γS-crystallin increase its aggregation during unfolding and oxidation.
      ,
      • Dasari S.
      • Wilmarth P.A.
      • Rustvold D.L.
      • Riviere M.A.
      • Nagalla S.R.
      • David L.L.
      Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses.
      ). These are common modifications known to proceed through a succinimide intermediate with known kinetics and reaction products that are often resistant to spontaneous biochemical degradation or proteolytic cleavage in living cells (
      • Truscott R.J.W.
      • Schey K.L.
      • Friedrich M.G.
      Old proteins in man: a field in its infancy.
      ,
      • Zhao Z.
      • Yue J.
      • Ji X.
      • Nian M.
      • Kang K.
      • Qiao H.
      • et al.
      Research progress in biological activities of succinimide derivatives.
      ,
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ). 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 (
      • Masters P.M.
      • Bada J.L.
      • Zigler Jr., J.S.
      Aspartic acid racemization in heavy molecular weight crystallins and water insoluble protein from normal human lenses and cataracts.
      ,
      • Hooi M.Y.
      • Truscott R.J.
      Racemisation and human cataract. D-Ser, D-Asp/Asn and D-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses.
      ,
      • Norton-Baker B.
      • Mehrabi P.
      • Kwok A.O.
      • Roskamp K.W.
      • Sprague-Piercy M.A.
      • Stetten D.v.
      • et al.
      Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging.
      ,
      • Wilmarth P.A.
      • Tanner S.
      • Dasari S.
      • Nagalla S.R.
      • Riviere M.A.
      • Bafna V.
      • et al.
      Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?.
      ,
      • Vetter C.J.
      • Thorn D.C.
      • Wheeler S.G.
      • Mundorff C.
      • Halverson K.
      • Wales T.E.
      • et al.
      Cumulative deamidations of the major lens protein γS-crystallin increase its aggregation during unfolding and oxidation.
      ,
      • Dasari S.
      • Wilmarth P.A.
      • Rustvold D.L.
      • Riviere M.A.
      • Nagalla S.R.
      • David L.L.
      Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses.
      ,
      • Truscott R.J.W.
      • Schey K.L.
      • Friedrich M.G.
      Old proteins in man: a field in its infancy.
      ,
      • Zhao Z.
      • Yue J.
      • Ji X.
      • Nian M.
      • Kang K.
      • Qiao H.
      • et al.
      Research progress in biological activities of succinimide derivatives.
      ,
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ). In long-lived biological cells and tissues, racemization and deamidation can be an accurate measure of age, comparable to radiocarbon dating (
      • Robinson N.E.
      • Robinson A.B.
      Deamidation of human proteins.
      ). While their resistance to degradation or proteolysis can account for their accumulation with age, there may be a threshold for their protective effect in vivo. Perhaps, such a threshold could equate to a tipping point, which could occur when the balance between favorable and unfavorable interactions is disrupted, leading to an increase in the rate of change in CL (Fig. 5; (
      • Uwineza A.
      • Kalligeraki A.A.
      • Hamada N.
      • Jarrin M.
      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      )). This threshold could occur prior to changes in the PTM landscape of lens proteins for an individual lens (
      • Schey K.L.
      • Wang Z.
      • Friedrich M.G.
      • Garland D.L.
      • Truscott R.J.W.
      Spatiotemporal changes in the human lens proteome: critical insights into long-lived proteins.
      ). It should be noted that the PTM profile of aged transparent lenses is reported to show little variation between different people (
      • Schey K.L.
      • Wang Z.
      • Friedrich M.G.
      • Garland D.L.
      • Truscott R.J.W.
      Spatiotemporal changes in the human lens proteome: critical insights into long-lived proteins.
      ), suggesting that a PTM signature for aging transparent lenses could be produced. This will help identify, for example, which succinimide intermediates are protective against loss of function in cell and molecular aging. The predicted tipping point may coincide with a collective change in CL (
      • Schey K.L.
      • Wang Z.
      • Friedrich M.G.
      • Garland D.L.
      • Truscott R.J.W.
      Spatiotemporal changes in the human lens proteome: critical insights into long-lived proteins.
      ) when the predominant size of scatterers approaches or exceeds λ/2, where λ is the wavelength of visible light (380–740 nm; (
      • Clark J.I.
      Biology of the transparent lens and changes with age.
