BBA - General Subjects (v.1860, #1PB)
Editorial Board (i).
Crystallin biochemistry in health and disease by K. Krishna Sharma (147-148).
Keywords: Crystallin; Chaperone; Small heat shock protein; Retina; Cataract;
Structure and function of α-crystallins: Traversing from in vitro to in vivo by Martin Haslbeck; Jirka Peschek; Johannes Buchner; Sevil Weinkauf (149-166).
The two α-crystallins (αA- and αB-crystallin) are major components of our eye lenses. Their key function there is to preserve lens transparency which is a challenging task as the protein turnover in the lens is low necessitating the stability and longevity of the constituent proteins. α-Crystallins are members of the small heat shock protein family. αB-crystallin is also expressed in other cell types.The review summarizes the current concepts on the polydisperse structure of the α-crystallin oligomer and its chaperone function with a focus on the inherent complexity and highlighting gaps between in vitro and in vivo studies.Both α-crystallins protect proteins from irreversible aggregation in a promiscuous manner. In maintaining eye lens transparency, they reduce the formation of light scattering particles and balance the interactions between lens crystallins. Important for these functions is their structural dynamics and heterogeneity as well as the regulation of these processes which we are beginning to understand. However, currently, it still remains elusive to which extent the in vitro observed properties of α-crystallins reflect the highly crowded situation in the lens.Since α-crystallins play an important role in preventing cataract in the eye lens and in the development of diverse diseases, understanding their mechanism and substrate spectra is of importance. To bridge the gap between the concepts established in vitro and the in vivo function of α-crystallins, the joining of forces between different scientific disciplines and the combination of diverse techniques in hybrid approaches are necessary.This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Crystallin; Small heat shock protein; Eye lens; Protein dynamics; Molecular chaperone;
Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions by Raman Bakthisaran; Kranthi Kiran Akula; Ramakrishna Tangirala; Ch. Mohan Rao (167-182).
αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known.The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases.Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate.Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: AlphaB-crystallin; Small heat shock protein; Molecular chaperone; Phosphorylation; Stress, aging and diseases; Desmin-related myopathy;
Isomerization of aspartyl residues in crystallins and its influence upon cataract by Noriko Fujii; Takumi Takata; Norihiko Fujii; Kenzo Aki (183-191).
Age-related cataracts, which probably form due to insolubilization of lens proteins, can lead to loss of vision. Although the exact reason is unknown, lens protein aggregation may be triggered by increases in PTMs such as d-β-, l-β- and d-α-Asp isomers. These isomers have been observed in aged lens; however, there have been few quantitative and site-specific studies owing to the lack of a quick and precise method for distinguishing between d- and l-Asp in a peptide or protein.We describe a new method for detecting peptides containing Asp isomers at individual sites in any protein by using an LC–MS/MS system combined with commercial enzymes that specifically react with different isomers. We also summarize current data on the effect of Asp isomerization on lens crystallins.The new technique enabled the analysis of isomers of Asp residues in lens proteins precisely and quickly. An extensive proportion of Asp isomerization was observed at all Asp sites of crystallins in the insoluble fraction of aged lens. In addition, d-amino acid substitutions in crystallin-mimic peptides showed altered structural formation and function. These results indicate that isomerization of Asp residues affects the stability, structure and inter-subunit interaction of lens crystallins, which will induce crystallin aggregation and insolubilization, disrupt the associated functions, and ultimately contribute to the onset of senile cataract formation.The mechanism underlying the onset of age-related diseases may involve isomerization, whereby d-amino acids are incorporated in the l-amino acid world of life.This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.Display Omitted
Keywords: d-Amino acid; Aging; Isoaspartate (β-aspartate); Protein; Cataract; LC–MS/MS;
The etiology of human age-related cataract. Proteins don't last forever by Roger J.W. Truscott; Michael G. Friedrich (192-198).
It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification.Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses.The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein–protein interactions are disrupted and lens opacification ensues.Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Human aging; Protein degradation; Lens; Lifespan; Crystallins;
Differential role of arginine mutations on the structure and functions of α-crystallin by Alok Kumar Panda; Sandip Kumar Nandi; Ayon Chakraborty; Ram H. Nagaraj; Ashis Biswas (199-210).
