ßA3/A1-crystallin regulates apical polarity and EGFR endocytosis in retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.

ßA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity.

ßA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as ßA3 and ßA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that ßA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of ßA3/A1-crystallin in metabolism of retinal astrocytes. We found that ßA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but ßA3-crystallin does not. Loss of ßA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited ßA1-knockdown (KD) mice, but not in ßA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified ßA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced ßA1-crystallin and higher levels of PTP1B in the vitreous humor.

Modulating EGFR-MTORC1-autophagy as a potential therapy for persistent fetal vasculature (PFV) disease.

Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway. ABBREVIATIONS: ACTB: actin, beta; CCND3: cyclin 3; CDK6: cyclin-dependent kinase 6; CHQ: chloroquine; COL4A1: collagen, type IV, alpha 1; CRYBA1: crystallin, beta A1; DAPI: 4'6-diamino-2-phenylindole; EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; KDR: kinase insert domain protein receptor; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MKI67: antigen identified by monoclonal antibody Ki 67; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP: poly (ADP-ribose) polymerase family; PCNA: proliferating cell nuclear antigen; PFV: persistent fetal vasculature; PHPV: persistent hyperplastic primary vitreous; RPE: retinal pigmented epithelium; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SQSTM1/p62: sequestome 1; TUBB: tubulin, beta; VCL: vinculin; VEGFA: vascular endothelial growth factor A; WT: wild type.

The identification and characterization of the p.G91 deletion in CRYBA1 in a Chinese family with congenital cataracts.

BACKGROUND: Mutations in more than 52 genes have been identified in isolated congenital cataracts, the majority of which are located in crystalline and connexin (gap junction) genes. An in-frame one amino acid deletion in the beta-crystalline gene CRYBA1 has been reported in several different Chinese, Caucasian and Iranian families of congenital cataracts. Further functional studies are needed to confirm the variant pathogenicity. METHODS: The purpose of this study is to identify the genetic causes that contribute to congenital cataracts with esotropia and nystagmus in a Chinese family. Whole-exome sequencing was performed on samples from all five family members. The two brothers of the father and their daughters were then enrolled in the study, and 40 suspected variants were sequenced among the 9 subjects using Sanger sequencing. The mRNA and protein levels of CRYBA1 in the lens epithelium from cataract patients and normal controls were compared using quantitative polymerase chain reaction (qPCR) and Western blot analyses. The wild-type and mutated forms (p.G91del) of CRYBA1 cDNA were transfected into two types of cell lines, and the expression level of exogenous CRYBA1 was measured by Western blot analysis. The exogenous CRYBA1 proteins were visualized by immunofluorescence staining. RESULTS: In this two-generation family, all three descendants inherited congenital cataracts with esotropia and nystagmus from the father, while the mother's lens was normal. After two rounds of sequencing, CRYBA1 (c. 269-271 del, p.G91del) was identified as the mutation responsible for the autosomal dominant congenital cataract in the Chinese family. CRYBA1 showed lower expression in cataract lenses than in control lenses. The deleted form (p.G91del) of CRYBA1 showed lower expression and was more aggregate to the cell membrane than the wild-type CRYBA1. CONCLUSIONS: We performed molecular experiments to confirm that the p.G91del mutation in CRYBA1 results in abnormal expression and distribution of CRYBA1 protein, and this study could serve as an example of the pathogenicity of an in-frame small deletion in an inherited eye disorder.

Photoreceptor-induced RPE phagolysosomal maturation defects in Stargardt-like Maculopathy (STGD3).

For many neurodegenerative disorders, expression of a pathological protein by one cell type impedes function of other cell types, which in turn contributes to the death of the first cell type. In transgenic mice modelling Stargardt-like (STGD3) maculopathy, human mutant ELOVL4 expression by photoreceptors is associated with defects in the underlying retinal pigment epithelium (RPE). To examine how photoreceptors exert cytotoxic effects on RPE cells, transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates were studied one month prior (preclinical stage) to onset of photoreceptor loss (two months). TG photoreceptor outer segments presented to human RPE cells are recognized and internalized into phagosomes, but their digestion is delayed. Live RPE cell imaging pinpoints decreased numbers of acidified phagolysomes. In vivo, master regulator of lysosomal genes, transcription factor EB (TFEB), and key lysosomal enzyme Cathepsin D are both unaffected. Oxidative stress, as ruled out with high-resolution respirometry, does not play a role at such an early stage. Upregulation of CRYBA1/A3 and phagocytic cells (microglia/macrophages) interposed between RPE and photoreceptors support adaptive responses to processing delays. Impaired phagolysosomal maturation is observed in RPE of mice expressing human mutant ELOVL4 in their photoreceptors prior to photoreceptor death and associated vision loss.

Mutation Analysis of Families with Autosomal Dominant Congenital Cataract: A Recurrent Mutation in the CRYBA1/A3 Gene Causing Congenital Nuclear Cataract.

PURPOSE: To identify the CRYBA1/A3 mutation spectrum and analyze the genotype-phenotype correlations in Chinese families with congenital cataract. METHODS: Family history and clinical data of 47 unrelated families with autosomal dominant congenital cataract (ADCC) were recorded. CRYBA1/A3 gene sequencing was applied to identify the causative mutation. Haplotypes were constructed using closely linked microsatellite markers and intragenic single-nucleotide polymorphisms (SNPs) to compare the affected haplotype in three families. RESULTS: Nuclear cataract was the most common type of ADCC in Chinese families, accounting for 42.6% (20/47). A recurrent CRYBA1/A3 deletion mutation (ΔG91) was identified in three families (6.4%) with nonprogressive nuclear congenital cataract. Different haplotypes segregated with the mutation in each family. CONCLUSIONS: A recurrent ΔG91CRYBA1/A3 mutation occurs independently in 6.4% of the Chinese families with autosomal dominant nuclear cataracts and most likely represents a mutational hot spot, which underscores the relations between nonprogressive nuclear cataract and CRYBA1/A3.

Congenital microcornea-cataract syndrome-causing mutation X253R increases ßB1-crystallin hydrophobicity to promote aggregate formation.

The high solubility and lifelong stability of crystallins are crucial to the maintenance of lens transparency and optical properties. Numerous crystallin mutations have been linked to congenital cataract, which is one of the leading causes of newborn blindness. Besides cataract, several crystallin mutations have also been linked to syndromes such as congenital microcornea-cataract syndrome (CMCC). However, the molecular mechanism of CMCC caused by crystallin mutations remains elusive. In the present study, we investigated the mechanism of CMCC caused by the X253R mutation in ßB1-crystallin. The exogenously expressed X253R proteins were prone to form p62-negative aggregates in HeLa cells, strongly inhibited cell proliferation and induced cell apoptosis. The intracellular X253R aggregates could be successfully redissolved by lanosterol but not cholesterol. The extra 26 residues at the C-terminus of ßB1-crystallin introduced by the X253R mutation had little impact on ßB1-crystallin structure and stability, but increased ßB1-crystallin hydrophobicity and decreased its solubility. Interestingly, the X253R mutant fully abolished the aggregatory propensity of ßB1- and ßA3/ßB1-crystallins at high temperatures, suggesting that X253R was an aggregation-inhibition mutation of ß-crystallin homomers and heteromers in dilute solutions. Our results suggest that an increase in hydrophobicity and a decrease in solubility might be responsible for cataractogenesis induced by the X253R mutation, while the cytotoxic effect of X253R aggregates might contribute to the defects in ocular development. Our results also highlight that, at least in some cases, the aggregatory propensity in dilute solutions could not fully mimic the behaviours of mutated proteins in the crowded cytoplasm of the cells.

ßA3/A1-crystallin and persistent fetal vasculature (PFV) disease of the eye.

BACKGROUND: 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. SCOPE OF REVIEW: 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 ßA3/A1-crystallin. In this review we focus on the possible mechanisms whereby loss of functional ßA3/A1-crystallin might lead to PFV. MAJOR CONCLUSIONS: Cryba1 is abundantly expressed in the lens, but is also expressed in certain other ocular cells, including astrocytes. In animal models lacking ßA3/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 ßA3/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. GENERAL SIGNIFICANCE: 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.

Interaction of ßA3-Crystallin with Deamidated Mutants of αA- and αB-Crystallins.

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and ß-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with ßA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with ßA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for ßA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for ßA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with ßA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ßA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with ßA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor ßA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Effect of Ca2+ and Mg2+ ions on oligomeric state and chaperone-like activity of αB-crystallin in crowded media.

The main function of small heat shock proteins acting as suppressors of aggregation of non-native proteins is greatly influenced by crowded environment in the cell and the presence of divalent metal ions. The goal of the present work was to study the effects of Ca(2+) and Mg(2+) ions on the quaternary structure and anti-aggregation activity of αB-crystallin under crowding conditions. We showed that Ca(2+) and Mg(2+) ions induced formation of suboligomeric forms of αB-crystallin. This effect was retained in the presence of crowder (polyethylene glycol), although to a lesser degree. The chaperone-like activity of αB-crystallin was analyzed using heat-induced aggregation of myosin subfragment 1 (S1) at 40°C. In the absence of crowding agents chaperone-like activity of αB-crystallin exhibited low sensitivity to the presence of Ca(2+) and Mg(2+) ions. The addition of the crowding agents (polyethylene glycol 20000, Ficoll 70) dramatically increased S1 aggregation rates and significantly depressed anti-aggregation activity of αB-crystallin. Low concentrations of Ca(2+) (0.1mM) and Mg(2+) (10mM) partially restored the chaperone-like activity of αB-crystallin in the presence of crowders. This effect was observed at relatively low values of [αB-crystallin]/[S1] molar ratio, however, at [αB-crystallin]/[S1]>0.2 the stimulating effect of Ca(2+) became less pronounced. These findings might indicate that under crowded cell conditions different factors, including divalent cations, can effectively modulate chaperone-like activity of protein chaperones, which otherwise cannot properly cope with crowding-provoked accelerated rates of substrates aggregation.

Alpha B- and ßA3-crystallins containing d-aspartic acids exist in a monomeric state.

Crystallin stability and subunit-subunit interaction are essential for eye lens transparency. There are three types of crystallins in lens, designated as α-, ß-, and γ-crystallins. Alpha-crystallin is a hetero-polymer of about 800kDa, consisting of 35-40 subunits of two different αA- and αB-subunits, each of 20kDa. The ß/γ-crystallin superfamily comprises oligomeric ß-crystallin (2-6 subunits) and monomeric γ-crystallin. Since lens proteins have very long half-lives, they undergo numerous post-translational modifications including racemization, isomerization, deamidation, oxidation, glycation, and truncation, which may decrease crystallin solubility and ultimately cause cataract formation. Racemization and isomerization of aspartyl (Asp) residues have been detected only in polymeric α- and oligomeric ß-crystallin, while the situation in monomeric γ-crystallin has not been studied. Here, we investigated the racemization and isomerization of Asp in the γ-crystallin fraction of elderly donors. The results show that Asp residues of γS-, γD- and γC-crystallins were not racemized and isomerized. However, strikingly, we found that a portion of αB-crystallin and ßA3-crystallin moved to the lower molecular weight fraction which is the same size of γ-crystallin. In those fractions, Asp-96 of αB-crystallin and Asp-37 of ßA3-crystallin were highly inverted, which do not occur in the native lens higher molecular weight fraction. Our results indicate the possibility that the inversion of Asp residues may induce dissociation of αB- and ßA3-crystallins from the polymeric and oligomeric states. This is the first report that stereoinversion of amino acids disturbs lens protein assembly in aged human lens.

Alpha beta-crystallin expression and presentation following infection with murine gammaherpesvirus 68.

Alpha beta-crystallin (CRYAB) is a small heat shock protein that can function as a molecular chaperone and has protective effects for cells undergoing a variety of stressors. Surprisingly, CRYAB has been identified as one of the dominant autoantigens in multiple sclerosis. It has been suggested that autoimmune mediated destruction of this small heat shock protein may limit its protective effects, thereby exacerbating inflammation and cellular damage during multiple sclerosis. It is not altogether clear how autoimmunity against CRYAB might develop, or whether there are environmental factors which might facilitate the presentation of this autoantigen to CD4+ T lymphocytes. In the present study, we utilized an animal model of an Epstein Barr Virus (EBV)-like infection, murine gammaherpesvirus 68 (HV-68), to question whether such a virus could modulate the expression of CRYAB by antigen presenting cells. Following exposure to HV-68 and several other stimuli, in vitro secretion of CRYAB and subsequent intracellular accumulation were observed in cultured macrophages and dendritic cells. Following infection of mice with this virus, it was possible to track CRYAB expression in the spleen and in antigen presenting cell subpopulations, as well as its secretion into the blood. Mice immunized with human CRYAB mounted a significant immune response against this heat shock protein. Further, dendritic cells that were exposed to HV-68 could stimulate CD4+ T cells from CRYAB immunized mice to secrete interferon gamma. Taken together these studies are consistent with the notion of a gammaherpesvirus-induced CRYAB response in professional antigen presenting cells in this mouse model.

Is protein methylation in the human lens a result of non-enzymatic methylation by S-adenosylmethionine?

Since crystallins in the human lens do not turnover, they are susceptible to modification by reactive molecules over time. Methylation is a major post-translational lens modification, however the source of the methyl group is not known and the extent of modification across all crystallins has yet to be determined. Sites of methylation in human lens proteins were determined using HPLC/mass spectrometry following digestion with trypsin. The overall extent of protein methylation increased with age, and there was little difference in the extent of modification between soluble and insoluble crystallins. Several different cysteine and histidine residues in crystallins from adult lenses were found to be methylated with one cysteine (Cys 110 in γD crystallin) at a level approaching 70%, however, methylation of crystallins was not detected in fetal or newborn lenses. S-adenosylmethionine (SAM) was quantified at significant (10-50 µM) levels in lenses, and in model experiments SAM reacted readily with N-α-tBoc-cysteine and N-α-tBoc-histidine, as well as ßA3-crystallin. The pattern of lens protein methylation seen in the human lens was consistent with non-enzymatic alkylation. The in vitro data shows that SAM can act directly to methylate lens proteins and SAM was present in significant concentrations in human lens. Thus, non-enzymatic methylation of crystallins by SAM offers a possible explanation for this major human lens modification.

ßB1-crystallin: thermodynamic profiles of molecular interactions.

BACKGROUND: ß-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both ßA3 and ßB2 crystallins (ßA3 and ßB2) involves endothermic enthalpy of association (∼8 kcal/mol) mediated by hydrophobic interactions. METHODOLOGY/PRINCIPAL FINDINGS: Thermodynamic profiles of the associations of dimeric ßA3 and ßB1 and tetrameric ßB1/ßA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of ßB1 crystallin are dominated by exothermic enthalpy (-13.3 and -24.5 kcal/mol, respectively). CONCLUSIONS/SIGNIFICANCE: Global thermodynamics of ßB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those ß-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations.

The benefits of being ß-crystallin heteromers: ßB1-crystallin protects ßA3-crystallin against aggregation during co-refolding.

ß-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven ß-crystallins, ßA3-crystallin and ßB1-crystallin, an acidic and a basic ß-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic ß-crystallin facilitates the folding of acidic ß-crystallin. Equilibrium folding studies revealed that the ßA3-crystallin and ßB1-crystallin homomers and the ßA3/ßB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. ßA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of ßA3/ßB1-crystallin was close to that of ßB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the ß-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, ßA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by ßB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded ßB1-crystallin. These findings suggested that the formation of ß-crystallin heteromers not only stabilizes the unstable acidic ß-crystallin but also protects them against aggregation during refolding from the stress-denatured states.

A novel mutation in CRYBB1 associated with congenital cataract-microcornea syndrome: the p.Ser129Arg mutation destabilizes the ßB1/ßA3-crystallin heteromer but not the ßB1-crystallin homomer.

Congenital cataract-microcornea syndrome (CCMC) is a clinically and genetically heterogeneous condition characterized by lens opacities and microcornea. It appears as a distinct phenotype of heritable congenital cataract. Here we report a large Chinese family with autosomal dominant congenital cataract and microcornea. Evidence for linkage was detected at marker D22S1167 (LOD score [Z]=4.49, recombination fraction [θ]=0.0), which closely flanks the â-crystallin gene cluster locus. Direct sequencing of the candidate âB1-crystallin gene (CRYBB1) revealed a c.387C>A transversion in exon 4, which cosegregated with the disease in the family and resulted in the substitution of serine by arginine at codon 129 (p.Ser129Arg). A comparison of the biophysical properties of the recombinant ß-crystallins revealed that the mutation impaired the structures of both ßB1-crystallin homomer and ßB1/ßA3-crystallin heteromer. More importantly, the mutation significantly decreased the thermal stability of ßB1/ßA3-crystallin but not ßB1-crystallin. These findings highlight the importance of protein-protein interactions among ß-crystallins in maintaining lens transparency, and provide a novel insight into the molecular mechanism underlying the pathogenesis of human CCMC.

Expression of ßA3/A1-crystallin in the developing and adult rat eye.

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat ßA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). ßA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, ßA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that ßA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of ßA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.

A serine-type protease activity of human lens ßA3-crystallin is responsible for its autodegradation.

PURPOSE: The purpose of the study was to determine whether the autodegradation of human ßA3-crystallin is due to its intrinsic protease activity. METHODS: Recombinant His-tagged human ßA3-crystallin was expressed in E. coli and purified by a Ni+2-affinity column chromatographic method. To determine protease activity, the purified crystallin was incubated for 24 h with either sodium deoxycholate, Triton X-100, or CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate} and with benzoyl DL-arginine p-nitroanilide (BAPNA), a colorimetric protease substrate. The autodegradation of the crystallin at 0 h and 24 h on incubation at 37 °C with and without detergents (CHAPS/Triton X-100) was also determined by sodium dodecylsulfate-PAGE (SDS-PAGE) method. To examine whether the autodegradation of the crystallin was due to its protease activity, the crystallin was incubated with inhibitors of serine-, metallo- and cysteine-proteases. The binding of the intact ßA3-crystallin and its autodegradation products to FFCK [5-carboxyfluorescenyl-1-phenylalaninyl-chloromethyl ketone], an analog of TPCK [1-Chloro-3-tosylamido-4-phenyl-2-butanone, a chymotrypsin-type serine protease inhibitor] was also determined by their incubation followed by SDS-PAGE and scanning for fluorescence using a Typhoon 9400 scanner. RESULTS: ßA3-crystallin protease activity showed activation in the presence of CHAPS but not in presence of Triton X-100. Upon incubation of ßA3-crystallin for 24 h with CHAPS or sodium deoxycholate and BAPNA as a substrate, a time-dependent increase in the Arg-bond hydrolyzing activity was observed. SDS-PAGE analysis exhibited autodegradation products with Mr of 22, 27 and 30 kDa, which on partial NH2-terminal sequencing showed cleavage of Lys17-Met18, Gln4-Ala5 and Thr-Gly (in the NH2-terminal His-tag region) bonds, respectively. Almost no autodegradation of the ßA3-crystallin occurred during its incubation alone or with CHAPS plus serine protease inhibitors (phenylmethylsulfonyl fluoride [PMSF], approtinin, and chymostatin). In contrast, the autodegradation occurred in the presence of metallo-protease inhibitors (EDTA and EGTA) and cysteine protease inhibitors (E-64, N-methylmaleimide and iodoacetamide). The ßA3-crystallin also exhibited binding to FFCK, suggesting existence of a chymotrypsin-type active site in the ßA3-crystallin protease. CONCLUSIONS: The results suggested that a serine-type protease activity of ßA3-crystalllin was responsible for its autodegradation. The specific bonds cleaved during autodegradation (Gln4-Ala5 and Lys17-Met18), were localized in the NH2-terminal arm of ßA3-crystallin.

Decreasing the homodimer interaction: a common mechanism shared by the deltaG91 mutation and deamidation in betaA3-crystallin.

PURPOSE: Cataracts can be broadly divided into two types: congenital cataracts and age-related cataracts. DeltaG91 is a previously discovered congenital mutation in betaA3-crystallin that impairs protein solubility. On the other hand, the deamidation of beta-crystallin is a significant feature in aged and cataractous lenses. Several deamidation sites were also identified in betaA3-crystallin. The present study is to compare the functional consequence of DeltaG91 mutation and the deamidation of betaA3-crystallin in terms of folding properties and protein-protein interaction. METHODS: Protein secondary structure and hydrophobic properties were investigated by in silica analysis of the wild type and mutants sequences. Full-length betaA3-crystallin was cloned into a mammalian two-hybrid system in order to investigate protein-protein interactions. Deletion and deamidation were introduced by site-directed mutagenesis protocols. Both the Q85 and Q180 deamidation sites were substituted with glutamic acid residues to mimic deamidation. Different combinations of plasmid constructs were transfected in HeLa cells, and changes of protein-protein interactions were analyzed by the luciferase assay. RESULTS: Bioinformatics prediction suggested that DeltaG91 mutation alters both the predicted secondary structure and hydrophobic character of betaA3-crystallin, while deamidation only exhibits minimal effects. Mammalian two-hybrid results indicated that both DeltaG91 mutation and Q85/Q180 deamidation could significantly decrease the interaction of the betaA3-crystallin homodimer. CONCLUSION: Our results provided evidence that both mutations involved in congenital cataracts and deamidation in aged lenses commonly altered protein-protein interaction between human lens betaA3-crystallins, which may lead to protein insolubilization and contribute to cataracts.

Neuropathology of Parkinson's disease with the R1441G mutation in LRRK2.

We report the neuropathological findings in a patient with Parkinson's disease (PD) associated with Basque R1441G-LRRK2/dardarin mutation. The patient was a man with disease onset at 68 years of age, with unilateral rest tremor; the Parkinsonism was well controlled with medication for 15 years. He died at the age of 86, after 18 years of evolution. The neuropathological examination disclosed mild neuronal loss in the substantia nigra pars compacta without alpha-synuclein, tau, LRRK2, or ubiquitin cytoplasmic inclusions. Lewy bodies and Lewy neurites were absent. This is the first neuropathological study of PD associated with brain with the R1441G mutation in LRRK2.