      )) and initiate loss of transparency. It could be challenging to generate a PTM signature for age-related cataract (ARC) given the variation between different individuals. Quantitative data are needed to determine the importance of the major age-related PTMs in maintenance of lens transparency and optical function. It should be noted that the CL hypothesis is consistent with conceptual approaches for effective therapeutic protection against clinical cataract formation (
      • Benedek G.
      • Clark J.
      • Serrallach E.
      • Young C.
      • Mengel L.
      • Sauke T.
      • et al.
      Light scattering & reversible cataracts in calf and human lenses.
      ,
      • Benedek G.B.
      • Pande J.
      • Thurston G.M.
      • Clark J.I.
      Theoretical and experimental basis for the inhibition of cataract.
      ,
      • Kupfer C.
      Bowman lecture. The conquest of cataract: a global challenge.
      ,
      • Benedek G.B.
      Cataract as a protein condensation disease: the proctor lecture.
      ).
      Considerable effort has gone into defining PTMs involved with transparency and refraction for eye tissues. In the study of the cornea, there is agreement on the importance of PGs and GAGs in the control of the spatial order of the collagen fibril matrix that is necessary for SRO in the stroma (
      • Hassell J.R.
      • Birk D.E.
      The molecular basis of corneal transparency.
      ,
      • McCally R.L.
      • Farrell R.A.
      Light scattering from cornea and corneal transparency.
      ,
      • Lewis P.N.
      • Pinali C.
      • Young R.D.
      • Meek K.M.
      • Quantock A.J.
      • Knupp C.
      Structural interactions between collagen and proteoglycans are elucidated by three-dimensional electron tomography of bovine cornea.
      ,
      • Meek K.M.
      The cornea and sclera.
      ). SRO and transparency in the cornea are better understood, and similar biochemical interactions may also control SRO and transparency in the lens (
      • Clark J.I.
      Biology of the transparent lens and changes with age.
      ,
      • Piatigorsky J.
      Enigma of the abundant water-soluble cytoplasmic proteins of the cornea: the "refracton" hypothesis.
      ). For both systems though, the details of the changes in protein–protein interactions resulting from PTMs during aging in the establishment of SRO in differentiation of transparent lens fiber cells, remain to be characterized fully (
      • Clark J.I.
      Order and disorder in the transparent media of the eye.
      ,
      • Latina M.
      • Chylack Jr., L.T.
      • Fagerholm P.
      • Nishio I.
      • Tanaka T.
      • Palmquist B.M.
      Dynamic light scattering in the intact rabbit lens. Its relation to protein concentration.
      ,
      • Slingsby C.
      • Wistow G.J.
      • Clark A.R.
      Evolution of crystallins for a role in the vertebrate eye lens.
      ,
      • Clark J.I.
      • Giblin F.J.
      • Reddy V.N.
      • Benedek G.B.
      Phase separation of X-irradiated lenses of rabbit.
      ,
      • Takemoto L.
      • Sorensen C.M.
      Protein-protein interactions and lens transparency.
      ,
      • Ponce A.
      • Sorensen C.
      • Takemoto L.
      Role of short-range protein interactions in lens opacifications.
      ,
      • Klevit R.E.
      Peeking from behind the veil of enigma: emerging insights on small heat shock protein structure and function.
      ).
      If a single PTM were responsible for SRO and transparency in the lens, then its characterization and spatial distribution would be a relatively simple task (e.g., Fig. 4B). Thus far, no direct link between a single PTM in an individual lens protein and the onset of cataracts has been found, but some potential associations have been identified (
      • Vetter C.J.
      • Thorn D.C.
      • Wheeler S.G.
      • Mundorff C.
      • Halverson K.
      • Wales T.E.
      • et al.
      Cumulative deamidations of the major lens protein γS-crystallin increase its aggregation during unfolding and oxidation.
      ,
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ,
      • Hooi M.Y.
      • Raftery M.J.
      • Truscott R.J.
      Age-dependent racemization of serine residues in a human chaperone protein.
      ,
      • Hooi M.Y.
      • Raftery M.J.
      • Truscott R.J.
      Age-dependent deamidation of glutamine residues in human γS crystallin: deamidation and unstructured regions.
      ,
      • Takata T.
      • Murakami K.
      • Toyama A.
      • Fujii N.
      Identification of isomeric aspartate residues in βB2-crystallin from aged human lens.
      ,
      • Nandi S.K.
      • Rakete S.
      • Nahomi R.B.
      • Michel C.
