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Posner M, Garver T, Kaye T, Brdicka S, Suttle M, Patterson B, Farnsworth DR. Loss of αBa-crystallin, but not αA-crystallin, increases age-related cataract in the zebrafish lens. Exp Eye Res 2024; 244:109918. [PMID: 38705506 DOI: 10.1016/j.exer.2024.109918] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2024] [Revised: 04/26/2024] [Accepted: 04/29/2024] [Indexed: 05/07/2024]
Abstract
The vertebrate eye lens is an unusual organ in that most of its cells lack nuclei and the ability to replace aging protein. The small heat shock protein α-crystallins evolved to become key components of this lens, possibly because of their ability to prevent aggregation of aging protein that would otherwise lead to lens opacity. Most vertebrates express two α-crystallins, αA- and αB-crystallin, and mutations in each are linked to human cataract. In a mouse knockout model only the loss of αA-crystallin led to early-stage lens cataract. We have used the zebrafish as a model system to investigate the role of α-crystallins during lens development. Interestingly, while zebrafish express one lens-specific αA-crystallin gene (cryaa), they express two αB-crystallin genes, with one evolving lens specificity (cryaba) and the other retaining the broad expression of its mammalian ortholog (cryabb). In this study we used individual mutant zebrafish lines for all three α-crystallin genes to determine the impact of their loss on age-related cataract. Surprisingly, unlike mouse knockout models, we found that the loss of the αBa-crystallin gene cryaba led to an increase in lens opacity compared to cryaa null fish at 24 months of age. Loss of αA-crystallin did not increase the prevalence of cataract. We also used single cell RNA-Seq and RT-qPCR data to show a shift in the lens expression of zebrafish α-crystallins between 5 and 10 days post fertilization (dpf), with 5 and 6 dpf lenses expressing cryaa almost exclusively, and expression of cryaba and cryabb becoming more prominent after 10 dpf. These data show that cryaa is the primary α-crystallin during early lens development, while the protective role for cryaba becomes more important during lens aging. This study is the first to quantify cataract prevalence in wild-type aging zebrafish, showing that lens opacities develop in approximately 25% of fish by 18 months of age. None of the three α-crystallin mutants showed a compensatory increase in the expression of the remaining two crystallins, or in the abundant βB1-crystallin. Overall, these findings indicate an ontogenetic shift in the functional importance of individual α-crystallins during zebrafish lens development. Our finding that the lens-specific zebrafish αBa-crystallin plays the leading role in preventing age-related cataract adds a new twist to our understanding of vertebrate lens evolution.
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Affiliation(s)
- Mason Posner
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA.
| | - Taylor Garver
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Taylor Kaye
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Stuart Brdicka
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Madison Suttle
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Bryce Patterson
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Dylan R Farnsworth
- The RNA Institute, University at Albany, State University of New York, Albany, NY, USA
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Posner M, Garver T, Kaye T, Brdicka S, Suttle M, Patterson B, Farnsworth DR. Loss of αBa-crystallin, but not αA-crystallin, increases age-related cataract in the zebrafish lens. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.03.574085. [PMID: 38260567 PMCID: PMC10802301 DOI: 10.1101/2024.01.03.574085] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2024]
Abstract
The vertebrate eye lens is an unusual organ in that most of its cells lack nuclei and the ability to replace aging protein. The small heat shock protein α-crystallins evolved to become key components of this lens, possibly because of their ability to prevent aggregation of aging protein that would otherwise lead to lens opacity. Most vertebrates express two α-crystallins, αA- and αB-crystallin, and mutations in each are linked to human cataract. In a mouse knockout model only the loss of αA-crystallin led to early-stage lens cataract. We have used the zebrafish as a model system to investigate the role of α-crystallins during lens development. Interestingly, while zebrafish express one lens-specific αA-crystallin gene (cryaa), they express two αB-crystallin genes, with one evolving lens specificity (cryaba) and the other retaining the broad expression of its mammalian ortholog (cryabb). In this study we used individual mutant zebrafish lines for all three α-crystallin genes to determine the impact of their loss on age-related cataract. Surprisingly, unlike mouse knockout models, we found that the loss of the αBa-crystallin gene cryaba led to an increase in lens opacity compared to cryaa null fish at 24 months of age. Loss of αA-crystallin did not increase the prevalence of cataract. We also used single cell RNA-Seq and RT-qPCR data to show a shift in the lens expression of zebrafish α-crystallins between 5 and 10 days post fertilization (dpf), with 5 and 6 dpf lenses expressing cryaa almost exclusively, and expression of cryaba and cryabb becoming more prominent after 10 dpf. These data show that cryaa is the primary α-crystallin during early lens development, while the protective role for cryaba becomes more important during lens aging. This study is the first to quantify cataract prevalence in wild-type zebrafish, showing that lens opacities develop in approximately 25% of fish by 18 months of age. None of the three α-crystallin mutants showed a compensatory increase in the expression of the remaining two crystallins, or in the abundant βB1-crystallin. Overall, these findings indicate an ontogenetic shift in the functional importance of individual α-crystallins during zebrafish lens development. Our finding that the lens-specific zebrafish αBa-crystallin plays the leading role in preventing age-related cataract adds a new twist to our understanding of vertebrate lens evolution.