α-Crystallin is a major protein of the eye lens in vertebrates. It is composed of two subunits, αA- and αB-crystallin. α-Crystallin is an oligomeric protein having these two subunits in 3:1 ratio. It belongs to small heat shock protein family and exhibits molecular chaperone function, which plays an important role in maintaining the lens transparency. Apart from chaperone function, both subunits also exhibit anti-apoptotic property. Comparison of their primary sequences reveals that αA- and αB-crystallin posses 13 and 14 arginine residues, respectively. Several of them undergo mutations which eventually lead to various eye diseases such as congenital cataract, juvenile cataract, and retinal degeneration. Interestingly, many arginine residues of these subunits are modified during glycation and even some are truncated during aging. All these facts indicate the importance of arginine residues in α-crystallin.In this review, we will emphasize the recent in vitro and in vivo findings related to congenital cataract causing arginine mutations in α-crystallin.Congenital cataract causing arginine mutations alters the structure and decreases the chaperone function of α-crystallin. These mutations also affect the lens morphology and phenotypes. Interestingly, non-natural arginine mutations (generated for mimicking the glycation and truncation environment) improve the chaperone function of α-crystallin which may play an important role in maintaining the eye lens transparency during aging.The neutralization of positive charge on the guanidino group of arginine residues is not always detrimental to the functionality of α-crystallin.This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Congenital cataract; α-Crystallin; Arginine mutations; Chaperone; Post-translational modifications;
Interaction of α-crystallin with some small molecules and its effect on its structure and function by A. Biswas; S. Karmakar; A. Chowdhury; K.P. Das (211-221).
α-Crystallin acts like a molecular chaperone by interacting with its substrate proteins and thus prevents their aggregation. It also interacts with various kinds of small molecules that affect its structure and function.In this article we will present a review of work done with respect to the interaction of ATP, peptide generated from lens crystallin and other proteins and some bivalent metal ions with α-crystallin and discuss the role of these interactions on its structure and function and cataract formation. We will also discuss the interaction of some hydrophobic fluorescence probes and surface active agents with α-crystallin.Small molecule interaction controls the structure and function of α-crystallin. ATP and Zn+ 2 stabilize its structure and enhance chaperone function. Therefore the depletion of these small molecules can be detrimental to maintenance of lens transparency. However, the accumulation of small peptides due to protease activity in the lens can also be harmful as the interaction of these peptides with α-crystallin and other crystallin proteins in the lens promotes aggregation and loss of lens transparency. The use of hydrophobic probe has led to a wealth of information regarding the location of substrate binding site and nature of chaperone–substrate interaction. Interaction of surface active agents with α-crystallin has helped us to understand the structural stability and oligomeric dissociation in α-crystallin.These interactions are very helpful in understanding the mechanistic details of the structural changes and chaperone function of α-crystallin. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Small molecule interaction; Chaperone function of alpha crystallin; ATP binding; Hydrophobic probe; Lens peptide;
Multidimensional significance of crystallin protein–protein interactions and their implications in various human diseases by Kumarasamy Anbarasu; Jeyarajan Sivakumar (222-233).
Crystallins are the important structural and functional proteins in the eye lens responsible for refractive index. Post-translational modifications (PTMs) and mutations are major causative factors that affect crystallin structural conformation and functional characteristics thus playing a vital role in the etiology of cataractogenesis.The significance of crystallin protein–protein interactions (PPIs) in the lens and non-lenticular tissues is summarized.Aberrancy of PPIs between crystallin, its associated protein and metal ions has been accomplished in various human diseases including cataract. A detailed account on multidimensional structural and functional significance of crystallin PPI in humans must be brought into limelight, in order to understand the biochemical and molecular basis augmenting the aberrancies of such interaction. In this scenario, the present review is focused to shed light on studies which will aid to expand our present understanding on disease pathogenesis related to loss of PPI thereby paving the way for putative future therapeutic targets to curb such diseases.The interactions with α-crystallins always aid to protect their structural and functional characteristics. The up-regulation of αB-crystallin in the non-lenticular tissues always decodes as biomarker for various stress related disorders. For better understanding and treatment of various diseases, PPI studies provide overall outline about the structural and functional characteristics of the proteins. This information not only helps to find out the route of cataractogenesis but also aid to identify potential molecules to inhibit/prevent the further development of such complicated phenomenon. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Crystallin; Cataract; Mutation; Protein–protein interaction;
Autophagy and UPR in alpha-crystallin mutant knock-in mouse models of hereditary cataracts by Usha P. Andley; Joshua W. Goldman (234-239).
Knock-in mice provide useful models of congenital and age-related cataracts caused by α-crystallin mutations. R49C αA-crystallin and R120G αB-crystallin mutations are linked with hereditary cataracts. Knock-in αA-R49C +/− heterozygotes develop cataracts by 1–2 months, whereas homozygote mice have cataracts at birth. The R49C mutation drastically reduces lens protein water solubility and causes cell death in knock-in mouse lenses. Mutant crystallin cannot function as a chaperone, which leads to protein aggregation and lens opacity. Protein aggregation disrupts the lens fiber cell structure and normal development and causes cell death in epithelial and fiber cells. We determined what aspects of the wild-type phenotype are age-dependently altered in the mutant lens.Wild-type, heterozygote (αA-R49C +/−), and homozygote (αA-R49C +/+) mouse lenses were assessed pre- and postnatally for lens morphology (electron microscopy, immunohistochemistry), and autophagy or unfolded protein response markers (immunoblotting).Morphology was altered by embryonic day 17 in R49C +/+ lenses; R49C +/− lens morphology was unaffected at this stage. Active autophagy in the lens epithelium of mutant lenses was indicated by the presence of autophagosomes using electron microscopy. Protein p62 levels, which are degraded specifically by autophagy, increased in αA-R49C mutant versus wild-type lenses, suggesting autophagy inhibition in the mutant lenses. The unfolded protein response marker XBP-1 was upregulated in adult lenses of αB-R120G +/+ mice, suggesting its role in lens opacification.Mutated crystallins alter lens morphology, autophagy, and stress responses.Therapeutic modulation of autophagic pathways may improve protein degradation in cataractous lenses and reduce lens opacity. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Cataract; Crystallin; Mutation; Knock-in mouse; Unfolded protein response; Autophagy;
Functional sequences in human alphaB crystallin by John I. Clark (240-245).
Human alphaB crystallin (HspB5) contains the alpha crystallin core domain, a series of antiparallel beta-strands organized into the characteristic beta sandwich of small heat shock proteins (sHsps). The full 3-dimensional structure for alpha crystallin has not been determined and the mechanism for the biological activity remains elusive because sHsps participate in multiple interactions with a broad range of target proteins that favor self-assembly of polydisperse fibrils and complexes. We selected human alphaB crystallin to study interactive sequences because it is involved in many human condensation, amyloid, and aggregation diseases and it is very sensitive to the destabilization of unfolding proteins. Sophisticated methods are being used to analyze and complete the structure of alphaB crystallin with the expectation of understanding sHsp function. This review considers the identification of interactive sites on the surface of the alphaB crystallin, which may be the key to understanding the multifunctional activity of human alphaB crystallin.This review summarizes the research on the identification of the bioactive interactive sequences responsible for the function of human alphaB crystallin, an sHsp with chaperone-like activity.The multifunctional activity of human alphaB crystallin results from the interactive peptide sequences exposed on the surface of the molecule. The multiple, non-covalent, interactive sequences can account for the selectivity and sensitivity of alphaB crystallin to the initiation of protein unfolding.Human alphaB crystallin may be an important part of an endogenous protective mechanism in aging cells and tissues. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Crystallin; Lens; Cataract; Human; Pin array; Amyloid;
Alpha-crystallin-derived peptides as therapeutic chaperones by Murugesan Raju; Puttur Santhoshkumar; K. Krishna Sharma (246-251).
The demonstration of chaperone-like activity in peptides (mini-chaperones) derived from α-crystallin's chaperone region has generated significant interest in exploring the therapeutic potential of peptide chaperones in diseases of protein aggregation. Recent studies in experimental animals show that mini-chaperones could reach intended targets and alter the disease phenotype. Although mini-chaperones show potential benefits against protein aggregation diseases, they do tend to form aggregates on storage. There is thus a need to fine-tune peptide chaperones to increase their solubility, pharmacokinetics, and biological efficacy.This review summarizes the properties and the potential therapeutic roles of mini-chaperones in protein aggregation diseases and highlights some of the refinements needed to increase the stability and biological efficacy of mini-chaperones while maintaining or enhancing their chaperone-like activity against precipitation of unfolding proteins.Mini-chaperones suppress the aggregation of proteins, block amyloid fibril formation, stabilize mutant proteins, sequester metal ions, and exhibit antiapoptotic properties. Much work must be done to fine-tune mini-chaperones and increase their stability and biological efficacy. Peptide chaperones could have a great therapeutic value in diseases associated with protein aggregation and apoptosis.Accumulation of misfolded proteins is a primary cause for many age-related diseases, including cataract, macular degeneration, and various neurological diseases. Stabilization of native proteins is a logical therapeutic approach for such diseases. Mini-chaperones, with their inherent antiaggregation and antiapoptotic properties, may represent an effective therapeutic molecule to prevent the cascade of protein conformational disorders. Future studies will further uncover the therapeutic potential of mini-chaperones. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Mini-chaperones; Crystallins; Protein aggregation; Peptide; Therapeutics;
Therapeutic potential of α-crystallin by Ram H. Nagaraj; Rooban B. Nahomi; Niklaus H. Mueller; Cibin T. Raghavan; David A. Ammar; J. Mark Petrash (252-257).
The findings that α-crystallins are multi-functional proteins with diverse biological functions have generated considerable interest in understanding their role in health and disease. Recent studies have shown that chaperone peptides of α-crystallin could be delivered into cultured cells and in experimental animals with beneficial effects against protein aggregation, oxidation, inflammation and apoptosis.In this review, we will summarize the latest developments on the therapeutic potential of α-crystallins and their functional peptides.α-Crystallins and their functional peptides have shown significant favorable effects against several diseases. Their targeted delivery to tissues would be of great therapeutic benefit. However, α-crystallins can also function as disease-causing proteins. These seemingly contradictory functions must be carefully considered prior to their therapeutic use.αA and αB-Crystallin are members of the small heat shock protein family. These proteins exhibit molecular chaperone and anti-apoptotic activities. The core crystallin domain within these proteins is largely responsible for these prosperities. Recent studies have identified peptides within the crystallin domain of both α- and αB-crystallins with remarkable chaperone and anti-apoptotic activities. Administration of α-crystallin or their functional peptides has shown substantial inhibition of pathologies in several diseases. However, α-crystallins have been shown to promote disease-causing pathways. These two sides of the proteins are discussed in this review.This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: α-crystallin; Chaperone; Apoptosis; Protein aggregation; Inflammation; Therapeutic potential;
Alpha crystallins in the retinal pigment epithelium and implications for the pathogenesis and treatment of age-related macular degeneration by Ram Kannan; Parameswaran G. Sreekumar; David R. Hinton (258-268).