      • Dunbar A.
      • Fritz K.S.
      • et al.
      Succinylation is a gain-of-function modification in human lens αb-crystallin.
      ). PTMs are accumulated in many lens proteins with age (
      • Ma Z.
      • Hanson S.R.
      • Lampi K.J.
      • David L.L.
      • Smith D.L.
      • Smith J.B.
      Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.
      ,
      • Hooi M.Y.
      • Truscott R.J.
      Racemisation and human cataract. D-Ser, D-Asp/Asn and D-Thr are higher in the lifelong proteins of cataract lenses than in age-matched normal lenses.
      ,
      • Vetter C.J.
      • Thorn D.C.
      • Wheeler S.G.
      • Mundorff C.
      • Halverson K.
      • Wales T.E.
      • et al.
      Cumulative deamidations of the major lens protein γS-crystallin increase its aggregation during unfolding and oxidation.
      ,
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ,
      • Friedrich M.G.
      • Wang Z.
      • Schey K.L.
      • Truscott R.J.W.
      Mechanism of protein cleavage at asparagine leading to protein-protein cross-links.
      ,
      • Warmack R.A.
      • Shawa H.
      • Liu K.
      • Lopez K.
      • Loo J.A.
      • Horwitz J.
      • et al.
      The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation.
      ,
      • Harrington V.
      • McCall S.
      • Huynh S.
      • Srivastava K.
      • Srivastava O.P.
      Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses.
      ,
      • Harrington V.
      • Srivastava O.P.
      • Kirk M.
      Proteomic analysis of water insoluble proteins from normal and cataractous human lenses.
      ,
      • Takemoto L.
      Increase in the intramolecular disulfide bonding of alpha-A crystallin during aging of the human lens.
      ). These previous studies were careful to identify the aging-associated PTMs (
      • Hanson S.R.
      • Smith D.L.
      • Smith J.B.
      Deamidation and disulfide bonding in human lens gamma-crystallins.
      ,
      • Ma Z.
      • Hanson S.R.
      • Lampi K.J.
      • David L.L.
      • Smith D.L.
      • Smith J.B.
      Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.
      ,
      • Hanson S.R.
      • Hasan A.
      • Smith D.L.
      • Smith J.B.
      The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage.
      ,
      • Lampi K.J.
      • Ma Z.
      • Hanson S.R.
      • Azuma M.
      • Shih M.
      • Shearer T.R.
      • et al.
      Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry.
      ), which can then allow the spatial and functional characterization of specific protein–protein interactions that control cytoplasmic SRO and cellular transparency to be identified.
      Global approaches have been used to study deamidation as a major PTM of lens proteins (
      • Vetter C.J.
      • Thorn D.C.
      • Wheeler S.G.
      • Mundorff C.
      • Halverson K.
      • Wales T.E.
      • et al.
      Cumulative deamidations of the major lens protein γS-crystallin increase its aggregation during unfolding and oxidation.
      ,
      • Norton-Baker B.
      • Mehrabi P.
      • Kwok A.O.
      • Roskamp K.W.
      • Rocha M.A.
      • Sprague-Piercy M.A.
      • et al.
      Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging.
      ). Deamidation of Asn and Gln residues is identified in proteins isolated from human cataractous and aged lenses. It is estimated to account for 66% of the total PTMs tabulated when water-soluble fractions and WIF were analyzed (
      • Wilmarth P.A.
      • Tanner S.
      • Dasari S.
      • Nagalla S.R.
      • Riviere M.A.
      • Bafna V.
      • et al.
      Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?.
      ). Modern technological advances in mass spectrometry will continue to develop our appreciation of PTM abundance and protein profiles in the aging lens (
      • Cantrell L.S.
      • Schey K.L.
      Data-independent acquisition mass spectrometry of the human lens enhances spatiotemporal measurement of fiber cell aging.
      ,
      • Harvey S.R.
      • O'Neale C.
      • Schey K.L.
      • Wysocki V.H.
      Native mass spectrometry and surface induced dissociation provide insight into the post-translational modifications of tetrameric AQP0 isolated from bovine eye lens.
      ). To follow dynamic changes in lens transparency, a measure of the collective and diverse protein interactions is needed, matched to the age-related PTMs for lens proteins. Such analyses would support the delivery of an effective and protective therapeutic capable of delaying the (age-related) loss of transparency.
      Crystallin PTMs start to accumulate even whilst the fetus is in the womb (
      • Hains P.G.
