ArticleLiterature Review

Ageing and vision: Structure, stability and function of lens crystallins

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Abstract

The alpha-, beta- and gamma-crystallins are the major protein components of the vertebrate eye lens, alpha-crystallin as a molecular chaperone as well as a structural protein, beta- and gamma-crystallins as structural proteins. For the lens to be able to retain life-long transparency in the absence of protein turnover, the crystallins must meet not only the requirement of solubility associated with high cellular concentration but that of longevity as well. For proteins, longevity is commonly assumed to be correlated with long-term retention of native structure, which in turn can be due to inherent thermodynamic stability, efficient capture and refolding of non-native protein by chaperones, or a combination of both. Understanding how the specific interactions that confer intrinsic stability of the protein fold are combined with the stabilizing effect of protein assembly, and how the non-specific interactions and associations of the assemblies enable the generation of highly concentrated solutions, is thus of importance to understand the loss of transparency of the lens with age. Post-translational modification can have a major effect on protein stability but an emerging theme of the few studies of the effect of post-translational modification of the crystallins is one of solubility and assembly. Here we review the structure, assembly, interactions, stability and post-translational modifications of the crystallins, not only in isolation but also as part of a multi-component system. The available data are discussed in the context of the establishment, the maintenance and finally, with age, the loss of transparency of the lens. Understanding the structural basis of protein stability and interactions in the healthy eye lens is the route to solve the enormous medical and economical problem of cataract.

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... 4,5 Crystallins are long-lived, soluble proteins of the eye lens and are essential for the optical properties of the lens and for the maintenance of transparency over a lifetime. [6][7][8] However, particularly with age, crystallins can become insolubilized, forming light-scattering aggregates which lead to cataract. There are three major classes of crystallins in most vertebrates: a-crystallins, which are small heat shock proteins, and the evolutionarily related band c-crystallins. ...
... 14,15 Posttranslational modifications, such as oxidation, deamidation and racemization, in genetically normal c-crystallins are also associated with cataract. 6,17,18 However, how these modifications lead to oligomerization and aggregation is not known. It has long been thought that intermolecular disulfide formation contributes, 6,[19][20][21] while other models, such as partial domain swapping of secondary structure elements in a mimic of an oxidized c-crystallin under low pH, have been proposed. ...
... 6,17,18 However, how these modifications lead to oligomerization and aggregation is not known. It has long been thought that intermolecular disulfide formation contributes, 6,[19][20][21] while other models, such as partial domain swapping of secondary structure elements in a mimic of an oxidized c-crystallin under low pH, have been proposed. [22][23][24] cS is the most abundant member of the ccrystallin family in adult human lens 25 and is widely expressed in vertebrates. ...
Article
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Misfolding and aggregation of proteins occur in many pathological states. Because of the inherent disorder involved, these processes are difficult to study. We attempted to capture aggregation intermediates of γ S-crystallin, a highly stable, internally symmetrical monomeric protein, by crystallization under mildly acidic and oxidizing conditions. Here we describe novel oligomerization through strained domain-swapping and partial intermolecular disulfide formation. This forms an octamer built from asymmetric tetramers, each of which comprises an asymmetric pair of twisted, domain-swapped dimers. Each tetramer shows patterns of acquired disorder among subunits, ranging from local loss of secondary structure to regions of intrinsic disorder. The octamer ring is tied together by partial intermolecular disulfide bonds, which may contribute to strain and disorder in the octamer. Oligomerization in this structure is self-limited by the distorted octamer ring. In a more heterogeneous environment, the disordered regions could serve as seeds for cascading interactions with other proteins. Indeed, solubilized protein from crystals retain many features observed in the crystal and are prone to further oligomerization and precipitation. This structure illustrates modes of loss of organized structure and aggregation that are relevant for cataract and for other disorders involving deposition of formerly well-folded proteins.
... The α-crystallins belong to the family of small heatshock proteins (HSPs) which act as molecular chaperones during embryonic development [8][9]. The α-crystallin family comprises two subunits, referred to as αAand αB-crystallins, which are encoded by the Cryaa and Cryab genes, respectively [10]. ...
... In mammals, these genes are not only organized as individual genes (Cryba1, Cryba2, Crygf, Crygs, CrygN), but also as duplets (Cryba4-Crybb1 and Crybb2-Crybb3) and into one major cluster (Cryga-Cryge) [11,16]. The γ-crystallins are monomeric proteins with molecular masses of about 20 kDa, whereas the βcrystallins are a heterogeneous mixture of dimers and higher oligomers with native molecular masses ranging from about 50 kDa to 200 kDa [8]. Like all members of the βγ-crystallin superfamily, β-crystallins comprise two domains connected by an 8-10 amino acid inter-domain connecting peptide [17]. ...
... The highly ordered, tightly packed crystallins make up the transparent structure of the lens and allow it to focus light onto the retina. To provide adequate lens structure and function, a protein must: (1) be highly soluble-high concentrations of soluble crystallins are responsible for the refractive index of the lens and maintain its transparency; (2) be extremely stable: the inherent stability of crystallins, arising from their native, compact structure, correlates tightly with their exceptional longevity; and (3) be able to have specific interactions with other crystallins: forming a stable protein matrix with a high degree of short-range order allows to increase resistance to oxidative stress and thermal denaturation, which is decisive to maintain lens transparency [8,38,39]. Therefore, the solubility and stability of βB2-crystallins are crucial determinants for the normal function of the lens: when these parameters are compromised, crystallin aggregation will affect lens transparency and reduce dioptric capacity [38][39][40][41]. Notable features of the molecular biology of the crystallin superfamily include the potential to be transported between cells via exosomes [42] and the ability of some of its members (e.g., αB-and βA3/A1crystallins) to regulate lens differentiation and epithelial-mesenchymal transition (EMT) in RPE and tumor cells [43][44][45]. ...
Article
Crystallins, the major constituent proteins of mammalian lenses, are significant not only for the maintenance of eye lens stability, transparency, and refraction, but also fulfill various physiopathological functions in extraocular tissues. βB2-crystallin, for example, is a multifunctional protein expressed in the human retina, brain, testis, ovary, and multiple tumors. Mutations in the βB2 crystallin gene or denaturation of βB2-crystallin protein are associated with cataracts, ocular pathologies, and psychiatric disorders. A prominent role for βB2-crystallins in axonal growth and regeneration, as well as in dendritic outgrowth, has been demonstrated after optic nerve injury. Studies in βB2-crystallin-null mice revealed morphological and functional abnormalities in testis and ovaries, indicating βB2-crystallin contributes to male and female fertility in mice. Interestingly, although pathogenic significance remains obscure, several studies identified a clear correlation between βB2 crystallin expression and the prognosis of patients with breast cancer, colorectal cancer, prostate cancer, renal cell carcinoma, and glioblastoma in the African American population. This review summarizes the physiological and pathological functions of βB2-crystallin in the eye and other organs and tissues and discusses findings related to the expression and potential role of βB2-crystallin in tumors.
... Crystallins are the main proteins in the human lens and are divided into two superfamilies: α-crystallins and βγ-crystallins [13][14][15]. The α-crystallins are members of the small heat-shock protein (sHsp) and function as chaperons. ...
... The βγ-crystallins are the main structural proteins of the lens and share a similar fold in their tertiary structures composed of two domains connected by a linker peptide, each one with two Greek-key motifs. The γ-crystallins have been described as monomers, while the β-crystallins can form dimers or larger oligomers [13,14,16]. ...
... The β-crystallins constitute~40% of the crystallins of the lens and historically are classified as acidic (βA) or basic (βB) [14,17]. The acidic isoforms have four members (βA1, βA2, βA3, βA4) while the basic ones contain three isoforms (βB1, βB2, βB3) [18]. ...
Article
Full-text available
Cataracts are defined as the clouding of the lens due to the formation of insoluble protein aggregates. Metal ions exposure has been recognized as a risk factor in the cataract formation process. The γ and β crystallins are members of a larger family and share several structural features. Several studies have shown that copper and zinc ions induce the formation of γ-crystallins aggregates. However, the interaction of metal ions with β-crystallins, some of the most abundant crystallins in the lens, has not been explored until now. Here, we evaluate the effect of Cu(II) and Zn(II) ions on the aggregation of HβA1, as a representative of the acidic form, and HβB2, as a representative of the basic β-crystallins. We used several biophysical techniques and computational methods to show that Cu(II) and Zn(II) induce aggregation following different pathways. Both metal ions destabilize the proteins and impact protein folding. Copper induced a small conformational change in HβA1, leading to high-molecular-weight light-scattering aggregates, while zinc is more aggressive towards HβB2 and induces a larger conformational change. Our work provides information on the mechanisms of metal-induced aggregation of β-crystallins.
... α Crystallin belongs to small heat shock proteins, whereas β and γ crystallins are from a superfamily relat ed to prokaryotic stress proteins [27]. The ratio between α , β , and γ crystallins in lens tissues changes with age due to the pattern of synthesis of these proteins in onto genesis [28]. ...
... Compared to proteins from other organs, lens pro teins undergo virtually no exchange [27], which means that proteins emerging during embryogenesis should serve for the entire organism lifetime. That implies that lens tissues possess the mechanisms counteracting post translational protein modifications or blocking their negative impact. ...
... 87 No. 2 2022 respectively [29]. αA and αB crystallins consist of 173 and 175 amino acid (a.a.) residues and have a molecular weight of 19.8 and 20.0 kDa, respectively [27]. These polypeptides create a pool of oligomeric molecules with a molecular weight of 160 1200 kDa [103]. ...
Article
The absence of cellular organelles in fiber cells and very high cytoplasmic protein concentration (up to 900 mg/ml) minimize light scattering in the lens and ensure its transparency. Low oxygen concentration, powerful defense systems (antioxidants, antioxidant enzymes, chaperone-like protein alpha-crystallin, etc.) maintain lens transparency. On the other hand, the ability of crystallins to accumulate age-associated post-translational modifications, which reduce the resistance of lens proteins to oxidative stress, is an important factor contributing to the cataract formation. Here, we suggest a mechanism of cataractogenesis common for the action of different cataractogenic factors, such as age, radiation, ultraviolet light, diabetes, etc. Exposure to these factors leads to the damage and death of lens epithelium, which allows oxygen to penetrate into the lens through the gaps in the epithelial layer and cause oxidative damage to crystallins, resulting in protein denaturation, aggregation, and formation of multilamellar bodies (the main cause of lens opacification). The review discusses various approaches to the inhibition of lens opacification (cataract development), in particular, a combined use of antioxidants and compounds enhancing the chaperone-like properties of alpha-crystallin. We also discuss the paradox of high efficiency of anti-cataract drugs in laboratory settings with the lack of their clinical effect, which might be due to the late use of the drugs at the stage, when the opacification has already formed. A probable solution to this situation will be development of new diagnostic methods that will allow to predict the emergence of cataract long before the manifestation of its clinical signs and to start early preventive treatment.
... Therefore, the refractive index of the human lens 4 increases from 1.38 (73-80% water) in the cortex to 1.42 (68% water) in the nucleus. 2 These proteins are mainly made of the crystallins protein family α-, -and λcrystallins. [14][15][16][17][18] The lens nucleus refer to the innermost part of the lens and its structure is similar to that of the cortex besides the fact that the cells are more tightly packed due to the accrual of cells overtime. 19 The very limited extracellular space between the fibre cells is critical for maintaining the lens transparency. ...
... 17 Effect of EGCNPs on the inhibition of the formation of advanced glycation end products (AGEs) for bovine serum albumin (BSA) when incubated with glucose (glu) for 72 h. ...