αA- and αB crystallins are principal members of the small heat shock protein family and elicit both a cell protective function and a chaperone function. α-Crystallins have been found to be prominent proteins in normal and pathological retina emphasizing the importance for in-depth understanding of their function and significance.Retinal pigment epithelial cells (RPE) play a vital role in the pathogenesis of age-related macular degeneration (AMD). This review addresses a number of cellular functions mediated by α-crystallins in the retina. Prominent expression of αB crystallin in mitochondria may serve to protect cells from oxidative injury. αB crystallin as secretory protein via exosomes can offer neuroprotection to adjacent RPE cells and photoreceptors. The availability of chaperone-containing minipeptides of αB crystallin could prove to be a valuable new tool for therapeutic treatment of retinal disorders.α-Crystallins are expressed in cytosol and mitochondria of RPE cells and are regulated during oxygen-induced retinopathy and during development. α-Crystallins protect RPE from oxidative-and ER stress-induced injury and autophagy. αB-Crystallin is a modulator of angiogenesis and vascular endothelial growth factor. αB Crystallin is secreted via exosomal pathway. Minichaperone peptides derived from αB Crystallin prevent oxidant induced cell death and have therapeutic potential.Overall, this review summarizes several novel properties of α-crystallins and their relevance to maintaining normal retinal function. In particular, the use of α-crystallin derived peptides is a promising therapeutic strategy to combat retinal diseases such as AMD. This article is part of a Special Issue entitled Crystallin biochemistry in health and disease.
Keywords: Crystallins; Subcellular localization; Apoptosis; Exosomes; Angiogenesis; Minichaperone peptide;
Role of crystallins in diabetic complications by Vadde Sudhakar Reddy; G. Bhanuprakash Reddy (269-277).
Crystallins are the major structural proteins of vertebrate eye lens responsible for maintaining the refractive index of the lens. However, recent studies suggest that they also have a functional significance in non-lenticular tissues. Prolonged uncontrolled diabetes results in the development of macro and microvascular complications that are the leading causes of morbidity and mortality in diabetic patients all over the world.Recent studies have shown that crystallins play an instrumental role in diabetes and its complications. Therefore, this review highlights the current data on the impact of chronic hyperglycemia on expression, distribution, glycation, phosphorylation, chaperone-like function and, anti-apoptotic activity of crystallins. Furthermore, we discussed the insights for developing therapeutic strategies for diabetic complications including natural agents, peptides, and pharmacological chaperones that modulate or mimic chaperone activity of α-crystallins.Upregulation of crystallins appears to be a common feature of chronic diabetes. Further, chronic hyperglycemia induces the glycation and phosphorylation of crystallins, mainly α-crystallins and thereby alters their properties. The disturbed interaction of αB-crystallin with various apoptotic mediators including Bax and caspases is also an important factor for increased cell death in diabetes. Numerous dietary agents, peptides, and chemical chaperones prevent apoptosis and the loss of chaperone activity in diabetes.Understanding the role of crystallins will aid in developing therapeutic strategies for alleviating pathophysiological conditions such as protein aggregation, inflammation, oxidative stress and apoptosis associated with chronic complications of diabetes including cataract, retinopathy, and cardiomyopathy. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Apoptosis; Chaperone-like activity; Crystallin; Glycation; Hyperglycemia; Phosphorylation;
Crystallins and neuroinflammation: The glial side of the story by Jennifer E. Dulle; Patrice E. Fort (278-286).
There is an abundance of evidence to support the association of damaging neuroinflammation and neurodegeneration across a multitude of diseases. One of the links between these pathological phenomena is the role of chaperone proteins as both neuroprotective and immune-regulatory agents.Chaperone proteins are highly expressed at sites of neuroinflammation both in glial cells and in the injured neurons that initiate the immune response. For this reason, the use of chaperones as treatment for various diseases associated with neuroinflammation is a highly active area of investigation. This review explores the various ways that the small heat shock protein chaperones, α-crystallins, can affect glial cell function with a specific focus on their implication in the inflammatory response associated with neurodegenerative disorders, and their potential as therapeutic treatment.Although the mechanisms are still under investigation, a clear link has now been established between alpha-crystallins and neuroinflammation, especially through their roles in microglial and macroglial cells. Interestingly, similar to inflammation in itself, crystallins can have a beneficial or detrimental impact on the CNS based on the context and duration of the condition.Overall this review points out the novel roles that chaperones such as alpha-crystallins can play outside of the classical protein folding pathways, and their potential in the development of new therapies for the treatment of neuroinflammatory/neurodegenerative diseases.This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Crystallins; Neuroinflammation; Microglia; Astrocytes; Central nervous system;
βA3/A1-crystallin and persistent fetal vasculature (PFV) disease of the eye by J. Samuel Zigler; Mallika Valapala; Peng Shang; Stacey Hose; Morton F. Goldberg; Debasish Sinha (287-298).