      • Truscott R.J.
      Proteome analysis of human foetal, aged and advanced nuclear cataract lenses.
      ,
      • Hains P.G.
      • Truscott R.J.
      Age-dependent deamidation of lifelong proteins in the human lens.
      ,
      • Hanson S.R.
      • Smith D.L.
      • Smith J.B.
      Deamidation and disulfide bonding in human lens gamma-crystallins.
      ,
      • Dasari S.
      • Wilmarth P.A.
      • Rustvold D.L.
      • Riviere M.A.
      • Nagalla S.R.
      • David L.L.
      Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses.
      ,
      • Takemoto L.
      Increase in the intramolecular disulfide bonding of alpha-A crystallin during aging of the human lens.
      ), long before any loss of transparency is observed in an aging lens. At the other end of the age spectrum, centenarians have a lower-than-expected incidence of ARC (
      • Evert J.
      • Lawler E.
      • Bogan H.
      • Perls T.
      Morbidity profiles of centenarians: survivors, delayers, and escapers.
      ,
      • Willcox D.C.
      • Willcox B.J.
      • Wang N.C.
      • He Q.
      • Rosenbaum M.
      • Suzuki M.
      Life at the extreme limit: phenotypic characteristics of supercentenarians in Okinawa.
      ). Although it might be expected that PTM would be progressive and similar in both lenses, ARC is not always bilateral (
      • Hong T.
      • Mitchell P.
      • Rochtchina E.
      • Fong C.S.
      • Chia E.M.
      • Wang J.J.
      Long-term changes in visual acuity in an older population over a 15-year period: the blue mountains eye study.
      ,
      • Maraini G.
      • Pasquini P.
      • Sperduto R.D.
      • Rosmini F.
      • Bonacini M.
      • Tomba M.C.
      • et al.
      Distribution of lens opacities in the Italian-American case-control study of age-related cataract. The Italian-American study group.
      ,
      • Schuster A.K.
      • Nickels S.
      • Pfeiffer N.
      • Schmidtmann I.
      • Wild P.S.
      • Münzel T.
      • et al.
      Frequency of cataract surgery and its impact on visual function-results from the German Gutenberg Health Study.
      ,
      • Yonova-Doing E.
      • Forkin Z.A.
      • Hysi P.G.
      • Williams K.M.
      • Spector T.D.
      • Gilbert C.E.
      • et al.
      Genetic and dietary factors influencing the progression of nuclear cataract.
      ), and many factors play a role in the loss of transparency (
      • Yonova-Doing E.
      • Forkin Z.A.
      • Hysi P.G.
      • Williams K.M.
      • Spector T.D.
      • Gilbert C.E.
      • et al.
      Genetic and dietary factors influencing the progression of nuclear cataract.
      ,
      • Taylor A.
      Nutritional and environmental influences on risk for cataract.
      ). It is important to acknowledge that life span and aging are not necessarily the same even for monozygotic human twins (
      • Fraga M.F.
      • Ballestar E.
      • Paz M.F.
      • Ropero S.
      • Setien F.
      • Ballestar M.L.
      • et al.
      Epigenetic differences arise during the lifetime of monozygotic twins.
      ,
      • Talens R.P.
      • Christensen K.
      • Putter H.
      • Willemsen G.
      • Christiansen L.
      • Kremer D.
      • et al.
      Epigenetic variation during the adult lifespan: cross-sectional and longitudinal data on monozygotic twin pairs.
      ,
      • van Dongen J.
      • Ehli E.A.
      • Slieker R.C.
      • Bartels M.
      • Weber Z.M.
      • Davies G.E.
      • et al.
      Epigenetic variation in monozygotic twins: a genome-wide analysis of DNA methylation in buccal cells.
      ), as lifestyle is a potential modifier (
      • Kilpeläinen T.O.
      • Bentley A.R.
      • Noordam R.
      • Sung Y.J.
      • Schwander K.
      • Winkler T.W.
      • et al.
      Multi-ancestry study of blood lipid levels identifies four loci interacting with physical activity.
      ,
      • Timmers P.R.
      • Mounier N.
      • Lall K.
      • Fischer K.
      • Ning Z.
      • Feng X.
      • et al.
      Genomics of 1 million parent lifespans implicates novel pathways and common diseases and distinguishes survival chances.
      ,
      • Vermeij W.P.
      • Dollé M.E.