Thesis
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Chronic diseases are rising in incidence and prevalence because of the increase in life expectancy in many parts of the world and the advances in medicine that manage disease progression, rather than curing and alleviating the causes. Cataract is one such chronic condition. Identifying a therapeutic intervention that is successful in reversing or preventing cataracts may have applications for other chronic diseases of protein misfolding, such as diabetes and Alzheimer's disease as these have similar causation factors, notably oxidative stress and/or glycation. To date, surgery remains the only effective treatment for cataract and the search for alternatives is still ongoing. Cerium oxide nanoparticles (nanoceria) which have antioxidant, radioprotective and enzyme-mimetic properties have the potential to lead to an effective non-surgical treatment. However, nanoceria stability in physiological media is poor thus hindering their effective use in biomedical applications. In the work described in this thesis, a highly efficient one-pot synthesis of nanoceria (2–5 nm) has been achieved. The nanoparticles were coated with a novel hybrid coating (ethylene glycol, ethylene glycol monoand di- acetates) providing the formulation with superior colloidal stability in physiological media. The ethylene glycol coated nanoceria formulation (EGCNPs), up to concentrations of 200 μg/ml, is not toxic to human lens epithelial cells and has no adverse effect on the cellular morphology, proliferation rate, mitochondrial morphology, mitochondrial membrane potential, ATP level, DNA integrity and basal reactive oxygen species (ROS) level. Exceeding the safe concentration of nanoceria leads to genotoxicity and apoptotic cell death mediated by ROS elevation and mitochondrial damage, a mechanism which is comprehensively investigated for the first time in human lens epithelial cells (HLECs). EGCNPs uptake in HLECs was found to be endocytosisdependent and the nanoparticles localised extensively in the mitochondria. This localisation has enabled the nanoparticles to protect HLECs against oxidative stress, act as a catalase mimetic and increase reduced glutathione/oxidised glutathione ratio (GSH/GSSG). Furthermore, it is shown for the first time that these nanoparticles can protect lens proteins against glucose-induced glycation that is a major cause of cataract particularly in diabetics. Together, these results demonstrate great potential for nanoceria in protecting against cataract and should be taken to in-vivo studies.
... Unlike other proteins in the cell, crystallins, once synthesized, remain in the lens for the individual's lifetime [13]. The primary function of crystallins is contributing to lens transparency and refractive properties [14]. α-crystallin accounts for up to 40% of the lens proteins [10,12]. ...
... αA-crystallin is found only in the lens, whereas αB-crystallin is found in the lens and many other tissues, including the heart, nervous system, striated muscles, and the kidney [17]. αA-and αB-crystallin consist of 173 and 175 amino acid residues, with 60% sequence similarity [14], and have a mass of 19.9 and 20.2 kDa, respectively [18]. The oligomeric form of α-crystallin has a mass range from 300 to 900 kDa [19]. ...
Article
Full-text available
α-crystallin is a major protein found in the mammalian eye lens that works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress in the eye lens. These functions of α-crystallin are significant for maintaining lens transparency. However, with age and cataract formation, the concentration of α-crystallin in the eye lens cytoplasm decreases with a corresponding increase in the membrane-bound α-crystallin, accompanied by increased light scattering. The purpose of this review is to summarize previous and recent findings of the role of the: 1) lens membrane components, i.e., the major phospholipids (PLs) and sphingolipids, cholesterol (Chol), cholesterol bilayer domains (CBDs), and the integral membrane proteins aquaporin-0 (AQP0; formally MIP26) and connexins, and 2) α-crystallin mutations and post-translational modifications (PTMs) in the association of α-crystallin to the eye lens’s fiber cell plasma membrane, providing thorough insights into a molecular basis of such an association. Furthermore, this review highlights the current knowledge and need for further studies to understand the fundamental molecular processes involved in the association of α-crystallin to the lens membrane, potentially leading to new avenues for preventing cataract formation and progression.
... The damaged proteins must be repaired, degraded or sequestered by lens chaperones as they have a propensity to form large insoluble cytotoxic aggregates (Moreau and King, 2012). Accumulation of insoluble protein aggregates results in eventual opacification and formation of cataract (Bloemendal et al., 2004;Moreau and King, 2012). ...
... The chaperone activity of alpha (α)-crystallin catalyzes the repair and re-activation of modified ζ-crystallin (Goenka et al., 2001;Malik et al., 2021). There is an inverse relationship between crystallin chaperone/oxidoreductase activity and aging, and the effects of this change are seen as increased protein aggregation, protein insolubility, light-scattering and loss of lens transparency (Bloemendal et al., 2004;Yang et al., 2015). The lenses of transgenic animals with lenses deficient for crystallins develop cataract, and this is thought to be caused at least in part by decreased resistance to oxidative stress (Brady et al., 1997;Zhang et al., 2008). ...
Article
Lens homeostasis and transparency are dependent on the function and intercellular communication of its epithelia. While the lens epithelium is uniquely equipped with functional repair systems to withstand reactive oxygen species (ROS)-mediated oxidative insult, ROS are not necessarily detrimental to lens cells. Lens aging, and the onset of pathogenesis leading to cataract share an underlying theme; a progressive breakdown of oxidative stress repair systems driving a pro-oxidant shift in the intracellular environment, with cumulative ROS-induced damage to lens cell biomolecules leading to cellular dysfunction and pathology. Here we provide an overview of our current understanding of the sources and essential functions of lens ROS, antioxidative defenses, and changes in the major regulatory systems that serve to maintain the finely tuned balance of oxidative signaling vs. oxidative stress in lens cells. Age-related breakdown of these redox homeostasis systems in the lens leads to the onset of cataractogenesis. We propose eight candidate hallmarks that represent common denominators of aging and cataractogenesis in the mammalian lens: oxidative stress, altered cell signaling, loss of proteostasis, mitochondrial dysfunction, dysregulated ion homeostasis, cell senescence, genomic instability and intrinsic apoptotic cell death.
... Meanwhile, their interactions ensure the lens' refractive properties and transparency. [5][6][7] Factors affecting the homeostasis of lens proteins may cause lens opacity. For example, the senescent lens may present with more protein modification and less proteasomal activity, which can disrupt protein homeostasis and cause aggregate formation. ...
... 14 15 These modifications may disrupt protein homeostasis and reduce lens protein stability by influencing protein interaction and promoting lens protein degradation. 5 Our data revealed higher concentration with higher stability, suggesting thermal stability of older lens proteins and lower concentration of soluble lens proteins. Additionally, the proportion of various lens proteins and lens protein oligomeric states are essential for protein haemostasis. ...
Article
Background Age-related cataract, which presents as a cloudy lens, is the primary cause of vision impairment worldwide and can cause more than 80% senile blindness. Previous studies mainly explored the profile of lens proteins at a low concentration because of technical limitations, which could not reflect physiological status. This study focuses on protein stability changes with ageing under physiological conditions using a novel equipment, Unchained Labs (Uncle), to evaluate protein thermal stability. Methods Samples were assessed through Unchained Labs, size-exclusion chromatography, western blot and biophysics approaches including the Thioflavin T, ultraviolet and internal fluorescence. Results With age, the melting temperature value shifted from 67.8°C in the young group to 64.2°C in the aged group. Meanwhile, crystallin may form more isomeric oligomers and easy to be degraded in aged lenses. The spectroscopic and size-exclusion chromatography results show a higher solubility after administrated with lanosterol under the environmental stress. Conclusion We are the first to explore rabbit lens protein stability changes with ageing using biophysical methods under physiological conditions, and this study can conclude that the structural stability and solubility of lens proteins decrease with ageing. Additionally, lanosterol could aid in resolving protein aggregation, making it a potential therapeutic option for cataracts. So, this study provides cataract models for anti-cataract drug developments
... KNA exhibits a high triplet state yield (82% [65]) independent of viscosity [66], and the triplet state is able to react not only with oxygen [17,18] with rate constant 2.3 × 10 9 M − 1 s − 1 [15], but also with amino acid residues of tryptophan and tyrosine with rate constants 2.5 × 10 9 M − 1 s − 1 and 0.8 × 10 9 M − 1 s − 1 [15,67], respectively, leading to the formation of corresponding radicals [15,67,68]. Due to high concentrations of proteins in an eye lens (up to 400 mg/ml [69]) and low concentration of oxygen, even under air-saturated conditions, we expect the main reactions of KNA triplet state to be with proteins and not with oxygen, (see Section S4 of SI for additional details). Thus, these reactions are expected to initiate Type I photodamage. ...
... Finally, we should mention that the lipid composition of plasma membranes of human eye lenses significantly differs from porcine samples, mainly by higher content of cholesterol and dihydrosphingomyelins present in human lens membranes [25,26,32]. The composition of intracellular milieu is also somewhat different, for proteins [69] and metabolites [12,46]. However, our results clearly show that changes in viscosity upon Type I and II damage to porcine eye lens membranes correlate well with changes observed within the model DOPC membranes [29], which are much less ordered and exhibit lower viscosity as compared to porcine lens membranes. ...
Article
An eye lens is constantly exposed to the solar UV radiation, which is considered the most important external source of age-related changes to eye lens constituents. The accumulation of modifications of proteins and lipids with age can eventually lead to the development of progressive lens opacifications, such as cataracts. Though the impact of solar UV radiation on the structure and function of proteins is actively studied, little is known about the effect of photodamage on plasma membranes of lens cells. In this work we exploit Fluorescence Lifetime Imaging Microscopy (FLIM), together with microviscosity-sensitive fluorophores termed molecular rotors, to study the changes in viscosity of plasma membranes of porcine eye lens resulting from two different types of photodamage: Type I (electron transfer) and Type II (singlet oxygen) reactions. We demonstrate that these two types of photodamage result in clearly distinct changes in viscosity – a decrease in the case of Type I damage and an increase in the case of Type II processes. Finally, to simulate age-related changes that occur in vivo, we expose an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts.
... 1 The disease is caused by light-scattering aggregation of the extremely long-lived crystallin proteins in the lens. 2 This aggregation is associated with accumulation of post-translational modifications, including disulfide formation, UV-or metal-catalyzed Trp oxidation, deamidation, Asp isomerization, Lys derivatization, truncation, and various others. [3][4][5] Most age-onset cataract forms in the oldest, nuclear (or core) region of the lens. 2 The cells in this region lack all organelles and thus protein synthesis and degradation capacity, and lens crystallins comprise ~90% of their protein mass. ...
... [3][4][5] Most age-onset cataract forms in the oldest, nuclear (or core) region of the lens. 2 The cells in this region lack all organelles and thus protein synthesis and degradation capacity, and lens crystallins comprise ~90% of their protein mass. 6 The most thermodynamically stable in humans is γD-crystallin (HγD), which, at neutral pH, has a Tm above 80 °C, 7 resistance to 8M urea, 8 and unfolding half-life estimated to be on the order of decades. ...
Preprint
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Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo-inositol's molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol's primary target was neither the native nor the unfolded state of the protein, nor the final aggregated state, but rather the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.
... When the lens ages, it loses its transparency and becomes cloudy, a condition known as cataract 5 . Cataract affects the refractive property of lens leading to decreased vision 5 . ...
... When the lens ages, it loses its transparency and becomes cloudy, a condition known as cataract 5 . Cataract affects the refractive property of lens leading to decreased vision 5 . An estimated 51% blindness globally is attributed to cataract, 32.65% of these cases belong to South Asia 6,7 . ...
Article
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Aim: Comparative immunohistochemical study of expression of α A Crystallin in non-cataract lenses and age-related cataract lenses in humans. Methodology: This was an observational cross sectional study. There are two groups in this study. Group A comprised of 121 senile degenerative cataract lenses from diagnosed patients. Group B included of 10 non-cataract lenses from patients who underwent surgeries for enucleation due to trauma and retinoblastoma. Lenses were fixed in 10% Buffered Neutral Formalin and processed to make paraffin blocks. Immunohistochemistry (IHC) staining was performed on sections using primary antibody for α A crystallin. Data was analyzed through SPSS software version 24. Results: Immunohistochemical staining of group A showed 80.2% Strong Positive expression while 19.8% showed Intermediate Positive expression of α A Crystallin. 100% Strong Positive expression of α A Crystallin was seen in group B. Comparison of expression of α A Crystallin in two groups showed significant decrease (p<0.001) in expression. Conclusion: Decreased expression of α A Crystallin in IHC stained senile cataract lens indicates the role of structural alterations of lens fibers in pathogenesis of senile cataract. If mechanism involved in causing these alterations can be identified and targeted so that progression of senile cataract may be delayed. Keywords: Immunohistochemistry, α A crystallin expression, senile cataract, Human eye Lens, Lens Fiber.
... T he crystallin family of proteins is essential for lens transparency, and altered short-range interactions of crystallins are thought to be responsible for cataract-associated light scattering. 1 Distributions and classes of crystallin proteins in the lens have been linked to the gradient of refractive index (GRIN) profile and refractive index magnitude. 2 A high concentration of crystallin proteins provides transparency and creates the unique GRIN profile within the lens that is needed to provide the high level of image quality required for vision. 1 Because the lens has limited capacity for new protein synthesis, crystallins must remain soluble and stable for decades 1 , a remarkable feature facilitated in part by the chaperone α-crystallin, which helps maintain protein stability and preserve lens transparency. ...
... 2 A high concentration of crystallin proteins provides transparency and creates the unique GRIN profile within the lens that is needed to provide the high level of image quality required for vision. 1 Because the lens has limited capacity for new protein synthesis, crystallins must remain soluble and stable for decades 1 , a remarkable feature facilitated in part by the chaperone α-crystallin, which helps maintain protein stability and preserve lens transparency. 3,4 It has been shown that α-crystallin accounts for nearly 50% of the protein mass in human lenses and is known to interact with non-native proteins to prevent their irreversible aggregation and insolubilization in vitro. ...
Article
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Purpose: To investigate how cataract-linked mutations affect the gradient refractive index (GRIN) and lens opacification in mouse lenses and whether there is any effect on the optics of the lens from treatment with an oxysterol compound. Methods: A total of 35 mice including wild-type and knock-in mutants (Cryaa-R49C and Cryab-R120G) were used in these experiments: 26 mice were treated with topical VP1-001, an oxysterol, in one eye and vehicle in the other, and nine mice were untreated controls. Slit lamp biomicroscopy was used to analyze the lens in live animals and to provide apparent cataract grades. Refractive index in the lenses of 64 unfixed whole mouse eyes was calculated from measurements with X-ray phase tomography based on X-ray Talbot interferometry with a synchrotron radiation source. Results: Heterozygous Cryaa-R49C lenses had slightly irregularly shaped contours in the center of the GRIN and distinct disturbances of the gradient index at the anterior and posterior poles. Contours near the lens surface were denser in homozygous Cryab-R120G lenses. Treatment with topical VP1-001, an oxysterol, showed an improvement in refractive index profiles in 61% of lenses and this was supported by a reduction in apparent lens opacity grade by 1.0 in 46% of live mice. Conclusions: These results indicate that α-crystallin mutations alter the refractive index gradient of mouse lenses in distinct ways and suggest that topical treatment with VP1-001 may improve lens transparency and refractive index contours in some lenses with mutations.
... The eye lenses of mammals are composed of fibre cells [21,50] with unique properties in terms of transparency, due to the very high concentration of proteins expressed in the fibre cells [51][52][53]. Over 90% of lens proteins belong to crystallin family [54]. ...
Article
Silk fibroin (SF) is a non-pathological amyloidogenic protein prone, in solution, to the formation of amyloid-like aggregated species, displaying similarities in fibrillation kinetics with pathological amyloids, as widely reported in the literature. We show here, on the basis of different biophysical approaches (turbidity, Congo Red assays, CD, DLS and fluorescence), that fusidic acid (FA), a well-known antibiotic, acts on SF as an anti-aggregating agent in a dose-dependent manner, being also able to revert SF aggregation. FA binds to SF inducing changes in the environment of SF aromatic residues. We further provide the proof of principle that FA, already approved as drug on humans and used in ophthalmic preparations, displays its anti-aggregation properties also on lens material derived from cataract surgery and is capable of reducing aggregation. Thus it is suggested that FA can be foreseen as a therapeutic treatment for cataract and other protein aggregation disorders.
... In adulthood, the lens undergoes very little protein turnover (Lynnerup et al., 2008). Crystallins are the predominant protein species in the lens, comprising over 90% of dry mass and reaching concentrations above 400 mg/mL (Bloemendal et al., 2004;Vendra et al., 2016;Wistow, G. J.;Piatigorsky, 1988). Crystallins were originally characterized by their transparency and presence in the "crystalline lens," and are divided into two superfamilies, α-crystallins and βγ-crystallins (Slingsby et al., 2013). ...
Preprint
Cataract disease, a clouding of the eye lens due to precipitation of lens proteins, affects millions of people every year worldwide. The proteins that comprise the lens, the crystallins, show extensive post-translational modifications (PTMs) in aged and cataractous lenses, most commonly deamidation and oxidation. Although surface-exposed glutamines and asparagines show the highest rates of deamidation, multiple modifications can accumulate over time in these long-lived proteins, even for buried residues. Both deamidation and oxidation have been shown to promote crystallin aggregation in vitro ; however, it is not clear precisely how these modified crystallins contribute to insolubilization. Here, we report six novel crystal structures of a major human lens protein, γS-crystallin (γS): one of the wild-type in a monomeric state, and five of deamidated γS variants, ranging from three to nine deamidation sites, after varying degrees of sample aging. Consistent with previous work that focused on single-to triple-site deamidation, the deamidation mutations do not appear to drastically change the fold of γS; however, increasing deamidation leads to accelerated oxidation and disulfide bond formation. Successive addition of deamidated sites progressively destabilized protein structure as evaluated by differential scanning fluorimetry. Light scattering showed the deamidated variants display an increased propensity for aggregation compared to the wild-type protein. The results suggest the deamidated variants are useful as models for accelerated aging; the structural changes observed over time provide support for redox activity of γS-crystallin in the human lens. Highlights Novel structures of cataract-associated variants of human eye lens protein γS-crystallin reported Increasing deamidation of γS-crystallin decreases stability and affects aggregation propensity Overall fold of γS-crystallin maintained among deamidated and disulfide-bonded variants Deamidated γS variants form disulfide bonds more rapidly than wild-type γS Potential functional advantage of disulfide bonding in the CXCXC motif proposed
... Being an ATP-independent chaperone, it readily interacts with partially unfolded proteins and sequesters them to avoid protein aggregation. It occurs as a hetero oligomer composed of two subunits known as αАand αB-crystallins [18,19]. αА-crystallin is exclusively found in the eye lens, while αB is present in other tissues, including different parts of the brain and heart [20]. ...
Article
The loss of crystallins solubility with aging and the formation of amyloid-like aggregates is considered the hallmark characteristic of cataract pathology. The present study was carried out to assess the effect of temperature on the soluble lens protein and the formation of protein aggregates with typical amyloid characteristics. The soluble fraction of lens proteins was subjected for heat treatment in the range of 40–60 °C, and the nature of protein aggregates was assessed by using Congo red (CR), thioflavin T (ThT), and 8-anilinonaphthalene-1-sulfonic acid (ANS) binding assays, circular dichroism (CD), Fourier-transform infrared (FT-IR) spectroscopy, and transmission electron microscopy (TEM). The heat-treated protein samples displayed a substantial bathochromic shift (≈15 nm) in the CR's absorption maximum (λmax) and increased ThT and ANS binding. The heat treatment of lens soluble proteins results in the formation of nontoxic, β-sheet rich, non-fibrillar, protein aggregates similar to the structures evident in the insoluble fraction of proteins isolated from the cataractous lens. The data obtained from the present study suggest that the exposure of soluble lens proteins to elevated temperature leads to the formation of non-fibrillar aggregates, establishing the role of amyloid in the heat-induced augmentation of cataracts pathology.
... 29 Although our observations do not inform us on how age alters the interaction between water and lens proteins that affects overall lens optics, we can speculate on two possible mechanisms. In the first, we envisage that the lifelong accumulation of posttranslational modifications to lens proteins [69][70][71] directly impairs their ability to bind water, causing an increase in free water content in the deeper areas of the lens where long-lived crystallin proteins are concentrated. In line with this, several protein modifications including truncation, oxidation, and deamidation have been identified in water-insoluble protein fractions of both clear and cataractous aged lenses. ...
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Purpose: To use magnetic resonance imaging (MRI) to measure age-dependent changes in total and free water in human lenses in vivo. Methods: Sixty-four healthy adults aged 18 to 86 years were recruited, fitted with a 32-channel head receiver coil, and placed in a 3 Tesla clinical MR scanner. Scans of the crystalline lens were obtained using a volumetric interpolated breath-hold examination sequence with dual flip angles, which were corrected for field inhomogeneity post-acquisition using a B1-map obtained using a turbo-FLASH sequence. The spatial distribution and content of corrected total (ρlens) and free (T1) water along the lens optical axis were extracted using custom-written code. Results: Lens total water distribution and content did not change with age (all P > 0.05). In contrast to total water, a gradient in free water content that was highest in the periphery relative to the center was present in lenses across all ages. However, this initially parabolic free water gradient gradually developed an enhanced central plateau, as indicated by increasing profile shape parameter values (anterior: 0.067/y, P = 0.004; posterior: 0.050/y, P = 0.020) and central free water content (1.932 ms/y, P = 0.022) with age. Conclusions: MRI can obtain repeatable total and free water measurements of in vivo human lenses. The observation that the lens steady-state free, but not total, water gradient is abolished with age raises the possibility that alterations in protein-water interactions are an underlying cause of the degradation in lens optics and overall vision observed with aging.
... Crystallins are proteins kept at high concentrations in the lens of the eye, whose aggregation results in cataract disease. [192][193][194] Such assemblies have translational ordering as a function of protein concentration -a sharp change to high local density, like the freezing of solids or the crystallization of salts in solution. The process is generic; many different proteins do it. ...
Article
The protein folding problem was first articulated as question of how order arose from disorder in proteins: How did the various native structures of proteins arise from interatomic driving forces encoded within their amino acid sequences, and how did they fold so fast? These matters have now been largely resolved by theory and statistical mechanics combined with experiments. There are general principles. Chain randomness is overcome by solvation-based codes. And in the needle-in-a-haystack metaphor, native states are found efficiently because protein haystacks (conformational ensembles) are funnel-shaped. Order-disorder theory has now grown to encompass a large swath of protein physical science across biology.
... In this section, we study the aggregation status of human γS-crystallin (HγS), a major structural component of the human eye lens. HγS is noteworthy for its ability to remain in solution at the extremely high concentrations necessary to give the lens its refractive power, while resisting aggregation; indeed, as the lens contains no mechanisms to either remove or replace aggregated HγS, it must remain in solution for one's entire life [50][51][52]. Crystallin aggregation leads to cataract, the leading cause of blindness worldwide [53], and is hence of considerable scientific importance. ...
Article
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Static light scattering is a popular physical chemistry technique that enables calculation of physical attributes such as the radius of gyration and the second virial coefficient for a macromolecule (e.g., a polymer or a protein) in solution. The second virial coefficient is a physical quantity that characterizes the magnitude and sign of pairwise interactions between particles, and hence is related to aggregation propensity, a property of considerable scientific and practical interest. Estimating the second virial coefficient from experimental data is challenging due both to the degree of precision required and the complexity of the error structure involved. In contrast to conventional approaches based on heuristic ordinary least squares estimates, Bayesian inference for the second virial coefficient allows explicit modeling of error processes, incorporation of prior information, and the ability to directly test competing physical models. Here, we introduce a fully Bayesian model for static light scattering experiments on small-particle systems, with joint inference for concentration, index of refraction, oligomer size, and the second virial coefficient. We apply our proposed model to study the aggregation behavior of hen egg-white lysozyme and human γ S-crystallin using in-house experimental data. Based on these observations, we also perform a simulation study on the primary drivers of uncertainty in this family of experiments, showing in particular the potential for improved monitoring and control of concentration to aid inference.
... Aging, which causes age-related changes in the eye lens, is one of the primary causes of cataract development [2][3][4]. Water-soluble crystallin proteins, i.e., α-, β-, and γ-crystallins, account for more than 90% of the lens proteins and function constructively to lens transparency and refractive properties [5][6][7]. α-crystallin accounts for 40% of the lens proteins [6]. The aggregation of crystallin proteins in the lens into the higher molecular weight complexes (HMWCs) [8][9][10][11] accounts for the hazy or foggy vision through the cataractous lens [12][13][14]. ...
Article
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α-crystallin-membrane association increases with age and cataracts, with the primary association site of α-crystallin being phospholipids. However, it is unclear if phospholipids’ acyl chain length and degree of unsaturation influence α-crystallin association. We used the electron paramagnetic resonance approach to investigate the association of α-crystallin with phosphatidylcholine (PC) membranes of different acyl chain lengths and degrees of unsaturation and with and without cholesterol (Chol). The association constant (Ka) of α-crystallin follows the trends, i.e., Ka (14:0–14:0 PC) > Ka (18:0–18:1 PC) > Ka (18:1–18:1 PC) ≈ Ka (16:0–20:4 PC) where the presence of Chol decreases Ka for all membranes. With an increase in α-crystallin concentration, the saturated and monounsaturated membranes rapidly become more immobilized near the headgroup regions than the polyunsaturated membranes. Our results directly correlate the mobility and order near the headgroup regions of the membrane with the Ka, with the less mobile and more ordered membrane having substantially higher Ka. Furthermore, our results show that the hydrophobicity near the headgroup regions of the membrane increases with the α-crystallin association, indicating that the α-crystallin-membrane association forms the hydrophobic barrier to the transport of polar and ionic molecules, supporting the barrier hypothesis in cataract development.
... This experiment was performed twice using a mouse in each experiment. 34,35 . The transparency of the lens depends on maintaining the tertiary structures and solubility of lens crystallin proteins. ...
Article
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Autophagy is a degradation process of cytoplasmic proteins and organelles trafficked to degradation vesicles known as autophagosomes. The conversion of LC3-I to LC3-II is an essential step of autophagosome formation, and FYCO1 is a LC3-binding protein that mediates autophagosome transport. The p62 protein also directly binds to LC3 and is degraded by autophagy. In the present study, we demonstrated that disrupting the FYCO1 gene in mice resulted in cataract formation. LC3 conversion decreased in eyes from FYCO1 knockout mice. Further, FYCO1 interacted with αA- and αB-crystallin, as demonstrated by yeast two-hybrid screening and immunoprecipitation analyses. In eyes from knockout mice, the soluble forms of αA- and αB-crystallin, the lens’s major protein components, decreased. In addition, p62 accumulated in eyes from FYCO1 knockout mice. Collectively, these findings suggested that FYCO1 recruited damaged α-crystallin into autophagosomes to protect lens cells from cataract formation.
... It adjusts the refractive power by changing the diopter so that objects at different distances are imaged on the retina (Fisher, 1977;Hejtmancik and Shiels, 2015). Protein denaturation reduces the transparency of the lens and is caused by the increase of age or the influence of other factors such as trauma, genetics, and metabolic diseases (Bloemendal et al., 2004). This pathological condition is called a cataract. ...
Article
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Advances in cataract surgery have increased the demand for intraocular lens (IOL) materials. At present, the progress of IOL materials mainly contains further improving biocompatibility, providing better visual quality and adjustable ability, reducing surgical incision, as well as dealing with complications such as posterior capsular opacification (PCO) and ophthalmitis. The purpose of this review is to describe the research progress of relevant IOL materials classified according to different clinical purposes. The innovation of IOL materials is often based on the common IOL materials on the market, such as silicon and acrylate. Special properties and functions are obtained by adding extra polymers or surface modification. Most of these studies have not yet been commercialized, which requires a large number of clinical trials. But they provide valuable thoughts for the optimization of the IOL function.
... CRYAA, the responsible gene for encoding the 173 amino acid residues of human αA-crystallin, exists on chromosome 21 while its counterpart protein, αB-crystallin, with 175 amino acids is coded by CRYAB gene on chromosome 11 [6]. αA-crystallin, known as HSPB4, is almost exclusively expressed in the eye lens where its interaction with αB-crystallin in a ratio of 3:1 allocates 40% of the whole protein combination to itself [8][9][10]. However, HSPB5 (αB-crystallin) can be found in a wide range of the body organs and tissues such as heart, brain, muscle, kidney and liver [8,11,12]. ...
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αB-crystallin (heat shock protein β5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein ( CRYAB ) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.
... Delivery of any therapeutic compound to the lens tissue is uniquely challenging, as even the smallest drugs have been shown not to permeate into the nuclear region of the lens (Heikkinen et al., 2019), precisely the region where most cataract occurs (Bloemendal et al., 2004). The nuclear region, the oldest and densest of the lens, is largely sealed off from the supply of glutathione from the lens periphery by the diffusion barrier discussed above. ...
Article
The nuclear region of the lens is metabolically quiescent, but it is far from inert chemically. Without cellular renewal and with decades of environmental exposures, the lens proteome, lipidome, and metabolome change. The lens crystallins have evolved exquisite mechanisms for resisting, slowing, adapting to, and perhaps even harnessing the effects of these cumulative chemical modifications to minimize the amount of light-scattering aggregation in the lens over a lifetime. Redox chemistry is a major factor in these damages and mitigating adaptations, and as such, it is likely to be a key component of any successful therapeutic strategy for preserving or rescuing lens transparency, and perhaps flexibility, during aging. Protein redox chemistry is typically mediated by Cys residues. This review will therefore focus primarily on the Cys-rich γ-crystallins of the human lens, taking care to extend these findings to the β- and α-crystallins where pertinent.
... Also, they function to increase the refractive index while not obstructing light. Second, α-crystallin which is constituted by two polypeptides of αA and αB possesses chaperone-like properties (Bloemendal et al., 2004). In this context, α-crystallin works also to maintain lens transparency by preventing the precipitation of denatured proteins. ...
Article
The BALB.NCT-Cpoxnct is a mutant mouse model for hereditary cataracts. We previously uncovered that the primary cause of the cataracts of BALB.NCT-Cpoxnct is a mutation in the coproporphyrinogen oxidase (Cpox) gene. Because of the mutation, excessive coproporphyrin is accumulated in the BALB.NCT-Cpoxnct lens. In this study, we analyzed the changes in transcriptome and proteins in the lenses of 4- and 12-week-old BALB.NCT-Cpoxnct to further elucidate the molecular etiology of cataracts in this mouse strain. Transcriptome analysis revealed that endoplasmic reticulum (ER) stress was increased in the BALB.NCT-Cpoxnct lens that induced persistent activation of the PERK signaling pathway of the ER stress response. Also, levels of crystallin transcripts and proteins were reduced in the BALB.NCT-Cpoxnct lens. Analysis of proteins disclosed aggregation of crystallins and keratins prior to the manifestation of cataracts in 4-week-old BALB.NCT-Cpoxnct mice. At 12 weeks of age, insoluble crystallins were accumulated in the cataractous BALB.NCT-Cpoxnct lens. Overall, our data suggest the following sequence of events in the BALB.NCT-Cpoxnct lens: accumulated coproporphyrin induces the aggregation of proteins including crystallins. Aggregated proteins increase ER stress that, in turn, leads to the repression of global translation of proteins including crystallins. The decline in the molecular chaperone crystallin aggravates aggregation and insolubilization of proteins. This vicious cycle would eventually lead to cataracts in BALB.NCT-Cpoxnct.
... These changes are especially important for the tissues in which proteins have little or no turnover, 1 for example, the eye lens. 2 With age the eye lens proteins accumulate numerous post-translational modifi cations leading to macroscopic changes in the properties of the whole tissue; namely, an increase of stiffness, yellow coloration, and light scattering. 3 A frequent consequence of these age-related changes is the formation of light scattering domains inside the eye lens, i.e., the development of cataract. Molecular mechanisms of the initiation and progression of this diseas eare still unclear. ...
Article
Under UV-A light and anaerobic conditions, the photodegradation of kynurenic acid and tryptophan significantly decreases with an increase in the viscosity of the medium without significant changes in the composition of the formed products. The recombination products of tryptophan radicals (covalently linked dimeric forms) exhibit the most pronounced decrease. The observed effects are due to restrictions of mobility of the formed radicals and a longer stay in the radical cage as compared to the behavior in aqueous solutions. This leads to an increase in the efficiency of the back electron transfer with the restoration of the initial reagents, which is the main decay pathway for the radicals. The backside of cage effect is an increase in quantities of covalently bound adducts of kynurenic acid and tryptophan. In the case of a living cell, similar changes in the composition of the radical reaction products can be expected.
... With ageing, eye lens proteins including α crystallin undergo various post translational modifications (PTM) most of which lead to their aggregation [10,16,17]. Many of these modifications that have been shown to occur with aging are accelerated under hyperglycemic conditions due to diabetes. ...
Article
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Abstract Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure–function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.
... Highly enriched cholesterol content in membrane is a unique characteristic of the lens tissue compared to other tissues 15 . Previous studies suggested that sterols are required for the proper function of membrane-embedded α-crystallins, which serve as chaperones to facilitate the folding of lens proteins, including βand γ-crystallins 18,75,76 . In addition, cholesterol is essential for maintaining membrane integrity, and loss of cholesterol in the cytoplasmic membrane will likely cause influx of Ca 2+ , which has been shown to activate calpain, a protease that aberrantly cleaves crystallins, thereby leading to crystallin aggregation 77 . ...
Article
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Defective cholesterol biosynthesis in eye lens cells is often associated with cataracts; however, how genes involved in cholesterol biosynthesis are regulated in lens cells remains unclear. Here, we show that Quaking (Qki) is required for the transcriptional activation of genes involved in cholesterol biosynthesis in the eye lens. At the transcriptome level, lens-specific Qki-deficient mice present downregulation of genes associated with the cholesterol biosynthesis pathway, resulting in a significant reduction of total cholesterol level in the eye lens. Mice with Qki depletion in lens epithelium display progressive accumulation of protein aggregates, eventually leading to cataracts. Notably, these defects are attenuated by topical sterol administration. Mechanistically, we demonstrate that Qki enhances cholesterol biosynthesis by recruiting Srebp2 and Pol II in the promoter regions of cholesterol biosynthesis genes. Supporting its function as a transcription co-activator, we show that Qki directly interacts with single-stranded DNA. In conclusion, we propose that Qki-Srebp2–mediated cholesterol biosynthesis is essential for maintaining the cholesterol level that protects lens from cataract development.
... The βB1-crystallin amino acid sequence alignment revealed that the 93rd serine residue remained highly conserved during the evolution of vertebrates ( Figure 1A). Furthermore, previous studies of sequence alignment of the β/γ-crystallins reported that the S93 residue is conserved in the β/γ-crystallin superfamily in many species (Bloemendal et al., 2004;Jin et al., 2020). From the dimeric structure of the βB1-WT, S93 is located in the first Greek key motif and structurally close to the second Greek key motif ( Figure 1B), implying that this site probably plays an important role in maintaining structural stability of βB1-crystallin. ...
Article
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Cataract, opacity of the eye lens, is the leading cause of visual impairment worldwide. The crucial pathogenic factors that cause cataract are misfolding and aggregation of crystallin protein. βB1‐crystallin, which is the most abundant water‐soluble protein in mammalian lens, is essential for lens transparency. A previous study identified the missense mutation βB1‐S93R being responsible for congenital cataract. However, the exact pathogenic mechanism causing cataract remains unclear. The S93 residue, which is located at the first Greek‐key motif of βB1‐crystallin, is highly conserved, and its substitution to Arginine severely impaired hydrogen bonds and structural conformation, which were evaluated via Molecular Dynamic Simulation. The βB1‐S93R was also found to be prone to aggregation in both human cell lines and Escherichia coli. Then, we isolated the βB1‐S93R variant from inclusion bodies by protein renaturation. The βB1-S93R mutation exposed more hydrophobic residues, and the looser structural mutation was prone to aggregation. Furthermore, the S93R mutation reduced the structural stability of βB1-crystallin when incubated at physiological temperature and made it more sensitive to environmental stress, such as UV irradiation or oxidative stress. We also constructed a βB1-S93R cellular model and discovered that βB1-S93R was more sensitive to environmental stress, causing not only aggregate formation but also cellular apoptosis and impaired cellular viability. All of the results indicated that lower solubility and structural stability, sensitivity to environmental stress, vulnerability to aggregation, and impaired cellular viability of βB1-S93R might be involved in cataract development.
Article
The chaperone activity of human αA-crystallin (HAA) against aggregation of human γD-crystallin (HGD) was enhanced by gold nanoparticles (AuNPs). Chaperone activity of HAA was almost doubled in the presence of 5.5 nM gold nanoparticles (AuNPs). To decipher this effect at molecular level, interactions between HAA and AuNPs were studied using fluorescence and circular dichroism spectroscopic techniques. The nanoparticles were synthesized and characterized by using TEM and dynamic light scattering (DLS). TEM and DLS studies revealed that bioconjugation of AuNPs with HAA did not cause any significant change in the size of the nanoparticles. AuNPs had caused static quenching of tryptophan (Trp) fluorescence, which was confirmed through determination of excited state lifetime of Trp residue of HAA in absence and the presence of AuNPs. The association and quenching constant for HAA-AuNPs conjugation were ∼ 10⁹ M⁻¹. Hydrogen bonding and van der Waals interactions were found to be involved in HAA-AuNPs complex. Polarity of Trp microenvironment in HAA was not perturbed by AuNPs as supported by synchronous and three-dimensional fluorescence spectroscopy. Far-UV CD spectra suggested that the secondary structure of HAA was not significantly affected by AuNPs.
Article
Fetal examinations in embryo-fetal developmental (EFD) studies are based on macroscopic and dissecting microscopic evaluations, and histopathology is rarely performed other than to confirm macroscopic findings. Fetal lens examination is therefore generally limited to the presence, size, shape, and color of any abnormality. In a Sprague-Dawley rat EFD study with the fatty acid amide hydrolase (FAAH) inhibitor JNJ-42165279, an unusually high incidence of macroscopic granular foci was noted within the lens of gestation day 21 fetuses across all groups including controls, with higher incidence in the high-dose group. On histological evaluation of the lenses from fetuses with/without gross findings, primary lens fiber hypertrophy (swelling) and degeneration were observed across vehicle- and JNJ-42165279-exposed fetuses. In a follow-up study to investigate the progression or resolution of the fetal lens changes, animals exposed to suprapharmacological doses of JNJ-42165279 in utero had higher incidence of nuclear cataracts as detected via slit-lamp ophthalmic examinations on postnatal days 18 to 21 and 35 to 41. No histologic correlates for these cataracts were identified. We conclude that fetal primary lens fiber hypertrophy and nuclear cataracts at ophthalmology, are common background changes in this rat strain that are exacerbated by in utero exposure to the FAAH inhibitor JNJ-42165279.
Article
Protein misfolding and aggregation are crucial pathogenic factors for cataracts, which are the leading cause of visual impairment worldwide. α-crystallin, as a small molecular chaperone, is involved in preventing protein misfolding and maintaining lens transparency. The chaperone activity of α-crystallin depends on its oligomeric state. Our previous work identified a natural compound, celastrol, which could regulate the oligomeric state of αB-crystallin. In this work, based on the UNcle and SEC analysis, we found that celastrol induced αB-crystallin to form large oligomers. Large oligomer formation enhanced the chaperone activity of αB-crystallin and prevented aggregation of the cataract-causing mutant βA3-G91del. The interactions between αB-crystallin and celastrol were detected by the FRET (Fluorescence Resonance Energy Transfer) technique, and verified by molecular docking. At least 9 binding patterns were recognized, and some binding sites covered the groove structure of αB-crystallin. Interestingly, αB-R120G, a cataract-causing mutation located at the groove structure, and celastrol can decrease the aggregates of αB-R120G. Overall, our results suggested celastrol not only promoted the formation of large αB-crystallin oligomers, which enhanced its chaperone activity, but also bound to the groove structure of its α-crystallin domain to maintain its structural stability. Celastrol might serve as a chemical and pharmacological chaperone for cataract treatment.
Article
Stable isotopes recorded in fish eye lenses are an emerging tool to track dietary shifts coincident with use of diverse habitats over the lifetime of individuals. Eye lenses are metabolically inert, sequentially deposited, archival tissues that can open avenues to chronicle contaminant exposures, diet histories, trophic dynamics and migratory histories of individual fishes. In this study, we demonstrated that trophic histories reconstructed using eye lenses can resolve key uncertainties regarding diet and trophic habitat shifts. Clear Lake Hitch Lavinia exilicauda chi, a threatened cyprinid, inhabits a single lake (Clear Lake, Lake County, California) and utilizes tributary streams for reproduction. Bayesian mixing models applied to δ13C and δ15N recorded in eye lenses uncovered ontogenetic diet shifts that corresponded with shifts in occupation of habitats providing spawning (tributary streams), rearing (littoral lake), and growth (pelagic lake) functions. The reconstruction of size-structured trophic and habitat information can provide vital information needed to manage and conserve imperiled species such as the Clear Lake Hitch.
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In human αB-crystallin or HspB5, the substitution of arginine residue at position 157 with histidine has been reported to cause cardiomyopathy. In this study, the impact of R157H mutation on the structure, stability and functional properties of human αB-crystallin was investigated using a variety of spectroscopic techniques and microscopic analyses. Our spectroscopic analyses revealed that this mutation has a negligible impact on the secondary and tertiary structures of HspB5 but its quaternary structure underwent fundamental changes. Although the chemical stability of the mutant protein remained largely unchanged, the differential scanning calorimetry (DSC) measurement suggested that its thermal stability was reduced. As examined with transmission electron microscopy, αB-crystallin and its mutant indicated a similar tendency for the amyloid fibril formation under thermochemical stress. Dynamic light scattering (DLS) analysis suggested important changes in the quaternary (oligomeric) structures of the mutant protein as compared with the native protein counterpart. Also, the mutant protein indicated an improved chaperone-like activity under in vitro assessment. In a pH-dependent manner, the side chains of arginine and histidine have different capabilities for establishing hydrogen bonds and electrostatic interaction (salt bridge) and this variation may be sufficient to produce the larger changes that ultimately alter the interaction of this protein with other target proteins. Overall, the pathogenic contribution of this mutation in cardiomyopathy can be explained by its role in quaternary structure/stability alteration of the mutated protein.
Article
In the human eye lens the endogenous chromophores of UV-A light (315-400 nm) are able to sensitize radical reactions leading to protein modifications during normal aging and the cataract progression. Kynurenic acid (KNA⁻) is the most photochemically active dye of the human eye lens reported to date with pKa(KNAH2•) 5.5 for its radical form. Cataract is thought to develop under oxidative stress which could be accompanied by acidosis, an acidification of the intracellular environment. Protonation of kynurenyl radicals at mildly acidic conditions may change the outcome of radical reactions leading to additional damage to proteins. In this work we investigated the influence of pH on the degradation of initial reagents and the formation of products in photoinduced radical reactions between KNA⁻ and amino acids tryptophan (Trp) and tyrosine (Tyr) in free states. Our results have shown that pH variation has minor influence on kinetics of reagent decay and accumulation of products in reactions between tyrosyl and kynurenic acid radicals. However in the case of Trp a two-fold decrease of the reagent degradation without visible changes in the composition of formed products was observed with pH decrease from 7 to 3. Time-resolved measurements have shown similar acidification-induced two-fold acceleration of decay of kynurenyl and tryptophanyl radicals via Back Electron Transfer (BET) with the restoration of initial reagents. Experiments with tryptophan derivatives with different pKa values for their radical forms point out the protonation of tryptophanyl radical as the driving force for BET acceleration at low pH. Our results demonstrate that the protonation of kynurenyl radical does not change its reactivity towards amino acids radicals but the total yield of radical photodamage decreases with the protonation of tryptophanyl radicals. It could be expected that radical induced damage to proteins will depend on the pKa of tryptophanyl radicals within a protein globule.
Article
Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. These results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens.
Article
An acidosis, a decrease of pH within a living tissue, may alter yields of radical reactions if participating radicals undergo partial or complete protonation. One of photosensitizers found in the human eye lens, kynurenic acid (KNA⁻), possesses the pKa 5.5 for its radical form that is close to physiological pH 6.89 for a healthy lens. In this work we studied the influence of pH on the mechanisms and products of photoinduced radical reactions between KNA⁻ and amino acids tryptophan (Trp) and tyrosine (Tyr) within a globule of model protein, Hen White Egg Lysozyme (HEWL). Our results show that the rate constant of back electron transfer from kynurenyl to HEWL• radicals with the restoration of initial reagents – the major decay pathway for these radicals – does not change in the pH 3–7. The quantum yield of HEWL degradation is also pH independent, however a shift of pH from 7 to 5 completely changes the outcome of photoinduced damage to HEWL from intermolecular cross-linking to oxygenation. HPLC-MS analysis has shown that four of six Trp and all Tyr residues of HEWL are modified in different extents at all pH, but the lowering of pH from 7 to 5 significantly changes the direction of main photodamage from Trp⁶² to Trp¹⁰⁸ located at the entrance and bottom of enzymatic center, respectively. A decrease of intermolecular cross-links via Trp⁶² is followed by an increase in quantities of intramolecular cross-links Tyr²⁰-Tyr²³ and Tyr²³-Tyr⁵³. The obtained results point out the competence of cross-linking and oxygenation reactions for Trp and Tyr radicals within a protein globule and significant increase of oxygenation to the total damage of protein in the case of cross-linking deceleration by coulombic repulsion of positively charged protein globules.
Article
Congenital cataracts, which are genetically heterogeneous eye disorders, lead to visual impairment in childhood. In our previous study, we identified a novel mutation in exon 4 of the CRYBA1/BA3 gene, which resulted in the deletion of a highly conserved glycine at codon 91 (G91del) and perinuclear zonular cataract. The G91del variant is one of the most frequent pathogenic mutations in CRYBA1/BA3; however, its pathogenic mechanism remains unclear. In this study, we purified βA3-crystallin and the βA3-G91del variant. βA3-G91del was prone to proteolysis and exhibited very low solubility and low structural stability. Next, we constructed a CRYBA1/BA3 mutant cell model and observed that G91del mutant proteins were more sensitive to environmental stress and prone to form aggregates. Size-exclusion chromatography and molecular dynamics simulation showed that the G91del mutation impaired the ability of βA3 to form homo-oligomers. In addition, the protein folding process of βA3-G91del was complicated and showed more intermediate states, resulting in amyloid fiber aggregation and induction of cellular apoptosis. Finally, we investigated intervention strategies for congenital cataract caused by the CRYBA1/A3-G91del variant. The addition of lanosterol reversed the negative effects of the G91del mutation under external stress. This study may help explore potential treatment strategies for related cataracts.
Article
Background/aims Congenital cataracts, which are genetically heterogeneous eye disorders, result in visual loss in childhood around the world. CRYBA1/BA3 serves as an abundant structural protein in the lens, and forms homomers and heteromers to maintain lens transparency. In previous study, we identified a common cataract-causing mutation, βA3-glycine at codon 91 (G91del) (c.271–273delGAG), which deleted a highly conserved G91del and led to perinuclear zonular cataract. In this study, we aimed to explore the underlying pathogenic mechanism of G91del mutation. Methods Protein purification, size-exclusion chromatography, spectroscopy and molecular dynamics simulation assays were used to investigate the effects on the heteromers formation and the protein structural properties of βA3-crystallin caused by G91del mutation. Intracellular βA3-G91del overexpression, MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) and cell apoptosis were used to investigate the cellular functions of βA3-G91del. Results βA3-crystallin and βB2-crystallin could form heteromers, which have much more stable structures than βA3 homomers. Interestingly, βA3/βB2 heteromers improved their resistance against the thermal stress and the guanidine hydrochloride treatment. However, the pathogenic mutation βA3-G91del destroyed the interaction with βB2, and thereby decreased its structural stability as well as the resistance of thermal or chemical stress. What’s more, the βA3-G91del mutation induced cell apoptosis and escaped from the protection of βB2-crystallin. Conclusions βA3/βB2 heteromers play an indispensable role in maintaining lens transparency, while the βA3-G91del mutation destabilises heteromers formation with βB2-crystallin, impairs cellular viability and induces cellular apoptosis. These all might contribute to cataract development.
Article
Kynurenic acid (KNA) and 4-hydroxyquinoline (4HQN) are photochemically active products of tryptophan catabolism that readily react with tryptophan (Trp) and tyrosine (Tyr) after optical excitation. Recently, transient absorption experiments have shown that at neutral pH Trp reacts with triplet KNA via proton-coupled electron transfer (PCET), and not via electron transfer (ET) as it was suggested before. PCET includes the stepwise transition of both electrons and protons from Trp to triplet KNA. In this work, we confirmed that PCET is the reaction mechanism by the alternative method of time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP). Further studies by TR-CIDNP revealed hydrogen transfer as the mechanism of the reaction between triplet KNA and Tyr in neutral solutions and a transition of both PCET and H-transfer mechanisms to ET under acidic conditions. 4HQN, being the chromophoric core of KNA, exhibits different spectral and photophysical properties from KNA but employs the same mechanisms for the reactions of its triplet state with Trp and Tyr at neutral and acidic pH.
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The G98R mutation in αA-crystallin is associated with presenile cataract development in humans. Previous studies have indicated that mutant proteins altered structure, decreased stability, increased oligomeric size, loss of chaperone-like activity, and susceptibility to proteolysis could be contributing factors to cataract formation. To evaluate the effect of substrate protein interactions with the mutant protein on cataract formation, we have performed chaperone assays with alcohol dehydrogenase (ADH), citrate synthase (CS), and βB2-crystallin (βB2), and analyzed the reaction mixtures by multi-angle light scattering (MALS) analysis. It appears that αAG98R protein initially gets stabilized upon interaction with substrate proteins. Analysis of the chaperone-client protein complexes revealed that wild-type αA-crystallin interacts with substrate proteins to form compact complexes leading to a slight increase in oligomeric mass, whereas αAG98R forms less compact and high molecular weight complexes with the substrate, and the resulting complexes continue to increase in size over time. As a result, the soluble complexes formed initially by the mutant protein begin to scatter light and precipitate. We found that the stability and chaperone activity of the αAG98R can be improved by modifying the protein with low concentrations (50 µM) of methylglyoxal (MGO). Incubation of αAG98R protein (1 mg/ml) under aseptic conditions for 30 days at 37°C resulted in precipitation of the mutant protein. In contrast, mutant protein incubations carried out with 50 µM MGO remained soluble and transparent. SDS-PAGE analysis showed gradual autolysis of the mutant protein in the absence of MGO. The average molar mass of the mutant protein oligomers changed from 7,258 ± 12 kDa to 3,950 ± 08 kDa within 60 min of incubation with MGO. There was no further significant change in the molar mass of mutant protein when tested on day 7 of MGO treatment. Our data suggest that the initial stabilization of αAG98R by substrate proteins could delay congenital cataracts’ appearance, and the uncontrolled long-term interaction amongst mutant subunits and substrate proteins could be the rationale behind presenile cataracts formation. The results also demonstrate the potential benefit of low concentrations of MGO in stabilizing mutant chaperone protein(s).
Article
Cataract is the most common pathogenic ophthalmic disease leading to blindness in children worldwide. Genetic disorder is the leading cause of congenital cataract, among which crystallin mutations have a high incidence. There are few reports on γA-crystallin, one critical member of crystallin superfamilies. In this study, we identified a novel pathogenic mutation (Ile82Met) in γA-crystallin from a three-generation Chinese family with cataract, and investigated the potential molecular mechanism in detail. To elucidate the pathogenic mechanism of I82M mutant, spectroscopic and solubility experiments were performed to determine the difference between the purified γA-crystallin wild type (WT) and I82M mutant under both physiological conditions and environmental stresses (UV irradiation, thermal denaturation or chemical denaturation). The I82M mutant did not affect the secondary/tertiary structure of monomeric γA-crystallin under physiological status, but decreased protein stability and increased aggregatory potency under the stressful treatment. Surprisingly, the chemical denaturation caused I82M to switch from the two-state unfolding of γA-crystallin to three-state unfolding involving an unfolding intermediate. This study expands the genetic variation map of cataract, and provides novel insights into the pathomechanism, in particular, filling in a gap in the understanding of γA-crystallin mutants causing cataract.
Article
Thin films prepared from protein sources are biodegradable, biocompatible and find application as biomaterials for slow-release drug carriers for skin treatment in burns, ulcers or infected wounds. Cataract eye lenses discarded after surgery have a significant protein content. Films have been prepared from the discarded cataract lens emulsions obtained after surgery. Sorbitol was used as a plasticizer to facilitate film formation that exhibited high water solubility. To enhance the mechanical properties and reduce the solubility of the films, cross-linking of the polypeptide chains was initiated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in presence of NHS (N-hydroxysuccinimide). As the effects of the cross-linker and plasticizer incorporate conflicting properties, an optimization was necessary. The plasticizer imparts flexibility to the film, while the cross-linker decreases solubility but increases hardness that imparts brittleness. A reduction in water solubility of the film for practical applicability was required in such a way that the flexibility was not compromised. The concentration of the cross-linker and the duration of cross-linking were varied to optimize the mechanical properties of the films. The films were characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and optical microscopy. Physical properties like thickness and total soluble matter were measured. The mechanical properties of the films were evaluated by performing nano-indentation and nano-tribology (NINT) experiments.
Article
Congenital cataract, a common disease with lens opacification, causes blindness in the newborn worldwide and is mainly caused by abnormal aggregation of crystallin. As the main structural protein in the mammalian lens, βB1-crystallin has an important role in the maintenance of lens transparency. Recently, the L116P mutation in βB1-CRY was found in a Chinese family with congenital nuclear cataracts, while its underlying pathogenic mechanism remains unclear. In the current study, the βB1 wild-type protein was purified, and the mutated form, βB1-L116P, was examined for examining the effect on structural stability and susceptibility against environmental stresses. Our results reveal low solubility and structural stability of βB1-L116P at physiological temperature, which markedly impaired the protein structure and the oligomerization of βB1-crystallin. Under guanidine hydrochloride-induced denaturing conditions, βB1-L116P mutation perturbed the protein unfolding process, making it prone to amyloid fibrils aggregation. More importantly, the L116P mutation increased susceptibility of βB1-crystallin against UV radiation. βB1-L116P overexpression led to the formation of more serious intracellular aggresomes under UV radiation or oxidative stress. Furthermore, the βB1-L116P mutation increased the sensitivity to the proteolysis process. These results indicate that the low structural stability, susceptibility to amyloid fibrils aggregation, and protease degradation of βB1-L116P may contribute to cataract development and associated symptoms.
Article
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Purpose: Crystallin protein mutations are associated with congenital cataract (CC), and several disease-causing mutations in the CRYGC gene have been identified. We present the location of a new mutation in CRYGC in members of a Chinese family who presented with CCs with or without microcornea. Design: Observational study. Participants: A Chinese family diagnosed with autosomal dominant (AD) CCs with or without microphthalmia. Methods: Because this was an observational study, it was not registered as a clinical trial. The proband and her 2 children were diagnosed with AD CCs and microcornea and were recruited for the study. Participants underwent complete ophthalmological examinations, and blood samples were used for genomic extraction. Main Outcome Measures: We detected 1 disease-associated variant using Exomiser analysis by matching the proband’s phenotype and the inheritance pattern. The variant was determined to be pathogenic according to American College of Medical Genetics and Genomics (ACMG) guidelines. Results: We detected 1 disease-associated variant using Exomiser analysis by matching the proband’s phenotype and the inheritance pattern. The variant was determined to be pathogenic according to the American College of Medical Genetics and Genomics guidelines. Next-generation sequencing was verified using Sanger sequencing, and we confirmed that the proband and her children carried the same mutation. We identified the heterozygous variant c.389_390insGCTG (p.C130fs), which includes a frameshift mutation. The residues in p.C130fs are all highly conserved across species. This disease-causing frameshift mutation in the CRYGC gene is not currently present in the ClinVar database. Conclusions: Our findings expand the repertoire of known mutations in the CRYGC gene that cause CCs and provide new insights into the etiology and molecular diagnosis of CCs; however, the molecular mechanism of this mutation warrants further investigation.
Article
Crystallin aggregation in the eye lens is one of the leading causes of cataract formation. The increase in the human γD-crystallin (HγDC) aggregation propensity has been associated with the oligomerization of its partially folded and fully unfolded structure. A recent study demonstrated that the binding of flavonoid morin (MOR) to HγDC inhibits the fibrillation of this protein. In this work, we carry out an exhaustive search for the possible binding site of MOR on HγDC by combining an ensemble docking approach with the Wrap ‘N' Shake protocol. In agreement with previous results, we found a potential MOR-binding site in the cleft formed between the N-terminal and C-terminal domains of HγDC. MOR preference for the cleft residues was observed even with the interface-opened intermediate conformers of HγDC. In addition, metadynamics simulations were carried out to corroborate the stabilizing activity of MOR on HγDC structure and to identify the structural regions implicated during the unfolding inhibition. Overall, this study provides relevant insights into the identification of new HγDC aggregation inhibitors.
Chapter
From the perspectives of molecular embryology, physiology, metabolism, and biophysics, there is much to learn from the lens about vision health and aging. The unique coordination of biological events that must synchronize the organization and functions of lens cells in the generation of symmetric, image-forming optics is remarkable. Biological optics in all species conform to the same set of physical laws to form effective, transparent, and refractive optics even in the eyes that have very diverse structures and simple nervous systems. Lens transparency is based on delicate interactions between cellular constituents that need to remain stable for a lifetime. This chapter is an introduction to basic biology of lens transparency in relation to both development and aging.
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Purpose To quantify the effect of cataract on colour vision as measured by the low vision Cambridge Colour Test (lvCCT). A secondary aim of our study was to understand whether different types and severities of cataract have different effects on colour vision. Design Cohort study Subjects Patients aged 18 - 95 years attending for routine cataract surgery at the Oxford Eye Hospital. Methods The lvCCT was performed to measure colour sensitivity in both eyes in a cohort of patients undergoing routine cataract surgery both pre-operatively and post-operatively. The crystalline lens was examined and graded according to the Lens Opacities Classification System III (LOCS III) to determine the type and severity of cataract. Measures of repeatability were performed for the data to explore test-retest bias using Bland-Altman analysis. The Wilcoxon signed-rank test was performed to assess the effect of cataract on colour vision by comparing control and surgical test measurements. Three multiple linear regressions were performed to relate cataract grading or severity to colour vision measurements. Main outcome measures Colour discrimination along each of the protan, deutan, and tritan confusion lines. Results The Wilcoxon signed-rank test showed a statistically significant difference in both the protan (P=0.024) and tritan (P=0.020) axes upon comparison of control and surgical test measurements. As severity of cataract increased colour vision sensitivity was more greatly affected, and nuclear sclerotic cataract had the most profound effect upon colour vision sensitivity in the lvCCT. The linear regression models though showed these observations did not reach statistical significance. Conclusions Cataract surgery has a statistically significant effect upon colour vision in both the protan and tritan axes. The effects of specific subtypes of cataract and different severities could not be elucidated due to the high prevalence of patients presenting with mixed cataract. LvCCT colour sensitivity measurements are regularly used as outcome measures in clinical gene therapy trials involving vitreoretinal surgery, and vitrectomy accelerates cataract formation. Therefore, it is important to quantify the effect of cataract upon colour vision measurements so it may be taken into account when used as an outcome measure in clinical trials. We were unable to derive a precise correction factor for cataract on color vision measurements.
Article
* The article is published as a part of the Special Issue "Protein Misfolding and Aggregation in Cataract Disorders" (Vol. 87, No. 2). ** To whom correspondence should be addressed. Cataract is a major cause of blindness. Due to the lack of protein turnover, lens proteins accumulate age-related and environmental modifications that alter their native conformation, leading to the formation of aggregation-prone intermediates, as well as insoluble and light-scattering aggregates, thus compromising lens transparency. The lens protein, α-crystallin, is a molecular chaperone that prevents protein aggregation, thereby maintaining lens transparency. However, mutations or post-translational modifications, such as oxidation, deamidation, truncation and crosslinking, can render α-crystallins ineffective and lead to the disease exacerbation. Here, we describe such mutations and alterations, as well as their consequences. Age-related modifications in α-crystallins affect their structure, oligomerization, and chaperone function. Mutations in α-crystallins can lead to the aggregation/intracellular inclusions attributable to the perturbation of structure and oligomeric assembly and resulting in the rearrangement of aggregation-prone regions. Such rearrangements can lead to the exposure of hitherto buried aggregation-prone regions, thereby populating aggregation-prone state(s) and facilitating amorphous/amyloid aggregation and/or inappropriate interactions with cellular components. Investigations of the mutation-induced changes in the structure, oligomer assembly, aggregation mechanisms, and interactomes of α-crystallins will be useful in fighting protein aggregation-related diseases.
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The chaperone activity and biophysical properties of recombinant human αA- and αB-crystallins were studied by light scattering and spectroscopic methods. While the chaperone function of αA-crystallin markedly improves with an increase in temperature, the activity of αB homopolymer appears to change very little upon heating. Compared with αB-crystallin, the αA-homopolymer is markedly less active at low temperatures, but becomes a more active species at high temperatures. At physiologically relevant temperatures, the αB homopolymer appears to be modestly (two times or less) more potent chaperone than αA homopolymer. In contrast to very similar thermotropic changes in the secondary structure of both homopolymers, αA- and αB-crystallins markedly differ with respect to the temperature-dependent surface hydrophobicity profiles. Upon heating, αA-crystallin undergoes a conformational transition resulting in the exposure of additional hydrophobic sites, whereas no such transition occurs for αB-crystallin. The correlation between temperature-dependent changes in the chaperone activity and hydrophobicity properties of the individual homopolymers supports the view that the chaperone activity of α-crystallin is dependent on the presence of surface-exposed hydrophobic patches. However, the present data also show that the surface hydrophobicity is not the sole determinant of the chaperone function of α-crystallin.
Article
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P-Crystallins are structural lens proteins with a conserved two-domain structure and variable N-and C-terminal extensions. These extensions are assumed to be involved in quaternary interactions within the p*-crystallin oligomers or with other lens proteins. Therefore, the production of PA3-and PAl-crystallin from the single PA3/A1 mRNA by dual translation initiation is of interest. These crystallins are identical, except that PAl has a much shorter N-terminal extension than PA3. This rare mechanism has been conserved for over 250 million years during the evolution of the (3A3/A1 gene, suggesting that the generation of different N-terminal extensions confers a selective advantage. We therefore compared the stability and association behaviour of recombinant pA3-and pAl-crystallin. Both proteins are equally stable in urea-and pH-induced denaturation experiments. Gel filtration and analytical ultracentrifuga-tion established that PA3 and PA1 both form homodimers. In the water-soluble proteins of bovine lens, PA3 and PA1 are present in the same molecular weight fractions, indicating that they oligomerize equally with other P-crystallins. 'H-NMR spectroscopy showed that residues Metl to Asn22 of the N-terminal extension of PA3 have great flexibility and are solvent exposed, excluding them from protein interactions in the homodimer. These results indicate that the different N-terminal extensions of pA3 and PA1 do not affect their homo-or heteromeric interactions.
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Lens α-crystallin subunits αA and αB are differentially expressed and have a 3-to-1 ratio in most mammalian lenses by intermolecular exchange. The biological significance of this composition and the mechanism of exchange are not clear. Preparations of human recombinant αA- and αB-crystallins provide a good system in which to study this phenomenon. Both recombinant αA- and αB-crystallins are folded and aggregated to the size of the native α-crystallin. During incubation together, they undergo an intermolecular exchange as shown by native isoelectric focusing. Circular dichroism measurements indicate that the protein with a 3-to-1 ratio of αA- and αB-crystallins has the same secondary structure but somewhat different tertiary structures after exchange: the near-UV CD increases after exchange. The resulting hybrid aggregate is more stable than the individual homogeneous aggregates: at 62 °C, αB-crystallin is more susceptible to aggregation and displays a greater light scattering than αA-crystallin. This heat-induced aggregation of αB-crystallin, however, was suppressed by intermolecular exchange with αA-crystallin. These phenomena are also observed by fast performance liquid chromatography gel filtration patterns. The protein structure of αB-crystallin is stabilized by intermolecular exchange with αA-crystallin.
Article
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A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as αA, αB, βA1, βA3, βA4, βB1, βB2, βB3, γS, γC, and γD-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human βA1, βA3, βA4, and βB3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of βA3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.
This book gives a comprehensive survey of the current knowledge of the expression and function of small stress proteins (sHsps) in different organisms, from prokaryotes to humans. It provides an overview of the diversity and complex evolutionary history of sHsps and describes their function and expression in different eukaryote models. Additional chapters discuss the role of sHsps in pathological conditions and gene therapy approaches towards a control of sHsp expression levels.
Chapter
The non-covalent physical-chemical interaction forces between polar polymers, biopolymers and/or various inert polar surfaces, particles or cells are of three fundamentally different classes: (1) apolar, electrodynamic, or Lifshitz-van der Waals (LW) interactions (Chaudhury, 1984); (2) polar, or Lewis acid-base (AB) interactions, which in aqueous media are mainly hydrogen-bonding interactions (van Oss et al., 1988a); (3) electrostatic (EL), or Coulombic interactions (van Oss et al., 1988a). In addition, thermal or Brownian movement (BR) interactions may play a role which, while quantitatively fairly unimportant in interactions between large molecules and particles, may not always be negligible when smaller molecules are involved. The first three interactions can be either attractive or repulsive, independent of the sign of the other two varieties, in any particular case (van Oss et al., 1988a); BR interactions are always considered repulsive. In liquids, LW interactions can only be repulsive when acting between two different solutes or particles; AB and EL interactions can be repulsive even when acting between identical solutes or particles.
Chapter
The molecular basis of eye lens transparency has been analyzed recently using osmotic pressure and solution X-ray scattering experiments, and the fundamental contribution of protein-protein interactions to the process was described (Vérétout et al., 1989). The aim of this chapter is to present the role of protein-protein interactions in a more pictorial way.
Article
Purpose: To determine which component of lens alpha-crystallin is responsible for heat-induced transition, conformational change and high molecular weight (HMW) aggregation. Methods: Recombinant alpha A- and alpha B-crystallins were used. Temperature dependent changes were probed by Trp fluorescence and circular dichroism (CD) measurements. HMW aggregates were induced by heating at 62 degrees C for 1-2 h and then cooling to room temperature. The nature of HMW aggregation was studied with fluorescent probes, 4,4'-dianilino-1,1'-binaphthalene-5,5'-disulfonic acid (bis-ANS) and thioflavin T (ThT). Results: CD and Trp fluorescence revealed that alpha B-crystallin was more susceptible than alpha A-crystallin to heat-induced conformational change and aggregation. At temperatures greater than 70 degrees C, alpha B-crystallin precipitated but alpha A-crystallin remained soluble. Both bis-ANS and ThT probes displayed increased fluorescence intensity with HMW aggregation, but the increase for bis-ANS was greater with alpha B-crystallin than with alpha A-crystallin, while the reverse was true for ThT. Conclusions: These results indicate that alpha B-crystallin is more susceptible than alpha A-crystallin to heat-induced conformational change and aggregation and are consistent with the notion that alpha A- and alpha B-crystallins have different biochemical and biophysical properties in spite of their high degree of homology.
Chapter
The first step toward unravelling the mechanism of an enzyme-catalyzed reaction is to specify the mechanisms available for the reaction concerned. Many of these reactions are not observed when the relevant groups are allowed to come together in bimolecular processes in aqueous solution. For mechanistic work involving intermolecular reactions, it is necessary to use activated substrates. An attractive alternative is to study intramolecular reactions. These are generally faster than the corresponding intermolecular processes. Thus, groups like carboxyl and imidazole are involved at the active sites of many enzymes hydrolyzing aliphatic esters and amides. Therefore, mechanistic studies of intramolecular reactions play an important part in elucidating the chemistry of the groups involved in enzyme catalysis and in defining the mechanisms available for particular reactions.
Article
Evidence of betaA2-crystallin expression has been detected in human lenses. The protein, which co-elutes with betaA1/A3 from reversed phase HPLC separation of beta(L)-crystallins, accounts for 1-2% of the lens crystallins. Its molecular mass, M, 22 006, is consistent with the cDNA deduced sequence with addition of acetylation at the N-terminal serine residue. Approximately 20% of the protein is phosphorylated at Ser30.
Article
Biological macromolecules in solution interact with each other through medium-range (from a few Å to a few nm) interaction potentials. These potentials control the macromolecular distribution in solution, the macromolecular phase diagram and the crystallization process. We have previously shown that small angle X-ray scattering (SAXS) is a convenient tool to characterize the resulting potential, either attractive or repulsive, and to follow the changes induced by the crystallizing agents. In the present paper SAXS and simulation methods derived from statistical mechanics are coupled to determine the best fit potentials from the comparison of experimental and theoretical intensity curves. The currently used models in the colloid field are derived from the DLVO (Derjaguin, Landau, Verwey, Overbeek) potential where three types of interactions play a major role: hard sphere and electrostatic are repulsive, van der Waals are attractive. A combination of a short-range attractive potential and a coulombic repulsive indeed correctly accounts at low ionic strength for the phase diagram as a function of pH and salt concentration. The origin of the ion specificities at high ionic strength associated with the so-called "Hofmeister series" remain, however, unclear. The whole of the data demonstrates that the colloidal approach may be applied with success to protein crystallization.
Article
During hominoid evolution the γ-crystallins of the lens have decreased in quantity as well as complexity, a change correlated with an increased water content of the lens. To trace the molecular basis for the decrease in γ-crystallin gene expression, we have characterized the structure and expression of the human γ-crystallin gene family. We show that the human γ-crystallin gene family consists of six complete genes (γA, γB, γC, γD, ψγE and ψγF) and one second exon fragment, the γG gene. Model experiments showed that, although the γG sequence is bordered by consensus splice sites, it is most likely transcriptionally inactive in the lens.In the human embryonic lens the γC and γD genes accounted for 81 % of the γ-crystallin transcripts, the γA gene contributed 14% and the γB gene only 5%. The composition of the γ-crystallin mRNA pool changed only after birth, with the γD transcript as the only detectable transcript at ten years of age.The relative activities of the γA, γC and γD promoters in a transient expression system were in agreement with the ratio of their in vivo RNA levels, suggesting that the difference in accumulation of these transcripts is due to differences in the rate of transcription. The γB promoter was much more active than expected and had lost its tissue-specificity. Model experiments showed that the low yield of the γB transcript is due to post-transcriptional processes, most likely RNA instability mediated by third exon sequences.Together with previous data, our results show that the decrease in expression of the γ-crystallin genes in the human lens is the consequence of gene loss (γG), inactivation of coding sequences (ψγE and ψγF), decrease in rate of transcription (γA), increase in rate of RNA turn-over (γB) and a delay in the onset of transcription during development.
Article
Three classes of β-crystallin, which have previously been named β1, β2 and β3-crystallins, have been isolated from normal human lenses by gel filtration on Sephadex G-200. They have been compared to each other in terms of physico-chemical, immunochemical and conformational parameters. All three β-crystallins have similar secondary and tertiary structures as seen by ultraviolet circular dichroism. They appear to be very closely related immunochemically. All three are quite heterogeneous but have similar isoelectric point ranges which are more acidic than that of bovine β-crystallin. β1, β2 and β3 appear to be composed in large part of identical subunits, but there are differences in the distributions of several minor polypeptides as visualized by electrophoresis in the presence of sodium dodecyl sulfate. The major distinction among the three β-crystallin classes is in molecular weight.All three classes contain a minor polypeptide or group of polypeptides of approximately 43 000 daltons which have not been seen in the β-crystallin from other species. The 43 000 dalton polypeptides were isolated from each β-crystallin class. Preliminary analysis of these preparations by isoelectric focusing and immunodiffusion was done.
Article
Eyes are the preeminent source of sensory information for the brain in most species, and many features of eyes reflect evolutionary solutions to particular selective pressures, both from the nonbiological environment and from other animals. As a result, the evolution of eyes, among all the sense organs, has attracted considerable attention from scientists. Paired eyes in the three major phyla, vertebrates, arthropods and mollusks, have long been considered to be classic examples of evolutionary convergence. At the macroscopic level, this must be true since they arise from different tissues and have evolved radically different solutions to the common problem of collecting and focusing light. However, opsin, the light-absorbing receptor protein, has a significant amount of shared DNA sequence homology across the phyla, and recently it has been discovered that some part of ocular development in different phyla is coordinated by a homologous gene, Pax-6. So, although eyes from diverse phyla are clearly not homologous, neither can they be viewed as resulting solely from convergence. Instead, this shows that homology at the molecular level of organization does not predict homology at the organ or organismic level. The presence of homologous constituent molecules in nonhomologous structures reminds us that molecules are not eyes.
Article
The binary liquid phase separation of aqueous solutions of Γ-crystallins is utilized to gain insight into the microscopic interactions between these proteins. The interactions are modeled by a square-well potential with reduced range λ and depth &egr;. A comparison is made between the experimentally determined phase diagram and the results of a modified Monte Carlo procedure which combines simulations with analytic techniques. The simplicity and economy of the procedure make it practical to investigate the effect on the phase diagram of an essentially continuous variation of λ in the domain 1.05≤λ≤2.40. The coexistence curves are calculated and are in good agreement with the information available from previous standard Monte Carlo simulations conducted at a few specific values of λ. Analysis of the experimental data for the critical volume fractions of the Γ-crystallins permits the determination of the actual range of interaction appropriate for these proteins. A comparison of the experimental and calculated widths of the coexistence curves suggests a significant contribution from anisotropy in the real interaction potential of the Γ-crystallins. The dependence of the critical volume fraction ϕc and the reduced critical energy &egr;ˆc upon the reduced range λ is also analyzed in the context of two ‘‘limiting’’ cases; mean field theory (λ↠∞) and the Baxter adhesive sphere model (λ↠1). Mean field theory fails to describe both the value of ϕc and the width of the coexistence curve of the Γ-crystallins. This is consistent with the observation that mean field is no longer applicable when λ≤1.65. In the opposite case, λ↠1, the critical parameters are obtained for ranges much shorter than those investigated in the literature. This allows a reliable determination of the critical volume fraction in the adhesive sphere limit, ϕc(λ=1)=0.266±0.009.
Article
The interaction between two spherical colloids immersed in an electrolyte of screening constant κ is studied using the recent charge regulation primitive model and the hypernetted chain integral equation. The charge of the particles is not fixed a priori but results from the adsorption of positive and/or negative ions onto the colloidal surfaces. In the case of symmetrical adsorption, the model exhibits a long-range attraction between the globally neutral colloids. At large distance, the effective colloid–colloid potential behaves as −exp(−2κr)/r2 for small colloids where r is the center to center interparticles distance and as −exp(−2κh)/h for large colloids where h is the surface to surface distance. For nonsymmetrical cases, such an attraction adds to the usual screened Coulombic repulsion between the globally charged colloids. The numerical results and the physical origin of the attraction are explained in terms of elementary diagrams. The attraction arises from the ion exchange between the adsorbed layers and the bulk and from the non mean-field ion–ion correlations.
Article
New results obtained from a two-dimensional sequence analysis of the small heat shock protein (shsp) family are described. It is confirmed that the conserved C-terminal α-crystallin domain is essentially made of β-strands, most probably two groups of β-strands separated by a large loop. A direct correspondence between the putative β-strands that have been identified in shsps and the seven β-strands of a classical immunoglobulin-like fold is proposed. The hypothesis that the shsp family could belong to the immunoglobulin superfamily (IgSF) is consistent with the ubiquitous distribution and the multifunctional properties of the crystallins that are now emerging.
Article
The chapter presents a study on the chaperone-like properties of lens α-crystalline. The most common source for lens α-crystallin has been cow or calf lens. The chapter presents a protocol for preparing calf or cow lens α-crystallin as well as the preparation and purification of recombinant human αB-crystallin. Several assays for the chaperone-like properties of α-crystallin are presented. α-crystallin is one of the abundant structural proteins of the vertebrate eye lens, where it can account for about 40% of the total soluble mass. The a-crystallin family consists of two genes, αA and αB. αB-crystallin has now been found in numerous tissues of the body, such as heart, skeletal muscle, brain, lung, skin, and kidney. αA-crystallin is much less abundant outside the eye lens. αB-crystallin is overexpressed in many degenerative diseases. The biochemical, biophysical, gene regulation, expression, and evolutionary properties of α-crystallin have been studied extensively. Recombinant α-crystallin has properties similar to those of native α-crystallin isolated from eye lens.
Article
This chapter provides an overview of the results and concepts that have been put forward in the field of in nitro misassembly (coagulation, aggregation). Considering the posttranslational fate of a protein molecule, polypeptide chains that fail to reach the native state are commonly discarded by appropriate cellular mechanisms, either protein turnover or deposition as aggregates. Over the past 30 years, protein chemists have not shown great interest in these side reactions. They merely tried to avoid them because what fully absorbed their curiosity was the structure function relationship of proteins, and function was exactly what aggregates or precipitates were lacking. Lately, the focus has changed for a number of reasons. This chapter summarizes experimental approaches to a deeper understanding of the physicochemical basis of protein misfolding in vitro and with the solution of the protein folding problem still ahead, one may easily predict that another generation of biochemists will be engaged in trying to find the solution to the misfolding problem, with its physical, cell biological, and medical implications.
Article
The goal of this chapter is to clarify the diversity within the heat shock proteins (sHsps) family and to describe evidence indicating that sHsps have many different substrates and affect a wide range of cellular functions. The diversity of sHsp structure and expression patterns is immense, and their activities in vivo may involve multiple mechanisms. The sHsps and the structurally related vertebrate eye lens α-crystallins are the poor cousins in the family of molecular chaperones, and remain the least understood both structurally and functionally. The chaperone model for sHsp function provides a basic framework to explain the many proposed sHsp/protein interactions and potential functions. The diversity of the sHsp family, however, indicates that care must be taken in generalizing biochemical properties and activities across different family members. Nonetheless, the chapter has a firmer structural foundation on which to design future experiments to build a biochemical mechanism of action.
Article
β-Crystallin from calf lens cortex was fractionated in three different aggregates of increasing size: βL2, βL1 and βH, of which the subunit composition was revealed by 2-dimensional gel electrophoresis. While βL2 mainly consists of βBP, (the major polypeptide chain in all three aggregates), βL1 is characterized by the addition of a neutral and two acidic chains, and βH contains moreover two basic chains. Translation of calf lens polyribosomes in a reticulocyte cell-free system allowed the identification of six β-crystallin subunits as primary gene products. The distribution of these newly synthesized polypeptides over the three aggregates was established after gel filtration in the presence of carrier lens proteins. The aggregation behavior of the β-crystallin chains was studied by dissociation/reassociation experiments. The three separate aggregates could be reversibly dissociated. Reassociation of basic, neutral and acidic polypeptides, isolated by ion-exchange chromatography of β-crystallin, produced a βH-like aggregate. The neutral and acidic polypeptides reassociated into a βL1-like aggregate, while the neutral polypeptides gave dimers like βL2. A βH-like aggregate could also be obtained by reaggregation of βL2 with the acidic and basic chains of βH. On the basis of these results a preliminary model for the formation of β-crystallin aggregates is discussed.
Article
α-Crystallin, a major lens protein of ≈ 800 kDa with subunits of ≈ 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced aggregation. Here it is demonstrated that α-crystallin can bind to partially denatured enzymes at 42–43 °C and prevent their irreversible aggregation, but cannot prevent loss of enzyme activity. However, the α-crystallin-bound enzymes regain activity on interaction with other chaperones. The data indicate that the re-activated enzymes are no longer associated with the α-crystallin, and ATP is required for re-activation. When inactive luciferase bound to α-crystallin was treated with reticulocyte lysate, a rich source of chaperones, up to 60% of the original luciferase activity could be recovered. Somewhat less re-activation was observed when the α-crystallin-bound enzyme was treated with heat-shock protein (HSP)70, HSP40, HSP60 and an ATP-generating system. Similar results were also obtained with citrate synthase. The overall results suggest that α-crystallin acts to stabilize denaturing proteins so that they can later be re-activated by other chaperones.
Article
The betagamma-crystallins belong to a superfamily of two-domain proteins found in vertebrate eye lenses, with distant relatives occurring in microorganisms. It has been considered that an eukaryotic stress protein, spherulin 3a, from the slime mold Physarum polycephalum shares a common one-domain ancestor with crystallins, similar to the one-domain 3-D structure determined by NMR. The X-ray structure of spherulin 3a shows it to be a tight homodimer, which is consistent with ultracentrifugation studies. The (two-motif) domain fold contains a pair of calcium binding sites very similar to those found in a two-domain prokaryotic betagamma-crystallin fold family member, Protein S. Domain pairing in the spherulin 3a dimer is two-fold symmetric, but quite different in character from the pseudo-two-fold pairing of domains in betagamma-crystallins. There is no evidence that the spherulin 3a single domain can fold independently of its partner domain, a feature that may be related to the absence of a tyrosine corner. Although it is accepted that the vertebrate two-domain betagamma-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage. The spherulin 3a protomer stability appears to be dependent on domain pairing. Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.
Article
Crystallins, the major structural proteins in the lens of the eye, are maintained with little turnover throughout the lifetime of the host. With time, lens crystallins undergo post-translational modifications that may play an important role in loss of vision during aging and cataract formation. Specific modifications include deamidation and truncation. Urea-induced denaturation was studied for recombinantly expressed wild-type βb1 (WT), the deamidated mutant (Q204E), an N-terminally truncated mutant (βb1(ΔN41)), and other truncated versions of these proteins generated by calpain II digestion. Tryptophan fluorescence was used to monitor loss of global tertiary structure. Loss of secondary structure was followed by circular dichroism, and electron paramagnetic resonance site-directed spin labeling was used to monitor loss of tertiary structure selectively in the N-terminal domain. Our results indicated that the deamidated mutant was significantly destabilized relative to WT. Q204E showed a two-step denaturation curve with transitions at 4.1 and 7.2 M urea, whereas denaturation of WT occurred in a cooperative single step with a transition midpoint of 5.9 M urea. Unfolding of WT was completely reversible, whereas Q204E failed to fully refold. Prolonged incubation under denaturing conditions led to aggregation, which was also more pronounced for Q204E dimers than for WT. Truncation of 41 residues from the N-terminus or 47 and 5 residues from the N- and C-termini did not affect stability. These studies indicated that a single-site deamidation could significantly diminish the stability of lens βB1-crystallin, supporting the idea that such modifications may play an important role in age-related cataract formation.
Article
The proteins of the eye lens, which do not turn over throughout life, undergo many modifications, some of which lead to senile cataract. We describe a modification, S-methylation of cysteine, that may serve to protect the lens from detrimental modifications. The modification was detected as a +14 Da peak in electrospray ionization mass spectra of human lens gammaS-crystallins. Derivatization of gammaS-crystallin with iodoacetamide showed reaction at only six of the seven cysteines, indicating the modification blocked reaction at one cysteine. Further analysis of the modified gammaS-crystallin as tryptic peptides located the modification primarily at Cys 26, with smaller amounts at Cys 24. Tandem mass spectrometry and exact mass measurements showed that the modification was S-methylation. Methylation of proteins has been documented at several other amino acid residues,but S-methylation of cysteine residues has previously been detected only as part of a methyltransferase DNA repair mechanism or at trace amounts in hemoglobin. The high levels of S-methylated cysteines in lens nuclei and the specificity for Cys 26 and Cys 24 suggest the reaction is enzymatically mediated. This modification is particularly important because it blocks disulfide bonding of gammaS-crystallins and, thereby, inhibits formation of the high-molecular weight assemblies associated with cataract: Evidence of more S-methylation in soluble than in insoluble gammaS-crystallins supports the contention that S-methylation of gammaS-crystallin inhibits protein insolubilization and may offer protection against cataract.
Article
The eye lens contains a structural protein, α crystallin, composed of two homologous primary gene products αA2 and αB2. In certain rodents, still another α crystallin polypeptide, αAIns, occurs, which is identical to αA2 except that it contains an insertion peptide between residues 63 and 64. In this paper we describe the complete αA crystallin gene that has been cloned from DNA isolated from Syrian golden hamster. Evidence is provided that the αA gene is present as a single copy in the hamster genome. The detailed organization of the gene has been established by means of DNA sequence analysis and S1 nuclease mapping, revealing that the gene consists of four exons. The first exon contains the information for the 68 base-pair long 5′ non-coding region as well as the coding information for the first 63 amino acids. The second exon encodes the 23 amino acid insertion sequence, the third exon codes for amino acid 87 to 127 of the αAIns chain, whereas the last exon encodes the C-terminal 69 amino acids and contains the information for the 523 base-pair long 3′ non-coding region. The second exon is bordered by a 3′ splice junction (A · G/G · C), which deviates from the consensus for donor splice sites (A · G/G · T). This deviation is found in both hamster and mouse. An internal duplication was detected in the first exon by using a DIAGON-generated matrix for comparison. By means of similar DIAGON-generated matrices it was confirmed that the amino acids coded for by the third and fourth exons are homologous to the small heat-shock proteins of Drosophila, Caenorhabditis and soyabean. The implications of the differential splicing and the evolutionary aspects of the detected homologies are discussed.
Article
Significant advances have been made in understanding the thermodynamics of the hydrophobic effect and of the reversible unfolding of proteins, although there is still considerable controversy. Recent analyses suggest that the predominant factor in stabilizing the folded states of proteins is intramolecular hydrogen bonding even though in the unfolded state the polar groups form intermolecular hydrogen bonds with water. Van der Waals interactions between non-polar groups also contribute substantially, but perhaps not as much as might have been expected if protein interiors were perfectly close-packed. The role of the solvent seems to be primarily destabilizing, and involves the favourable solvation of both polar and non-polar groups. The effects of added stabilizers and denaturants can be understood on the basis of their alterations of the properties of water and of their interactions with the surfaces of folded and unfolded proteins. Also, their electrostatic interactions between groups involved in salt bridges in folded proteins can be substantial.
Article
γ-Crystallin is reported to be conformationally stable because of its internal structural symmetry, and γF (γIVa) is the most stable among the various γ-crystallin gene products. However, there is no detailed report on its thermodynamic and kinetic stability. In the present study, detailed unfolding of γF-crystallin was investigated by equilibrium and kinetics methods with fluorescence and far-UV CD spectroscopic measurements. The GdnHCl-induced unfolding curves probed by Trp emission maximum and intensity showed a sharp single-step transition. Upon widening the unfolding transition with the use of urea in 1.5 M GdnHCl, a more proper fit for thermodynamic analysis was obtained. γF-Crystallin underwent a straightforward two-state process (N⇆U) without showing any measurable amount of intermediate. The conformational stability, as measured by ΔGH2OD (∼9 kcal/mol), indicates that γF-crystallin is a very stable protein. The high activation energy ΔG‡H2O (∼24 kcal/mol), calculated from unfolding kinetics monitored by far-UV CD at 218 nm, also indicates that the native and unfolded states are separated by a high activation energy barrier.
Article
A recent paper by Plater et al. [20], showed that the mutation of a single phenylalanine residue F27R in mouse αB completely abolished the chaperone-like property of α-crystallin when assayed with insulin at 25°C or with γ-crystallin at 66°C. We have produced the same mutation as well as some additional mutations in human αB-crystallin. Our data suggest that the F27R mutation effected the thermal stability of αB-crystallin making it unstable at temperatures ≥60°C. In agreement with the published work, at these temperatures the F27R human recombinant αB-crystallin does not protect the target protein from aggregation. When assayed with insulin or α-lactalbumin at 25 or 37°C, however, there were no differences in the protective abilities between the native αB-crystallin or the F27R mutated human αB-crystallin. Several other multiple mutations involving proline residues were also produced. These mutations did not effect the chaperone-like properties of human αB-crystallin, but some of them did effect the native molecular weight size as judged by gel filtration chromatography.