Persistent fetal vasculature (PFV) is a human disease in which the fetal vasculature of the eye fails to regress normally. The fetal, or hyaloid, vasculature nourishes the lens and retina during ocular development, subsequently regressing after formation of the retinal vessels. PFV causes serious congenital pathologies and is responsible for as much as 5% of blindness in the United States.The causes of PFV are poorly understood, however there are a number of animal models in which aspects of the disease are present. One such model results from mutation or elimination of the gene (Cryba1) encoding βA3/A1-crystallin. In this review we focus on the possible mechanisms whereby loss of functional βA3/A1-crystallin might lead to PFV. Cryba1 is abundantly expressed in the lens, but is also expressed in certain other ocular cells, including astrocytes. In animal models lacking βA3/A1-crystallin, astrocyte numbers are increased and they migrate abnormally from the retina to ensheath the persistent hyaloid artery. Evidence is presented that the absence of functional βA3/A1-crystallin causes failure of the normal acidification of endolysosomal compartments in the astrocytes, leading to impairment of certain critical signaling pathways, including mTOR and Notch/STAT3.The findings suggest that impaired endolysosomal signaling in ocular astrocytes can cause PFV disease, by adversely affecting the vascular remodeling processes essential to ocular development, including regression of the fetal vasculature. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Astrocytes; βA3/A1-crystallin; Fetal/hyaloid vasculature; Notch/STAT signaling; PI3K/Akt/mTOR signaling; Vascular remodeling;
Ca2 + and βγ-crystallins: An affair that did not last? by Amita Mishra; Bal Krishnan; Rajeev Raman; Yogendra Sharma (299-303).
During the last three decades, lens β- and γ-crystallins have found a huge number of kin from numerous taxonomical sources. Most of these proteins from invertebrates and microbes have been demonstrated or predicted to bind Ca2 + involving a distinct double-clamp motif, which is largely degenerated in lens homologues.The various aspects of transformation of βγ-crystallins from a quintessential Ca2 +-binding protein into a primarily structural molecule have been reviewed.In lens members of βγ-crystallins, the residues involved in Ca2 + binding have diverged considerably from the classical consensus with consequent reduction in their Ca2 +-binding properties. This evolutionary change is congenial to their new role as robust constituents of lens. The exact functions of the residual affinity for Ca2 + are yet to be established.This review highlights the significance of reduction in Ca2 +-binding ability of the βγ-crystallins for lens physiology and why this residual affinity may be functionally important. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Lens; β- and γ-crystallins; βγ-Crystallin domain; Ca2 +-binding; Lens transparency;
Differences in solution dynamics between lens β-crystallin homodimers and heterodimers probed by hydrogen–deuterium exchange and deamidation by Kirsten J. Lampi; Matthew R. Murray; Matthew P. Peterson; Bryce S. Eng; Eileen Yue; Alice R. Clark; Elisar Barbar; Larry L. David (304-314).
Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. βB2 crystallin in the lens of the eye readily forms dimers with other β-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study.Structures of βA3 and βB2 crystallin homodimers and the βA3/βB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen–deuterium exchange with mass spectrometry. We further mimicked deamidation in βB2 and probed the effect on the βA3/βB2 heterodimer. Results were confirmed with chemical crosslinking and NMR.Both βA3 and βB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of βB2 was supported by the identification of chemical crosslinks between lysines in βB2 within the heterodimer that were inconsistent with βB2's extended homodimeric structure. The compact structure of βA3 was supported by an overall decrease in mobility of βA3 in the heterodimer detected by NMR.In βB2, peptides 70–84 and 121–134 were exposed in the homodimer, but buried in the heterodimer with ≥ 50% decreases in deuterium levels. Homologous peptides in βA3, 97–109 and 134–149, had 25–50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between βB2 and βA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in βB2 at this predicted interface led to a less compact βB2 in the heterodimer.The more compact structure of the βA3/βB2 heterodimer was also more heat stable than either of the homodimers.The major structural proteins in the lens, the β-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of βB2, may facilitate formation of different size higher-ordered structures found in the transparent lens.Understanding complex hetero-oligomer interactions between β-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.Display Omitted
Keywords: β-Crystallins; Deamidation; Lens; Cataracts; Solution dynamics; Hydrogen–deuterium exchange; Mass spectrometry;
Deamidation of N76 in human γS-crystallin promotes dimer formation by Nicholas J. Ray; Damien Hall; John A. Carver (315-324).
Cataract formation is often attributed to the build-up of post-translational modifications in the crystallin proteins of the eye lens. One such modification, the deamidation of N76 in human γS-crystallin to D76, is highly correlated with age-related cataract (Hooi et al. Invest. Ophthalmol. Vis. Sci. 53 (2012) 3554–3561). In the current work, this modification has been extensively characterised in vitro. Biophysical characterisation was performed on wild type and N76D γS-crystallins using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using SEC–MALLS and analytical ultracentrifugation methods.Relative to the wild type protein, deamidation at N76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. We ascribe the apparent increase in thermal stability upon deamidation to the formation of dimer which prevents the unfolding of the inherently less stable monomer.Deamidation causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation.Deamidation at N76 in human γS-crystallin likely has a combinatorial effect with other post-translational crystallin modifications to induce age-related cataract. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Crystallin; Cataract; Lens; Ageing; Post-translational modification; Small heat-shock protein;
Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin by Domarin Khago; Eric K. Wong; Carolyn N. Kingsley; J. Alfredo Freites; Douglas J. Tobias; Rachel W. Martin (325-332).
The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin.We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting with the solution-state NMR structures of γS-WT and γS-G18V. γS-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in γS-G18V that are not present in γS-WT. Molecular docking predicts three binding sites that are specific to γS-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in γS-WT.Although both proteins display non-specific binding, more residues are involved in ANS binding to γS-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site.Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.Display Omitted
Keywords: Structural crystallin; Cataract; Protein aggregation; Chemical shift perturbation; ANS binding assay; Docking;
Gamma crystallins of the human eye lens by Venkata Pulla Rao Vendra; Ismail Khan; Sushil Chandani; Anbukkarasi Muniyandi; Dorairajan Balasubramanian (333-343).
Protein crystallins co me in three types (α, β and γ) and are found predominantly in the eye, and particularly in the lens, where they are packed into a compact, plastic, elastic, and transparent globule of proper refractive power range that aids in focusing incoming light on to the retina. Of these, the γ-crystallins are found largely in the nuclear region of the lens at very high concentrations (> 400 mg/ml). The connection between their structure and inter-molecular interactions and lens transparency is an issue of particular interest.We review the origin and phylogeny of the gamma crystallins, their special structure involving the use of Greek key supersecondary structural motif, and how they aid in offering the appropriate refractive index gradient, intermolecular short range attractive interactions (aiding in packing them into a transparent ball), the role that several of the constituent amino acid residues play in this process, the thermodynamic and kinetic stability and how even single point mutations can upset this delicate balance and lead to intermolecular aggregation, forming light-scattering particles which compromise transparency. We cite several examples of this, and illustrate this by cloning, expressing, isolating and comparing the properties of the mutant protein S39C of human γS-crystallin (associated with congenital cataract-microcornea), with those of the wild type molecule. In addition, we note that human γ-crystallins are also present in other parts of the eye (e.g., retina), where their functions are yet to be understood.There are several ‘crucial’ residues in and around the Greek key motifs which are essential to maintain the compact architecture of the crystallin molecules. We find that a mutation that replaces even one of these residues can lead to reduction in solubility, formation of light-scattering particles and loss of transparency in the molecular assembly.Such a molecular understanding of the process helps us construct the continuum of genotype–molecular structural phenotype–clinical (pathological) phenotype. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Keywords: Human gamma crystallins; Greek key motif; Structure–function correlation; Cataractogenic mutations; Congenital cataract;