      • Reiling E.
      • Jaarsma D.
      • Payan-Gomez C.
      • Bombardieri C.R.
      • et al.
      Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.
      ). It is, however, well established that crystallins are extensively modified during normal aging and in cataracts (
      • Hains P.G.
      • Truscott R.J.
      Age-dependent deamidation of lifelong proteins in the human lens.
      ,
      • Ma Z.
      • Hanson S.R.
      • Lampi K.J.
      • David L.L.
      • Smith D.L.
      • Smith J.B.
      Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.
      ,
      • Lampi K.J.
      • Ma Z.
      • Hanson S.R.
      • Azuma M.
      • Shih M.
      • Shearer T.R.
      • et al.
      Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry.
      ,
      • Wilmarth P.A.
      • Tanner S.
      • Dasari S.
      • Nagalla S.R.
      • Riviere M.A.
      • Bafna V.
      • et al.
      Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?.
      ,
      • Harrington V.
      • McCall S.
      • Huynh S.
      • Srivastava K.
      • Srivastava O.P.
      Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses.
      ,
      • Hains P.G.
      • Truscott R.J.
      Post-translational modifications in the nuclear region of young, aged, and cataract human lenses.
      ,
      • Hooi M.Y.
      • Raftery M.J.
      • Truscott R.J.
      Racemization of two proteins over our lifespan: deamidation of asparagine 76 in γS crystallin is greater in cataract than in normal lenses across the age range.
      ,
      • Srivastava O.P.
      • Srivastava K.
      Existence of deamidated alphaB-crystallin fragments in normal and cataractous human lenses.
      ,
      • Miesbauer L.R.
      • Zhou X.
      • Yang Z.
      • Yang Z.
      • Sun Y.
      • Smith D.L.
      • et al.
      Post-translational modifications of water-soluble human lens crystallins from young adults.
      ,
      • Takemoto L.J.
      Quantitation of asparagine-101 deamidation from alpha-A crystallin during aging of the human lens.
      ,
      • Zhang Z.
      • Smith D.L.
      • Smith J.B.
      Human beta-crystallins modified by backbone cleavage, deamidation and oxidation are prone to associate.
      ). The use of mass spectrometry has unambiguously identified deamidation as the cause for the increase in acidification of the lens crystallins in early studies of the human lens (
      • de Jong W.W.
      • Mulders J.W.
      • Voorter C.E.
      • Berbers G.A.
      • Hoekman W.A.
      • Bloemendal H.
      Post-translational modifications of eye lens crystallins: crosslinking, phosphorylation and deamidation.
      ,
      • Voorter C.E.
      • de Haard-Hoekman W.A.
      • van den Oetelaar P.J.
      • Bloemendal H.
      • de Jong W.W.
      Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate.
      ,
      • Van Kleef F.S.
      • De Jong W.W.
      • Hoenders H.J.
      Stepwise degradations and deamidation of the eye lens protein alpha-crystallin in ageing.
      ,
      • Groenen P.J.
      • van d.I.P.
      • Voorter C.E.
      • Bloemendal H.
      • de J.W.
      Site-specific racemization in aging alpha A-crystallin.
      ,
      • Voorter C.E.
      • Roersma E.S.
      • Bloemendal H.
      • de Jong W.W.
      Age-dependent deamidation of chicken alpha A-crystallin.
      ,
      • Fujii N.
      • Takemoto L.J.
      • Momose Y.
      • Matsumoto S.
      • Hiroki K.
      • Akaboshi M.
      Formation of four isomers at the asp-151 residue of aged human alphaA-crystallin by natural aging.
      ). Many of these studies analyzed deamidation in either purified crystallins or for a specific amide for all lens crystallins (
      • Hanson S.R.
      • Hasan A.
      • Smith D.L.
      • Smith J.B.
      The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage.
      ,
      • Lampi K.J.
      • Ma Z.
      • Hanson S.R.
      • Azuma M.
      • Shih M.
      • Shearer T.R.
      • et al.
      Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry.
      ,
      • Srivastava O.P.
      • Srivastava K.
      Existence of deamidated alphaB-crystallin fragments in normal and cataractous human lenses.
      ,
      • Miesbauer L.R.
      • Zhou X.
      • Yang Z.
      • Yang Z.
      • Sun Y.
      • Smith D.L.
      • et al.
      Post-translational modifications of water-soluble human lens crystallins from young adults.
