α-Crystallin chaperone mimetic drugs inhibit lens γ-crystallin aggregation: Potential role for cataract prevention.

Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency-approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket-binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the "NC pocket" (residues 50-150) of HγD and one spanning the "NC tail" (residues 56-61 to 168-174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.

Altered Protein Dynamics and Increased Aggregation of Human γS-Crystallin Due to Cataract-Associated Deamidations.

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (µs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Differences in solution dynamics between lens ß-crystallin homodimers and heterodimers probed by hydrogen-deuterium exchange and deamidation.

BACKGROUND: Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. ßB2 crystallin in the lens of the eye readily forms dimers with other ß-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study. METHODS: Structures of ßA3 and ßB2 crystallin homodimers and the ßA3/ßB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen-deuterium exchange with mass spectrometry. We further mimicked deamidation in ßB2 and probed the effect on the ßA3/ßB2 heterodimer. Results were confirmed with chemical crosslinking and NMR. RESULTS: Both ßA3 and ßB2 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 ßB2 was supported by the identification of chemical crosslinks between lysines in ßB2 within the heterodimer that were inconsistent with ßB2's extended homodimeric structure. The compact structure of ßA3 was supported by an overall decrease in mobility of ßA3 in the heterodimer detected by NMR. In ßB2, 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 ßA3, 97-109 and 134-149, had 25-50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between ßB2 and ßA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in ßB2 at this predicted interface led to a less compact ßB2 in the heterodimer. The more compact structure of the ßA3/ßB2 heterodimer was also more heat stable than either of the homodimers. CONCLUSIONS: The major structural proteins in the lens, the ß-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of ßB2, may facilitate formation of different size higher-ordered structures found in the transparent lens. GENERAL SIGNIFICANCE: Understanding complex hetero-oligomer interactions between ß-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.

Murein Hydrolase LytF of Streptococcus sanguinis and the Ecological Consequences of Competence Development.

The overall health of the oral cavity is dependent on proper homeostasis between health-associated bacterial colonizers and bacteria known to promote dental caries. Streptococcus sanguinis is a health-associated commensal organism, a known early colonizer of the acquired tooth pellicle, and is naturally competent. We have shown that LytF, a competence-controlled murein hydrolase, is capable of inducing the release of extracellular DNA (eDNA) from oral bacteria. Precipitated LytF and purified LytF were used as treatments against planktonic cultures and biofilms. Larger amounts of eDNA were released from cultures treated with protein samples containing LytF. Additionally, LytF could affect biofilm formation and cellular morphology. Biofilm formation was significantly decreased in the lytF-complemented strain, in which increased amounts of LytF are present. The same strain also exhibited cell morphology defects in both planktonic cultures and biofilms. Furthermore, the LytF cell morphology phenotype was reproducible in wild-type cells using purified LytF protein. In sum, our findings demonstrate that LytF can induce the release of eDNA from oral bacteria, and they suggest that, without proper regulation of LytF, cells display morphological abnormalities that contribute to biofilm malformation. In the context of the oral biofilm, LytF may play important roles as part of the competence and biofilm development programs, as well as increasing the availability of eDNA.IMPORTANCEStreptococcus sanguinis, a commensal organism in the oral cavity and one of the pioneer colonizers of the tooth surface, is associated with the overall health of the oral environment. Our laboratory showed previously that, under aerobic conditions, S. sanguinis can produce H2O2 to inhibit the growth of bacterial species that promote dental caries. This production of H2O2 by S. sanguinis also induces the release of eDNA, which is essential for proper biofilm formation. Under anaerobic conditions, S. sanguinis does not produce H2O2 but DNA is still released. Determining how S. sanguinis releases DNA is thus essential to understand biofilm formation in the oral cavity.

The α-crystallin Chaperones Undergo a Quasi-ordered Co-aggregation Process in Response to Saturating Client Interaction.

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined structural changes in αAc, αBc and native heteromeric lens α-crystallins (αLc) in their apo-states and at varying degree of chaperone saturation leading to co-aggregation, using lysozyme and insulin as model clients. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP oligomeric scaffold, whereby the native cage-like sHSP assembly displays a directional growth to accommodate saturating conditions of client sequestration. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP oligomers with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across αAc, αBc and αLc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of α-crystallins, carrying potential implications for a pathway toward cataract formation.

The α-crystallin chaperones undergo a quasi-ordered co-aggregation process in response to saturating client interaction.

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. This study investigates structural changes in αAc and αBc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined αAc and αBc in their apo-states and at various stages of client-induced co-aggregation, using lysozyme as a model client. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP scaffold. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP scaffolding with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across both αAc and αBc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of sHSPs, carrying potential implications for a pathway toward cataract formation.

Eye lens ß-crystallins are predicted by native ion mobility-mass spectrometry and computations to form compact higher-ordered heterooligomers.

Eye lens α- and ß-/γ-crystallin proteins are not replaced after fiber cell denucleation and maintain lens transparency and refractive properties. The exceptionally high (∼400-500 mg/mL) concentration of crystallins in mature lens tissue and multiple other factors impede precise characterization of ß-crystallin interactions, oligomer composition, size, and topology. Native ion mobility-mass spectrometry is used here to probe ß-crystallin association and provide insight into homo- and heterooligomerization kinetics for these proteins. These experiments include separation and characterization of higher-order ß-crystallin oligomers and illustrate the unique advantages of native IM-MS. Recombinantly expressed ßB1, ßB2, and ßA3 isoforms are found to have different homodimerization propensities, and only ßA3 forms larger homooligomers. Heterodimerization of ßB2 with ßA3 occurs ∼3 times as fast as that of ßB1 with ßA3, and ßB1 and ßB2 heterodimerize less readily. Ion mobility experiments, molecular dynamics simulations, and PISA analysis together reveal that observed oligomers are consistent with predominantly compact, ring-like topologies.

Changes in solvent accessibility of wild-type and deamidated ßB2-crystallin following complex formation with αA-crystallin.

Aberrant protein interactions can lead to aggregation and insolubilization, such as occurs during cataract formation. Deamidation, a prevalent age-related modification in the lens of the eye, decreases stability of the major lens proteins, crystallins. The mechanism of deamidation altering interactions between αA-crystallin and ßB2-crystallin was investigated by detecting changes in solvent accessibility upon complex formation during heating. Solvent accessibility was determined by measuring hydrogen/deuterium exchange levels of backbone amides by high-resolution mass spectrometry. Deuterium levels in wild type ßB2-crystallin increased 50-60% in both domains following complex formation with αA-crystallin. This increased solvent accessibility indicated a general loosening along the backbone amides. Peptides with the greatest deuterium increases were located at the buried monomer-monomer interface, suggesting that the ßB2 dimer was disrupted. The only region where the deuterium levels decreased was in ßB2 peptide 123-139, containing an outside loop, and may be a potential site of interaction with αA. Mimicking deamidation at the ßB2 dimer interface prevented complex formation with αA. When temperatures were lowered, an αA/ßB2 Q70E/Q162E complex formed with similar solvent accessibilities as αA/WT ßB2. Deamidation did not disrupt specific αA/ßB2 interactions but favored aggregation before complex formation with αA. We conclude that deamidation contributes to cataract formation through destabilization of crystallins before they can be rescued by α-crystallin.

New focus on alpha-crystallins in retinal neurodegenerative diseases.

The crystallin proteins were initially identified as structural proteins of the ocular lens and have been recently demonstrated to be expressed in normal retina. They are dramatically upregulated by a large range of retinal diseases including diabetic retinopathy, age-related macular degeneration, uveitis, trauma and ischemia. The crystallin family of proteins is composed of alpha-, beta- and gamma-crystallin. Alpha-crystallins, which are small heat shock proteins, have received substantial attention recently. This review summarizes the current knowledge of alpha-crystallins in retinal diseases, their roles in retinal neuron cell survival and retinal inflammation, and the regulation of their expression and activity. Their potential role in the development of new treatments for neurodegenerative diseases is also discussed.

Solvent accessibility of betaB2-crystallin and local structural changes due to deamidation at the dimer interface.

In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the beta/gamma-crystallin family share common beta-sheet rich domains and hydrophobic regions at the monomer-monomer or domain-domain interfaces. Disruption of these interfaces, due to post-translational modifications, such as deamidation, decreases the stability of the crystallins. Previous experiments have failed to define the structural changes associated with this decreased stability. Using hydrogen/deuterium exchange with mass spectrometry (HDMS), deamidation-induced local structural changes in betaB2-crystallin were identified. Deamidation was mimicked by replacing glutamines with glutamic acids at homologous residues 70 and 162 in the monomer-monomer interface of the betaB2-crystallin dimer. The exchange-in of deuterium was determined from 15 s to 24 h and the global and local changes in solvent accessibility were measured. In the wild type betaB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of betaB2-crystallin in solution. This is consistent with a tightly packed domain structure observed in the crystal structure. Deuterium levels were initially greater in N-terminal domain (N-td) peptides than in homologous peptides in the C-terminal domain (C-td). The more rapid incorporation suggests a greater solvent accessibility of the N-td. In the betaB2-crystallin crystal structure, interface Gln are oriented towards their opposite domain. When deamidation was mimicked at Gln70 in the N-td, deuterium levels increased at the interface peptide in the C-td. A similar effect in the N-td was not observed when deamidation was mimicked at the homologous residue, Gln162, in the C-td. This difference in the mutants can be explained by deamidation at Gln70 disrupting the more compact C-td and increasing the solvent accessibility in the C-td interface peptides. When deamidation was mimicked at both interface Gln, deuterium incorporation increased in the C-td, similar to deamidation at Gln70 alone. In addition, deuterium incorporation was decreased in the N-td in an outside loop peptide adjacent to the mutation site. This decreased accessibility may be due to newly exposed charge groups facilitating ionic interactions or to peptides becoming more buried when other regions became more exposed. The highly sensitive HDMS methods used here detected local structural changes in solution that had not been previously identified and provide a mechanism for the associated decrease in stability due to deamidation. Changes in accessibility due to deamidation at the interface led to structural perturbations elsewhere in the protein. The cumulative effects of multiple deamidation sites perturbing the structure both locally and distant from the site of deamidation may contribute to aggregation and precipitation during aging and cataractogenesis in the lens.

Aggregation of deamidated human betaB2-crystallin and incomplete rescue by alpha-crystallin chaperone.

Aging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deamidation has been shown in vitro to alter the structure and decrease the stability of human lens betaB1, betaB2 and betaA3-crystallin. Of particular interest, betaB2 mutants were constructed to mimic the effect of in vivo deamidations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C-terminal homologue. The double mutant was also constructed. We previously reported that deamidation at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine betaLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine alpha-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by alpha-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated beta-crystallin aggregates is discussed.

Evolution of multifunctionality through a pleiotropic substitution in the innate immune protein S100A9.

Multifunctional proteins are evolutionary puzzles: how do proteins evolve to satisfy multiple functional constraints? S100A9 is one such multifunctional protein. It potently amplifies inflammation via Toll-like receptor four and is antimicrobial as part of a heterocomplex with S100A8. These two functions are seemingly regulated by proteolysis: S100A9 is readily degraded, while S100A8/S100A9 is resistant. We take an evolutionary biochemical approach to show that S100A9 evolved both functions and lost proteolytic resistance from a weakly proinflammatory, proteolytically resistant amniote ancestor. We identify a historical substitution that has pleiotropic effects on S100A9 proinflammatory activity and proteolytic resistance but has little effect on S100A8/S100A9 antimicrobial activity. We thus propose that mammals evolved S100A8/S100A9 antimicrobial and S100A9 proinflammatory activities concomitantly with a proteolytic 'timer' to selectively regulate S100A9. This highlights how the same mutation can have pleiotropic effects on one functional state of a protein but not another, thus facilitating the evolution of multifunctionality.

A single protein sometimes does multiple jobs. For instance, our immune system uses a small number of multipurpose proteins to respond quickly to a large number of threats. One example is the protein S100A9. It acts as an antimicrobial by preventing microbes from getting the nutrients they need, while also stimulating inflammation by inducing the release of molecules that recruit white blood cells. S100A9, like all proteins, is made up of a chain of small building blocks. These building blocks interact with each other and with other molecules in the environment. The sequence of the building blocks thus determines what jobs the protein can do. Therefore, a single change to the sequence of building blocks can have a dramatic effect: one change might render the protein faulty, while another change might allow it to do a new job. Proteins face similar challenges humans do when trying to do several things at once. A person driving a car while using their phone will not do either task well. Likewise, a protein that does two jobs faces challenges a single-purpose protein does not. Harman et al. were interested in how S100A9 was able to evolve and maintain its dual functionality, despite this potential problem. They started by asking when S100A9 acquired its two purposes. They measured the antimicrobial and inflammatory activity of S100A9 proteins from humans, mice and opossums. The activities of S100A9 in these species was similar, suggesting that S100A9 acquired its different jobs in the ancestor of mammals, some 160 million years ago. Next, Harman et al. computationally reconstructed ancestral forms of S100A9 by comparing hundreds of similar proteins and building an evolutionary tree. They then measured the antimicrobial and inflammatory activity of these ancestral proteins. By comparing the last ancestor that did not have these activities to the first ancestor that did, they identified the sequence changes that gave S100A9 its dual activity. Importantly, these changes are located in separate regions of the protein, meaning they could occur independently, without affecting each other. Further, the same sequence change that converted S100A9 into an inflammatory signal also introduced a mechanism to regulate this activity. The results suggest that a small number of sequence changes ­ or even a single change ­ can make a protein more versatile. This means that evolving multipurpose proteins may not be as difficult as is often thought.

Cumulative deamidations of the major lens protein γS-crystallin increase its aggregation during unfolding and oxidation.

Age-related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long-lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface-exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen-deuterium exchange, and susceptibility to disulfide cross-linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light-scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide-linked aggregates. The lens-specific chaperone αA-crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS-crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.

Deamidation alters interactions of beta-crystallins in hetero-oligomers.

PURPOSE: Cataracts are a major cause of blindness worldwide. A potential mechanism for loss of visual acuity may be due to light scattering from disruption of normal protein-protein interactions. During aging, the lens accumulates extensively deamidated crystallins. We have previously reported that deamidation in the betaA3-crystallin (betaA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of betaA3 with other beta-crystallin subunits. METHODS: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between betaA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type betaA3 or the doubly deamidated mutant betaA3 Q85E/Q180E (DM betaA3) were mixed with either betaB1- or betaB2-crystallin (betaB1 or betaB2) subunits. After incubation at increasing temperatures, hetero-oligomers were resolved from individual subunits and their molar masses determined by size exclusion chromatography with in line multiangle laser light scattering. Structural changes of hetero-oligomers were analyzed with fluorescence spectroscopy and blue-native PAGE. RESULTS: Molar masses of the hetero-oligomer complexes indicated betaA3 formed a polydispersed hetero-tetramer with betaB1 and a mondispersed hetero-dimer with betaB2. Deamidation at the interface in the betaA3 dimer decreased formation of the hetero-oligomer with betaB1 and further decreased formation of the hetero-dimer with betaB2. During thermal-induced denaturation of the deamidated betaA3 dimer, betaB1 but not betaB2 was able to prevent precipitation of betaA3. CONCLUSIONS: Deamidation decreased formation of hetero-oligomers between beta-crystallin subunits. An excess accumulation of deamidated beta-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble beta-crystallins during aging and cataracts.

The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function.

Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4-5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1-6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4-7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

Quantitative measurement of young human eye lens crystallins by direct injection Fourier transform ion cyclotron resonance mass spectrometry.

PURPOSE: Human eye lenses at birth are primarily constructed of 12 distinct crystallins and two truncated crystallins. The molecular weights of these 14 proteins vary between about 20,000 and 30,000 Da. The relative amounts of these molecules and their post-synthetic changes with age are of substantial interest in the study of lens biochemistry and lens pathology. Fourier transform mass spectrometry of unfractionated lens homogenates now permits precise quantitative measurement of the relative amounts of lens crystallins. We report herein the measurement of the 14 crystallins in 10 pairs of lenses from humans between the ages of 2 and 300 days. METHODS: Eye lenses were obtained from human donors of various ages in the first year of life. These lenses were homogenized in 0.02 M phosphate buffer at pH 7.0 with 0.001 M EDTA, desalted by washing over a 3,000 Da filter, and injected directly into the nanospray source of a hybrid Fourier transform ion cyclotron resonance mass spectrometer, Qq-FT(ICR)MS, equipped with a 12 Tesla magnet. The crystallins were quantitatively ionized and mass analyzed in the ICR cell of the mass spectrometer. The detected signals of all of the isotopic and charge state species for each crystallin were normalized and summed to determine the protein quantities. RESULTS: The relative amounts of the 14 crystallins are found to be quite similar from individual to individual at birth. These amounts are in integer ratios to one another that suggest important structural relations within the lens. In two cases, the relative amounts of alphaA- and betaB2-crystallin change proportionally to the logarithm of age during the first year, with alphaA- decreasing and betaB2-crystallin increasing. The changes in alphaA- and betaB2-crystallin are mutually offsetting, with alphaA-crystallin decreasing from 30% to 18% and betaB2-increasing from 12% to 24%. CONCLUSIONS: These observations suggest that the human eye lens at birth is constructed of crystallins in which the numbers of crystallin molecules have regular integral relationships to each other. As the lens develops during the first year, some of these relationships change. While the functional significance of the reciprocal decrease in alphaA- and increase in betaB2-crystallin is not known, betaB2-crystallin may substitute for alphaA-crystallin in the lens structures synthesized during the year after birth. Direct injection FT(ICR)MS of unfractionated lens was found to be an excellent method for the quantitative measurement of lens crystallins.

Quantitative measurement of deamidation in lens betaB2-crystallin and peptides by direct electrospray injection and fragmentation in a Fourier transform mass spectrometer.

PURPOSE: Deamidation of lens crystallins and specific deamidation sites have been suggested to be associated with aging and cataracts. However, these studies have been hindered by the lack of suitable quantitative methods of measurement of protein deamidation. We demonstrate herein a method to quantitatively measure deamidation of proteins and peptides without prior sample preparation or separation in order to directly compare the amidated and deamidated forms. We have tested the hypothesis that the 19 mDa mass defect that distinguishes deamidated peptides and proteins from the ordinary natural isotopic species can be utilized for quantitative measurement of their rate and extent of deamidation. The measurement technique used was ion cyclotron resonance Fourier transform mass spectrometry (FTMS), alone with no prior sample preparation or separation. The amidated and deamidated species were recombinantly expressed human eye lens betaB2-crystallins and the peptides GlyIleAsnAlaGly and GlyAsnAsnAsnGly. FTMS measurements of lens proteins from a 1-month-old human donor were also carried out. METHODS: Wild type and mutant human eye lens betaB2-crystallins with Gln162 replaced by Glu162 were produced in bacteria, and GlyIleAsnAlaGly and GlyAsnAsnAsnGly were synthesized by Merrifield solid-phase peptide synthesis. The peptides were deamidated in pH 7.4, 37.00 degrees C, 0.15 M Tris-HCl aqueous solution for 18 successive time intervals before analysis. Mutant and wildtype betaB2-crystallin solutions at various compositional percentages were mixed and analyzed. The peptides were introduced by electrospray ionization and immediately analyzed in the ion cyclotron resonance (ICR) Fourier transform mass analyzer. Two mass defect analysis procedures were demonstrated for the proteins. In the first, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the +29 isotopic group was selectively introduced into the ICR mass analyzer, where 14 residue and 18 residue laser-induced fragments were separated and the extent of deamidation determined by mass defect analysis. In the second, betaB2-crystallin was introduced into the mass spectrometer by electrospray ionization and the entire sample was fragmented by collision ionization before introduction into the ICR mass analyzer, where 14 residue fragments were separated and the extent of deamidation determined by mass defect analysis. RESULTS: The betaB2-crystallin mass spectra showed a good quantitative dependence upon extent of deamidation. Direct injection by electrospray ionization followed by ion selection and laser fragmentation or by collision fragmentation produced fragments of amidated and deamidated betaB2-crystallin that were appropriate for FTMS quantitative analysis. The two peptides exhibited the expected four deamidation rate curves with acceptable precision. CONCLUSIONS: Mass defect FTMS quantitative analysis of protein deamidation, as reported for the first time herein and illustrated with betaB2-crystallin, should prove quite useful. This procedure omits gel separation, chromatography, enzymatic digestion, derivatization, and other procedures that currently add cost and time while degrading quantitative comparison of the amidated and deamidated forms. Mass defect FTMS is also well suited to quantitative deamidation rate studies of peptides. The substantial potential significance of this technique is evident, as example, for lens crystallins where it makes possible quantitative studies of age and disease-dependent deamidation that have heretofore been very difficult. This technique should allow convenient and reliable identification and quantitative measurement of specific deamidation sites that may play a role in aging and cataracts.

Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide.

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in beta B1-crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble beta B1 contained 13%-17% deamidation at Gln146 and Asn157. Static and quasi-elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild-type (wt) beta B1 and the deamidated beta B1 mutants, Q146E and N157D. Dimer formation occurred for wt beta B1, Q146E, and N157D in a concentration-dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of beta B1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.

Lens proteomics: analysis of rat crystallin sequences and two-dimensional electrophoresis map.

PURPOSE: To determine the sequence of four rat beta-crystallins, confirm the sequences by mass spectrometry, and produce a two-dimensional electrophoresis (2-DE) map of soluble crystallins in young rat lens. METHODS: New or additional sequences were determined for betaB1, betaB3, betaA3, and betaA4-crystallin cDNAs from Sprague-Dawley rats, and the deduced protein sequences confirmed by mass spectrometry. The identity and relative abundance of each crystallin was then determined by 2-DE of soluble protein from whole lenses of 12-day-old rats, image analysis, and tandem mass spectrometry (MS/MS) spectra of peptides from in-gel digests. RESULTS: The previously unreported sequence of rat betaA4 cDNA encoded a 195-amino-acid protein. Additional cDNA sequencing provided the previously unknown N-terminal sequence of rat betaA3, found two differences from the previous amino acid sequences of both rat betaB1 and betaB3, and detected a polymorphism at residue 54 in rat betaB3. These new sequences were then confirmed by whole protein masses and MS/MS spectra of proteolytic digests. 2-DE analysis provided a more detailed map of rat crystallins than previously available and allowed the composition of crystallins in young rat lens to be compared with that in young human lens. CONCLUSIONS: This report provides baseline data that will facilitate the analysis of posttranslational modifications in rat crystallins during cataract. Detection of a polymorphism in the sequence of rat betaB3 suggests that crystallins in humans could also exhibit polymorphisms. The unusual abundance of rat betaB3 and low abundance of betaB2 may account for the increased susceptibility of rat crystallins to insolubilization during aging and cataract.

Decreased heat stability and increased chaperone requirement of modified human betaB1-crystallins.

PURPOSE: To determine how deamidation and partial loss of the N- and C-terminal extensions alter the heat stability of betaB1-crystallin. METHODS: Human lens betaB1, a deamidated betaB1, Q204E, and alphaA-crystallins were expressed. Truncated betaB1 was generated by proteolytic removal of part of its terminal extensions. The aggregation and precipitation of these proteins due to heating was monitored by circular dichroism and light scattering. The effect of heat on the stability of both monomers and oligomers was investigated. The flexibility of the extensions in wild type and deamidated betaB1 was assessed by 1H NMR spectroscopy. RESULTS: With heat, deamidated betaB1 precipitated more readily than wild type betaB1. Similar effects were obtained for either monomers or oligomers. Flexibility of the N-terminal extension in deamidated betaB1 was significantly reduced compared to the wild type protein. Truncation of the extensions further increased the rate of heat-induced precipitation of deamidated betaB1. The presence of the molecular chaperone, alphaA-crystallin, prevented precipitation of modified betaB1s. More alphaA was needed to chaperone the truncated and deamidated betaB1 than deamidated betaB1 or truncated betaB1. CONCLUSIONS: Deamidation and truncation of betaB1 led to destabilization of the protein and decreased stability to heat. Decreased stability of lens crystallins may contribute to their insolubilization and cataract formation.