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Affiliation(s)
- Mason Posner
- Department of Biology and Toxicology, Ashland University, Ashland, OH
| | - Taylor Garver
- Department of Biology and Toxicology, Ashland University, Ashland, OH
| | - Taylor Kaye
- Department of Biology and Toxicology, Ashland University, Ashland, OH
| | - Stuart Brdicka
- Department of Biology and Toxicology, Ashland University, Ashland, OH
| | - Madison Suttle
- Department of Biology and Toxicology, Ashland University, Ashland, OH
| | - Bryce Patterson
- Department of Biology and Toxicology, Ashland University, Ashland, OH
| | - Dylan R. Farnsworth
- The RNA InsRtute, University at Albany, State University of New York, Albany, NY
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3
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Posner M, Murray KL, Andrew B, Brdicka S, Roberts A, Franklin K, Hussen A, Kaye T, Kepp E, McDonald MS, Snodgrass T, Zientek K, David LL. Impact of α-crystallin protein loss on zebrafish lens development. Exp Eye Res 2023; 227:109358. [PMID: 36572168 PMCID: PMC9918708 DOI: 10.1016/j.exer.2022.109358] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 11/27/2022] [Accepted: 12/19/2022] [Indexed: 12/24/2022]
Abstract
The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.
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Affiliation(s)
- Mason Posner
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA.
| | - Kelly L Murray
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Brandon Andrew
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Stuart Brdicka
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Alexis Roberts
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Kirstan Franklin
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Adil Hussen
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Taylor Kaye
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Emmaline Kepp
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Mathew S McDonald
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Tyler Snodgrass
- Department of Biology and Toxicology, Ashland University, Ashland, OH, USA
| | - Keith Zientek
- Department of Chemical Physiology & Biochemistry, Oregon Health and Science University, USA
| | - Larry L David
- Department of Chemical Physiology & Biochemistry, Oregon Health and Science University, USA
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Sprague-Piercy MA, Rocha MA, Kwok AO, Martin RW. α-Crystallins in the Vertebrate Eye Lens: Complex Oligomers and Molecular Chaperones. Annu Rev Phys Chem 2021; 72:143-163. [PMID: 33321054 PMCID: PMC8062273 DOI: 10.1146/annurev-physchem-090419-121428] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged β- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state nuclear magnetic resonance (NMR) and solution NMR, as well as cryo-electron microscopy.
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Affiliation(s)
- Marc A Sprague-Piercy
- Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA;
| | - Megan A Rocha
- Department of Chemistry, University of California, Irvine, California 92697, USA
| | - Ashley O Kwok
- Department of Chemistry, University of California, Irvine, California 92697, USA
| | - Rachel W Martin
- Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA;
- Department of Chemistry, University of California, Irvine, California 92697, USA
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What if? Mouse proteomics after gene inactivation. J Proteomics 2019; 199:102-122. [DOI: 10.1016/j.jprot.2019.03.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2018] [Revised: 03/09/2019] [Accepted: 03/10/2019] [Indexed: 12/17/2022]
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Malitan HS, Cohen AM, MacRae TH. Knockdown of the small heat-shock protein p26 by RNA interference modifies the diapause proteome of Artemia franciscana. Biochem Cell Biol 2019; 97:471-479. [PMID: 30620618 DOI: 10.1139/bcb-2018-0231] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Embryos of the crustacean Artemia franciscana may arrest as gastrulae, forming cysts that enter diapause, which is a state of reduced metabolism and enhanced stress tolerance. Diapausing cysts survive physiological stresses for years due, in part, to molecular chaperones. p26, a small heat-shock protein, is an abundant diapause-specific molecular chaperone in cysts, and it affects embryo development and stress tolerance. p26 is therefore thought to influence many proteins in cysts, and this study was undertaken to determine how the loss of p26 by RNA interference (RNAi) affects the diapause proteome of A. franciscana. The proteome was analyzed by shot-gun proteomics coupled to differential isotopic labeling and tandem mass spectrometry. Proteins in the diapause proteome included metabolic enzymes, antioxidants, binding proteins, structural proteins, transporters, translation factors, receptors, and signal transducers. Proteins within the diapause proteome either disappeared or were reduced in amount when p26 was knocked down, or conversely, proteins appeared or increased in amount. Those proteins that disappeared may be p26 substrates, whereas the synthesis of those proteins that appeared or increased may be regulated by p26. This study provides the first global characterization of the diapause proteome of A. franciscana and demonstrates that the sHsp p26 influences proteome composition.
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Affiliation(s)
| | - Alejandro M Cohen
- b Proteomics and Mass Spectrometry Core Facility, Life Sciences Research Institute, Dalhousie University, Halifax, NS B3H 4R2, Canada
| | - Thomas H MacRae
- a Department of Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada
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Proteomic analysis of protein homeostasis and aggregation. J Proteomics 2018; 198:98-112. [PMID: 30529741 DOI: 10.1016/j.jprot.2018.12.003] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2018] [Revised: 11/24/2018] [Accepted: 12/05/2018] [Indexed: 12/13/2022]
Abstract
Protein homeostasis (proteostasis) refers to the ability of cells to preserve the correct balance between protein synthesis, folding and degradation. Proteostasis is essential for optimal cell growth and survival under stressful conditions. Various extracellular and intracellular stresses including heat shock, oxidative stress, proteasome malfunction, mutations and aging-related modifications can result in disturbed proteostasis manifested by enhanced misfolding and aggregation of proteins. To limit protein misfolding and aggregation cells have evolved various strategies including molecular chaperones, proteasome system and autophagy. Molecular chaperones assist folding of proteins, protect them from denaturation and facilitate renaturation of the misfolded polypeptides, whereas proteasomes and autophagosomes remove the irreversibly damaged proteins. The impairment of proteostasis results in protein aggregation that is a major pathological hallmark of numerous age-related disorders, such as cataract, Alzheimer's, Parkinson's, Huntington's, and prion diseases. To discover protein markers and speed up diagnosis of neurodegenerative diseases accompanied by protein aggregation, proteomic tools have increasingly been used in recent years. Systematic and exhaustive analysis of the changes that occur in the proteomes of affected tissues and biofluids in humans or in model organisms is one of the most promising approaches to reveal mechanisms underlying protein aggregation diseases, improve their diagnosis and develop therapeutic strategies. Significance: In this review we outline the elements responsible for maintaining cellular proteostasis and present the overview of proteomic studies focused on protein-aggregation diseases. These studies provide insights into the mechanisms responsible for age-related disorders and reveal new potential biomarkers for Alzheimer's, Parkinson's, Huntigton's and prion diseases.
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8
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Hamilton PD, Andley UP. In vitro interactions of histones and α-crystallin. Biochem Biophys Rep 2018; 15:7-12. [PMID: 30023439 PMCID: PMC6047474 DOI: 10.1016/j.bbrep.2018.05.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2018] [Revised: 05/15/2018] [Accepted: 05/17/2018] [Indexed: 11/15/2022] Open
Abstract
The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone–α-crystallin binding with a Kd of 4 × 10−7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone–α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone–α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.
Histones are upregulated in α-crystallin mutant mice with hereditary cataracts. Isothermal titration calorimetry reveals histones and α-crystallin interact. These interactions are enthalpy and entropy driven. Histones affect the solubility and aggregation behavior of α-crystallin in vitro.
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Affiliation(s)
- Paul D Hamilton
- Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA
| | - Usha P Andley
- Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA
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9
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Andley UP, Tycksen E, McGlasson-Naumann BN, Hamilton PD. Probing the changes in gene expression due to α-crystallin mutations in mouse models of hereditary human cataract. PLoS One 2018; 13:e0190817. [PMID: 29338044 PMCID: PMC5770019 DOI: 10.1371/journal.pone.0190817] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2017] [Accepted: 12/20/2017] [Indexed: 11/30/2022] Open
Abstract
The mammalian eye lens expresses a high concentration of crystallins (α, β and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo.
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Affiliation(s)
- Usha P. Andley
- Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America
- * E-mail:
| | - Eric Tycksen
- Genome Technology Access Center, Washington University in St. Louis, St. Louis, Missouri, United States of America
| | - Brittney N. McGlasson-Naumann
- Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America
| | - Paul D. Hamilton
- Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America
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Koteiche HA, Claxton DP, Mishra S, Stein RA, McDonald ET, Mchaourab HS. Species-Specific Structural and Functional Divergence of α-Crystallins: Zebrafish αBa- and Rodent αA(ins)-Crystallin Encode Activated Chaperones. Biochemistry 2015; 54:5949-58. [PMID: 26378715 DOI: 10.1021/acs.biochem.5b00678] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
In addition to contributing to lens optical properties, the α-crystallins are small heat shock proteins that possess chaperone activity and are predicted to bind and sequester destabilized proteins to delay cataract formation. The current model of α-crystallin chaperone mechanism envisions a transition from the native oligomer to an activated form that has higher affinity to non-native states of the substrate. Previous studies have suggested that this oligomeric plasticity is encoded in the primary sequence and controls access to high affinity binding sites within the N-terminal domain. Here, we further examined the role of sequence variation in the context of species-specific α-crystallins from rat and zebrafish. Alternative splicing of the αA gene in rodents produces αA(ins), which is distinguished by a longer N-terminal domain. The zebrafish genome includes duplicate αB-crystallin genes, αBa and αBb, which display divergent primary sequence and tissue expression patterns. Equilibrium binding experiments were employed to quantitatively define chaperone interactions with a destabilized model substrate, T4 lysozyme. In combination with multiangle light scattering, we show that rat αA(ins) and zebrafish α-crystallins display distinct global structural properties and chaperone activities. Notably, we find that αA(ins) and αBa demonstrate substantially enhanced chaperone function relative to other α-crystallins, binding the same substrate more than 2 orders of magnitude higher affinity and mimicking the activity of fully activated mammalian small heat shock proteins. These results emphasize the role of sequence divergence as an evolutionary strategy to tune chaperone function to the requirements of the tissues and organisms in which they are expressed.
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Affiliation(s)
- Hanane A Koteiche
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States
| | - Derek P Claxton
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States
| | - Sanjay Mishra
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States
| | - Richard A Stein
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States
| | - Ezelle T McDonald
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States
| | - Hassane S Mchaourab
- Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States
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Haslbeck M, Peschek J, Buchner J, Weinkauf S. Structure and function of α-crystallins: Traversing from in vitro to in vivo. Biochim Biophys Acta Gen Subj 2015; 1860:149-66. [PMID: 26116912 DOI: 10.1016/j.bbagen.2015.06.008] [Citation(s) in RCA: 75] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2015] [Revised: 06/10/2015] [Accepted: 06/22/2015] [Indexed: 12/11/2022]
Abstract
BACKGROUND 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. SCOPE OF THE REVIEW 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. MAJOR CONCLUSIONS 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. GENERAL SIGNIFICANCE 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.
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Affiliation(s)
- Martin Haslbeck
- Center for Integrated Protein Science at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany
| | - Jirka Peschek
- Center for Integrated Protein Science at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany
| | - Johannes Buchner
- Center for Integrated Protein Science at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany.
| | - Sevil Weinkauf
- Center for Integrated Protein Science at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany.
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12
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Vedell PT, Townsend RR, You M, Malone JP, Grubbs CJ, Bland KI, Muccio DD, Atigadda VR, Chen Y, Vignola K, Lubet RA. Global molecular changes in rat livers treated with
RXR
agonists: a comparison using transcriptomics and proteomics. Pharmacol Res Perspect 2014. [DOI: 10.1002/prp2.74] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
Affiliation(s)
- Peter T. Vedell
- Department of Pharmacology Medical College of Wisconsin Cancer Center Milwaukee Wisconsin 53226
| | - Reid R. Townsend
- Department of Internal Medicine Washington University School of Medicine St. Louis Missouri 63110
| | - Ming You
- Department of Pharmacology Medical College of Wisconsin Cancer Center Milwaukee Wisconsin 53226
| | - James P. Malone
- Department of Internal Medicine Washington University School of Medicine St. Louis Missouri 63110
| | - Clinton J. Grubbs
- Department of Surgery University of Alabama at Birmingham Birmingham Alabama 35294
| | - Kirby I. Bland
- Department of Surgery University of Alabama at Birmingham Birmingham Alabama 35294
| | - Donald D. Muccio
- Department of Chemistry University of Alabama at Birmingham Birmingham Alabama 35294
| | - Venkatram R. Atigadda
- Department of Chemistry University of Alabama at Birmingham Birmingham Alabama 35294
| | - Yang Chen
- Department of Science Development Metabolon Research Triangle Park North Carolina 27709
| | - Katie Vignola
- Department of Science Development Metabolon Research Triangle Park North Carolina 27709
| | - Ronald A. Lubet
- Chemoprevention Agent Development Research Group National Cancer Institute Rockville Maryland 20892
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13
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Elsobky S, Crane AM, Margolis M, Carreon TA, Bhattacharya SK. Review of application of mass spectrometry for analyses of anterior eye proteome. World J Biol Chem 2014; 5:106-114. [PMID: 24921002 PMCID: PMC4050106 DOI: 10.4331/wjbc.v5.i2.106] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/19/2013] [Revised: 01/16/2014] [Accepted: 03/04/2014] [Indexed: 02/05/2023] Open
Abstract
Proteins have important functional roles in the body, which can be altered in disease states. The eye is a complex organ rich in proteins; in particular, the anterior eye is very sophisticated in function and is most commonly involved in ophthalmic diseases. Proteomics, the large scale study of proteins, has greatly impacted our knowledge and understanding of gene function in the post-genomic period. The most significant breakthrough in proteomics has been mass spectrometric identification of proteins, which extends analysis far beyond the mere display of proteins that classical techniques provide. Mass spectrometry functions as a “mass analyzer” which simplifies the identification and quantification of proteins extracted from biological tissue. Mass spectrometric analysis of the anterior eye proteome provides a differential display for protein comparison of normal and diseased tissue. In this article we present the key proteomic findings in the recent literature related to the cornea, aqueous humor, trabecular meshwork, iris, ciliary body and lens. Through this we identified unique proteins specific to diseases related to the anterior eye.
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Andley UP, Malone JP, Townsend RR. In vivo substrates of the lens molecular chaperones αA-crystallin and αB-crystallin. PLoS One 2014; 9:e95507. [PMID: 24760011 PMCID: PMC3997384 DOI: 10.1371/journal.pone.0095507] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2014] [Accepted: 03/26/2014] [Indexed: 12/11/2022] Open
Abstract
αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.
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Affiliation(s)
- Usha P. Andley
- Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States of America
- * E-mail:
| | - James P. Malone
- Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America
| | - R. Reid Townsend
- Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America
- Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America
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Shang F, Wilmarth PA, Chang ML, Liu K, David LL, Caceres MA, Wawrousek E, Taylor A. Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin. J Proteome Res 2014; 13:1177-89. [PMID: 24450463 PMCID: PMC3993935 DOI: 10.1021/pr400801v] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
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Ubiquitin is a tag that often initiates
degradation of proteins by
the proteasome in the ubiquitin proteasome system. Targeted expression
of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in
lens development and cataract formation, suggesting critical functions
for ubiquitin in lens. To study the developmental processes that require
intact ubiquitin, we executed the most extensive characterization
of the lens proteome to date. We quantified lens protein expression
changes in multiple replicate pools of P1 wild-type and K6W-Ub-expressing
mouse lenses. Lens proteins were digested with trypsin, peptides were
separated using strong cation exchange and reversed-phase liquid chromatography,
and tandem mass (MS/MS) spectra were collected with a linear ion trap.
Transgenic mice that expressed low levels of K6W-Ub (low expressers)
had normal, clear lenses at birth, whereas the lenses that expressed
high levels of K6W-Ub (higher expressers) had abnormal lenses and
cataracts at birth. A total of 2052 proteins were identified, of which
996 were reliably quantified and compared between wild-type and K6W-Ub
transgenic mice. Consistent with a delayed developmental program,
fiber-cell-specific proteins, such as γ-crystallins (γA,
γB, γC, and γE), were down-regulated in K6W-Ub higher
expressers. Up-regulated proteins were involved in energy metabolism,
signal transduction, and proteolysis. The K6W-Ub low expressers exhibited
delayed onset and milder cataract consistent with smaller changes
in protein expression. Because lens protein expression changes occurred
prior to lens morphological abnormalities and cataract formation in
K6W-Ub low expressers, it appears that expression of K6W-Ub sets in
motion a process of altered protein expression that results in developmental
defects and cataract.
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Affiliation(s)
- Fu Shang
- Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University , 711 Washington Steet, Boston, Massachusetts 02111, United States
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