      ,
      • Lapko V.N.
      • Purkiss A.G.
      • Smith D.L.
      • Smith J.B.
      Deamidation in human gamma S-crystallin from cataractous lenses is influenced by surface exposure.
      ,
      • Lund A.L.
      • Smith J.B.
      • Smith D.L.
      Modifications of the water-insoluble human lens alpha-crystallins.
      ,
      • Takemoto L.
      • Boyle D.
      Increased deamidation of asparagine during human senile cataractogenesis.
      ). When an eye lens is homogenized and the proportion of the various protein fractions (e.g., water soluble, HMW, WIF) is analyzed, increasing proportions of the proteins from the innermost and older “nuclear” region are found in the WIF in an age-dependent manner (Fig. 4A; (
      • Liu B.F.
      • Liang J.J.
      Protein-protein interactions among human lens acidic and basic beta-crystallins.
      )). The long-standing and widely held hypothesis is that the accumulation of crystallin PTMs is the cause of this insolubilization and subsequent cataract formation (
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ,
      • Harrington V.
      • McCall S.
      • Huynh S.
      • Srivastava K.
      • Srivastava O.P.
      Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses.
      ,
      • Truscott R.J.W.
      • Friedrich M.G.
      Molecular processes implicated in human age-related nuclear cataract.
      ,
      • Pande A.
      • Mokhor N.
      • Pande J.
      Deamidation of human γs-crystallin increases attractive protein interactions: implications for cataract.
      ,
      • Srivastava O.P.
      • Srivastava K.
      • Chaves J.M.
      • Gill A.K.
      Post-translationally modified human lens crystallin fragments show aggregation in vitro.
      ,
      • Budnar P.
      • Tangirala R.
      • Bakthisaran R.
      • Rao C.M.
      Protein aggregation and cataract: role of age-related modifications and mutations in α-crystallins.
      ). The experimental evidence suggests that we have been too quick to judge the role of PTMs in the loss of transparency and cataract formation (
      • Warmack R.A.
      • Shawa H.
      • Liu K.
      • Lopez K.
      • Loo J.A.
      • Horwitz J.
      • et al.
      The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation.
      ,
      • Truscott R.J.
      Age-related nuclear cataract-oxidation is the key.
      ,
      • Truscott R.J.
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      • Fan X.
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      ,
      • Srikanthan D.
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      ,
      • Grosas A.B.
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      ,
      • Schmid P.W.N.
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      ,
      • Liang J.N.
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      Interaction and aggregation of lens crystallins.
      ) rather than being connected to the formation, maintenance, and retention of SRO during lens aging. Lens protein PTMs are kept under constant review (
      • Lampi K.J.
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      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ,
      • Schey K.L.
      • Wang Z.
      • Friedrich M.G.
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      • Truscott R.J.W.
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      ,
      • Truscott R.J.W.
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      Molecular processes implicated in human age-related nuclear cataract.
      ,
      • Fan X.
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      ,
      • Serebryany E.
      • Thorn D.C.
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      Redox chemistry of lens crystallins: a system of cysteines.
      ,
      • Grosas A.B.
      • Carver J.A.
      Eye lens crystallins: remarkable long-lived proteins.
      ). Experimental data consistently demonstrate that most lens cytoplasmic protein PTMs identified in embryonic and young lenses can contribute to the development and maintenance of lens transparency (
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ,
      • Harrington V.
      • McCall S.
      • Huynh S.
      • Srivastava K.
      • Srivastava O.P.
      Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses.
      ,
      • Truscott R.J.W.
      • Friedrich M.G.
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      ,
      • Pande A.
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      • Pande J.
      Deamidation of human γs-crystallin increases attractive protein interactions: implications for cataract.
      ,
      • Srivastava O.P.
      • Srivastava K.
      • Chaves J.M.
      • Gill A.K.
      Post-translationally modified human lens crystallin fragments show aggregation in vitro.
      ,
      • Budnar P.
      • Tangirala R.
      • Bakthisaran R.
      • Rao C.M.
      Protein aggregation and cataract: role of age-related modifications and mutations in α-crystallins.
      ).
      We suggested previously (
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      • Hamada N.
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      • Quinlan R.A.
      Cataractogenic load - a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.
      ,
      • Berry V.
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      ,
      • Kalligeraki A.
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      Structural proteins | crystallins of the mammalian eye lens.
      ), and others (
      • Grosas A.B.
      • Carver J.A.
      Eye lens crystallins: remarkable long-lived proteins.
      ) have similarly speculated recently, an alternative hypothesis regarding the role of lens protein PTMs in lens transparency and optical function. When crystallin PTMs were first being identified, those associated with normal aging were included with those associated with cataracts (
      • Hanson S.R.
      • Smith D.L.
      • Smith J.B.
      Deamidation and disulfide bonding in human lens gamma-crystallins.
      ,
      • Hanson S.R.
      • Hasan A.
      • Smith D.L.
      • Smith J.B.
      The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage.
      ,
      • Lampi K.J.
      • Ma Z.
      • Hanson S.R.
      • Azuma M.
      • Shih M.
      • Shearer T.R.
      • et al.
      Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry.
      ). In the CL hypothesis, we propose that many lens protein PTMs have an active protective role in preserving transparency and optical function (Fig. 5). PTMs, such as deamidation (
      • Hanson S.R.
      • Smith D.L.
      • Smith J.B.
      Deamidation and disulfide bonding in human lens gamma-crystallins.
      ,
      • Hanson S.R.
      • Hasan A.
      • Smith D.L.
      • Smith J.B.
      The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage.
      ,
      • Dasari S.
      • Wilmarth P.A.
      • Rustvold D.L.
      • Riviere M.A.
      • Nagalla S.R.
      • David L.L.
      Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses.
      ), proteolysis (
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ), and disulphide bond formation (
      • Takemoto L.
      Increase in the intramolecular disulfide bonding of alpha-A crystallin during aging of the human lens.
      ), are necessary for the early development, establishment, and maintenance of SRO in the lens. It has been suggested previously that the proteolytic removal of the N-terminal extensions of β-crystallins has a potential stabilization/protection role (
      • Lampi K.J.
      • Wilmarth P.A.
      • Murray M.R.
      • David L.L.
      Lens β-crystallins: the role of deamidation and related modifications in aging and cataract.
      ) that facilitates crystallin SRO. The disulphide bonding and exchange that occurs because of the oxidoreductase activities of lens crystallins is also considered to be protective and could be a stabilizing influence in crystallin gels (
      • Serebryany E.
      • Thorn D.C.
      • Quintanar L.
      Redox chemistry of lens crystallins: a system of cysteines.
      ,
      • Quinlan R.A.
      • Hogg P.J.
      γ-Crystallin redox-detox in the lens.
      ). Others have recognized that “crystallin PTMs act in a complementary and synergistic manner, potentially coupled with some redundancy, such that they do not have a prejudicial effect on crystallin solubility and interactions (
      • Grosas A.B.
      • Thorn D.C.
      • Carver J.A.
      Crystallins, cataract, and dynamic lens proteostasis. A commentary on P.W.N. Schmid, N.C.H. Lim, C. Peters, K.C. Back, B. Bourgeois, F. Pirolt, B. Richter, J. Peschek, O. Puk, O.V. Amarie, C. Dalke, M. Haslbeck, S. Weinkauf, T. Madl, J. Graw, and J. Buchner (2021) Imbalances in the eye lens proteome are linked to cataract formation, Nat. Struct. Mol. Biol.28, 143-151. doi: 10.1038/s41594-020-00543-9.
      ).” We suggest that such aging-associated PTMs can be protective rather than passive in this role. Indeed, we note that the PTM profiles are different in the WIF, HMW, and membrane lens fractions (
      • Wilmarth P.A.
      • Tanner S.
      • Dasari S.
      • Nagalla S.R.
      • Riviere M.A.
      • Bafna V.
      • et al.
      Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?.
      ,
      • Dasari S.
      • Wilmarth P.A.
      • Rustvold D.L.
      • Riviere M.A.
      • Nagalla S.R.
      • David L.L.
      Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses.
      ,
      • Schey K.L.
      • Wang Z.
      • Friedrich M.G.
      • Garland D.L.
      • Truscott R.J.W.
      Spatiotemporal changes in the human lens proteome: critical insights into long-lived proteins.
      ,
      • Warmack R.A.
      • Shawa H.
      • Liu K.
      • Lopez K.
      • Loo J.A.
      • Horwitz J.
      • et al.
      The l-isoaspartate modification within protein fragments in the aging lens can promote protein aggregation.
      ,
      • Harrington V.
      • McCall S.
      • Huynh S.
      • Srivastava K.
      • Srivastava O.P.
      Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses.