αA and αB peptides from human cataractous lenses show antichaperone activity and enhance aggregation of lens proteins.

Purpose: To identify and characterize properties of αA- and αB-crystallins' low molecular weight peptides (molecular weight [Mr] < 5 kDa) that were present in a 62-year-old human nuclear cataract, but not in normal 62-year-old human lenses. Methods: Low molecular weight peptides (< 5 kDa) were isolated with a trichloroacetic acid (TCA) solubilization method from water-soluble (WS) and water-insoluble (WI) proteins of nuclear cataractous lenses of a 62-year-old donor and normal human lenses from an age-matched donor. Five commercially synthesized peptides (found only in cataractous lenses and not in normal lenses) were used to determine their chaperone and antichaperone activity and aggregation properties. Results: Mass spectrometric analysis showed 28 peptides of αA-crystallin and 38 peptides of αB-crystallin were present in the cataractous lenses but not in the normal lenses. Two αA peptides (named αAP1 and αAP2; both derived from the αA N-terminal domain (NTD) region) and three αB peptides (named αBP3, αBP4, and αBP5, derived from the αB NTD-, core domain (CD), and C-terminal extension (CTE) regions, respectively) were commercially synthesized. αAP1 inhibited the chaperone activity of αA- and αB-crystallins, but the other four peptides (αAP2, αBP3, αBP4, and αBP5) exhibited mixed effects on chaperone activity. Upon incubation with human WS proteins and peptides in vitro, the αBP4 peptide showed higher aggregation properties relative to the αAP1 peptide. During in vivo experiments, the cell-penetrating polyarginine-labeled αAP1 and αBP4 peptides showed 57% and 85% aggregates, respectively, around the nuclei of cultured human lens epithelial cells compared to only 35% by a scrambled peptide. Conclusions: The antichaperone activity of the αAP1 peptide and the aggregation property of the αBP4 peptide with lens proteins could play a potential role during the development of lens opacity.

Increased Association of Deamidated αA-N101D with Lens membrane of transgenic αAN101D vs. wild type αA mice: potential effects on intracellular ionic imbalance and membrane disorganization.

We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. METHODS: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice. RESULTS: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. CONCLUSIONS: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Lens epithelial cells-induced pluripotent stem cells as a model to study epithelial-mesenchymal transition during posterior capsular opacification.

The overall goal was to generate an epithelial-mesenchymal transition (EMT) model using lens epithelial cells-induced pluripotent stem cells to elucidate EMT-regulatory factors during posterior capsular opacification (PCO). For this purpose, the mouse lens epithelial cells-derived mesenchymal cells were reprogrammed to induced pluripotent stem cells (iPSC) and differentiated to lens epithelial cells to be used to determine regulatory factors during EMT. Lens epithelial cells from one-month-old C57BL/6 mice were transitioned to mesenchymal cells in culture, and were reprogrammed to iPSC by delivering reprogramming factors in a single polycistronic lentiviral vector (co-expressing four transcription factors, Oct 4, Sox2, Klf4, and Myc). iPSC were differentiated to epithelial cells by a three-step process using noggin, basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and Wnt-3. At various time points, the cells/clones were immunocytochemically analyzed for epithelial cell markers (Connexin-43 and E-cadherin), mesenchymal cell markers (Alpha-smooth muscle actin), stem cell markers (Sox1, Oct4, SSEA4 and Tra60) and lens-specific epithelial cell markers (αA- and ßA3/A1-crystallins). By increasing the number of genetic transductions, the time needed for generating iPSC from lens mesenchymal cells was reduced, successfully reprogrammed epithelial/mesenchymal cells into iPSC, and retransformed iPSC into lens epithelial cells by the growth factors' treatment. The epithelial cells could serve as a model system to elucidate regulatory factors involved during EMT to therapeutically stop it.

Human alpha A-crystallin missing N-terminal domain poorly complexes with filensin and phakinin.

The aim of this study was to determine relative importance of N-terminal domain and C-terminal extension of αA-crystallin during their in vitro complex formation with phakinin and filensin (the two lens-specific intermediate filament [IF] proteins). Cloned phakinin, filensin and vimentin were purified under a denaturing conditions by consecutive DEAE-cellulose-, hydroxyapatite- and Sephadex G-75-column chromatographic methods. WTαA-crystallin, αA-NT (N-terminal domain [residue number 1-63])-deleted and αA-CT (C-terminal terminal extension [residue number 140-173]-deleted), were cloned in pET 100 TOPO vector, expressed in BL-21 (DE3) cells using 1% IPTG, and purified using a Ni2+-affinity column. The following two in vitro methods were used to determine complex formation of WT-αA, αA-NT, or αA-CT with phakinin, filensin or both phakinin plus filensin together: an ultracentrifugation sedimentation (centrifugation at 80,000 × g for 30 min at 20 °C) followed by SDS-PAGE analysis, and an electron microscopic analysis. In the first method, the individual control proteins (WT-αA, αA-NT and αA-CT crystallin species) remained in the supernatant fractions whereas phakinin, filensin, and vimentin were recovered in the pellet fractions. On complex formation by individual WT-αA-, αA-NT or αA-CT-species with filensin, phakinin or both phakinin and filensin, WT-αA and αA-CT were recovered in the pellet fraction with phakinin, filensin or both filensin and phakinin, whereas αA-NT remained mostly in the supernatant, suggesting its poor complex formation property. EM-studies showed filamentous structure formation between WT-αA and αA-CT with phakinin or filensin, or with both filensin and phakinin together but relatively poor filamentous structures with αA-NT. Together, the results suggest that the N-terminal domain of αA-crystallin is required during in vitro complex formation with filensin and phakinin.

Molecular mechanism of formation of cortical opacity in CRYAAN101D transgenic mice.

PURPOSE: The CRYAAN101D transgenic mouse model expressing deamidated αA-crystallin (deamidation at N101 position to D) develops cortical cataract at the age of 7 to 9 months. The present study was carried out to explore the molecular mechanism that leads to the development of cortical opacity in CRYAAN101D lenses. METHODS: RNA sequence analysis was carried out on 2- and 4-month-old αA-N101D and wild type (WT) lenses. To understand the biologic relevance and function of significantly altered genes, Ingenuity Pathway Analysis (IPA) was done. To elucidate terminal differentiation defects, immunohistochemical, and Western blot analyses were carried out. RESULTS: RNA sequence and IPA data suggested that the genes belonging to gene expression, cellular assembly and organization, and cell cycle and apoptosis networks were altered in N101D lenses. In addition, the tight junction signaling and Rho A signaling were among the top three canonical pathways that were affected in N101D mutant. Immunohistochemical analysis identified a series of terminal differentiation defects in N101D lenses, specifically, increased proliferation and decreased differentiation of lens epithelial cells (LEC) and decreased denucleation of lens fiber cells (LFC). The expression of Rho A was reduced in different-aged N101D lenses, and, conversely, Cdc42 and Rac1 expressions were increased in the N101D mutants. Moreover, earlier in development, the expression of major membrane-bound molecular transporter Na,K-ATPase was drastically reduced in N101D lenses. CONCLUSIONS: The results suggest that the terminal differentiation defects, specifically, increased proliferation and decreased denucleation are responsible for the development of lens opacity in N101D lenses.

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin.

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.

Simultaneous ethambutol & isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

BACKGROUND & OBJECTIVE: There is a need to understand the nature of drug resistance patterns and predictors of emergence of drug resistance in Mycobacterium tuberculosis. There could be common factors/mechanisms for resistance to the drugs, isoniazid and ethambutol, both acting on cell wall. The present study was conducted to analyze the antimycobacterial susceptibility patterns of M. tuberculosis isolates to determine the minimum inhibitory concentrations (MICs) of ethambutol for M. tuberculosis; and to find out possible association of ethambutol resistance with isoniazid resistance. METHODS: A total of 380 M. tuberculosis isolates were tested for their susceptibilities to ethambutol at 2, 4, 6 microg/ml, isoniazid at 1 microg/ml and rifampicin at 64 microg/ml using MIC method. RESULTS: 44.21, 24.73 and 14.21 per cent isolates were resistant to ethambutol at concentrations of 2, 4 and 6 microg/ml respectively. At 6 microg/ml of ethambutol concentration, 85.18 per cent ethambutol resistant isolates were resistant to isoniazid also. At the same ethambutol concentration a fraction of 28.75 per cent isoniazid resistant isolates were ethambutol resistant. INTERPRETATION & CONCLUSION: Ethambutol resistance was accompanied with isoniazid resistance in a large percentage of isolates whereas ethambutol resistance was weakly linked with multidrug resistance. On the other hand, association between isoniazid and ethambutol resistance was weak showing one way linkage.

Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses.

PURPOSE: The aim of the study was to comparatively analyze crystallin fragments in the water soluble high molecular weight (WS-HMW) and in the water insoluble (WI) protein fractions of human cataractous (with nuclear opacity) and age matched normal lenses to determine the identity of crystallin species that show cataract specific changes such as truncation and post-translational modifications. Because these changes were cataract specific and not aging specific, the results were expected to provide information regarding potential mechanisms of age related cataract development. METHODS: The WS-alpha-crystallin, WS-HMW protein, and WI protein fractions were isolated from normal lenses of different ages and from cataractous lenses. The three fractions were subjected to two dimensional (2D) gel electrophoresis (IEF in the first dimension and SDS-PAGE in the second dimension). Individual spots from 2D gels were trypsin digested and the tryptic fragments were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. RESULTS: The 2D protein profiles of WS-alpha-crystallin fractions of normal human lenses showed an age related increase in the number of crystallin fragments. In young normal lenses, the WS-alpha-crystallin fragments were mostly C-terminally truncated, but in older lenses these were both N- and C-terminally truncated. The WS-HMW protein fraction from normal lenses contained mainly fragments of alphaA- and alphaB-crystallin, whereas additional fragments of betaB1- and betaA3-crystallin were present in this fraction from cataractous lenses. Similarly, the WI proteins in normal lenses contained fragments of alphaA- and alphaB-crystallin, but cataractous lenses contained additional fragments of betaA3- and betaB1-crystallin. The modifications identified in the WS-HMW and WI crystallin species of cataractous lenses were truncation, oxidation of Trp residues, and deamidation of Asn to Asp residues. CONCLUSIONS: The results show that the components of WS-HMW and WI protein fractions of cataractous lenses differed from normal lenses. Selective insolubilization of fragments of betaA3/A1- and betaB1-crystallin occurred during cataract development compared to normal lenses. Further, the crystallin species of cataractous lenses showed increased truncation, deamidation of Asn to Asp residues, and oxidation of Trp residue.

Characterization of covalent multimers of crystallins in aging human lenses.

The purpose of this study was to characterize covalent multimers with molecular mass of >90 kDa in the water-insoluble (WI) proteins of aging human lenses. The experimental approach was to first separate the multimers (molecular mass >90 kDa) as individual spots by two-dimensional gel electrophoresis and next analyze compositions of each multimers by matrix-assisted laser desorption ionization-time of flight and electrospray ionization-tandem mass spectrometric (ES-MS/MS) methods. The WI proteins from lenses of 25- and 41-year-old subjects showed distinct 5- and 16-multimer spots on two-dimensional gels, respectively, but the spots from 52- and 72-year-old lenses were non-descript and diffused. ES-MS/MS analyses showed two types of covalent multimers in 25- and 41-year-old lenses, i.e. the first type composed of fragments of eight different crystallins (i.e. alphaA, alphaB, betaA3, betaA4, betaB1, betaB2, gammaS, and gammaD), and the second type of alpha-, beta-, and gamma-crystallins (possibly fragments) and two beaded filament proteins (phakinin and filensin). The most commonly identified species in the complexes of 41-year-old lenses were: alphaA-fragment (C-terminally truncated, residues 1-157), alphaB-fragment (residues 83-90), betaB1-crystallin (residues 60-71), betaA3 (residues 33-44), betaA4 (residues 106-117), filensin (residues 78-90), and phakinin (residues 77-89). Three post-translational modifications (i.e. oxidation of Met and Trp, conversion of Ser to dehydroalanine, and formylation of His) were observed in alphaA-crystallin fragment, and the first two modifications could cross-link proteins. Together, the results suggested that covalent multimers appeared early in life (i.e. 25 years of age) and increased in number with aging, and the two beaded filament proteins form covalent complexes with crystallin fragments in vivo.

Crosslinking of human lens 9 kDa gammaD-crystallin fragment in vitro and in vivo.

PURPOSE: [corrected] The aims of this study were to determine in vitro crosslinking of a 9 kDa gammaD-crystallin fragment alone and with alpha-, beta-, or gamma-crystallins, the existence of covalent multimers of the polypeptide in vivo, and posttranslational modifications in the three isoforms of the polypeptide. METHODS: A mixture of crystallin fragments (3-14 kDa), a 9 kDa gammaD-crystallin polypeptide or the polypeptide and individual alpha-, beta-, or gamma-crystallins, were incubated at 37 degrees C for a desired length of time and the crosslinked species were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion Agarose A 1.5 gel chromatography, and western blot analysis. In addition, the existence of covalent multimers of the 9 kDa polypeptide in human lens water soluble (WS) and water insoluble (WI) protein fractions of normal and cataractous human lenses was determined by western blot analyses. The posttranslationally modified amino acids of three isofroms of the polypeptide were identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and ES-MS/MS mass spectrometric analyses. RESULTS: Following incubation of a mixture of the crystallin fragments or the 9 kDa polypeptide, covalently crosslinked species held via non-disulfide bonding were seen on SDS-PAGE analysis. The polypeptide also exhibited crosslinking with individual alpha-, beta-, and gamma-crystallins. After western blot analysis with site specific anti-9 kDa antibodies, both WS and WI protein fractions from normal and cataractous lenses showed immunoreactive 27 and 45 kDa multimers. The mass spectrometric analysis of the three isoforms of the polypeptide (with identical molecular weight but different charges) showed oxidized methionine and tryptophan residues, with the latter residue containing two oxygens. CONCLUSIONS: The data suggest that a 9 kDa gammaD-crystallin fragment demonstrated crosslinking properties, which might be due to oxidation of its methionine and tryptophan residues.

Existence of deamidated alphaB-crystallin fragments in normal and cataractous human lenses.

PURPOSE: The aims of this study were to characterize lens crystallin fragments having a molecular mass of <10 kDa, isolated by solubilization in trichloroacetic acid, in order to identify cleavage sites in the parent crystallins for their origin and determine post-translational modifications in the fragments. METHODS: The water-soluble (WS) and water-insoluble (WI) protein fractions were isolated from normal human lenses of 60 to 80 year old donors and from age-matched cataractous lenses. Both WS and WI protein fractions were treated with TCA at 60 degrees C for 2 h and the TCA-soluble fractions were recovered following centrifugation. The preparations were dialyzed against H2O to remove TCA, concentrated by lyophilization and subjected to two dimensional gel electrophoresis (2D-GE). The spots from 2D-gels were analyzed by western blot analysis, partial N-terminal sequencing, or excised for mass spectrometric analysis. RESULTS: SDS-PAGE analysis showed that TCA solubilized polypeptides having a molecular mass of <10 kDa from both WS and WI protein fractions of normal and cataractous lenses. Following 2D-GE of TCA-solubilized species from normal lenses, 8 and 5 polypeptides from the WS and WI protein fractions, respectively, were observed. Using similar 2D-GE analysis of TCA solubilized species from cataractous lenses, 9 and 5 polypeptides from WS and WI protein fractions, respectively, were seen. Partial N-terminal sequence analysis showed that the majority of the polypeptides from both WS and WI protein fractions of normal and cataractous lenses were derived from alphaB-crystallin following cleavage at the D129-P130 bond. Western blot and partial N-terminal sequence analyses identified three additional 4-kDa alphaA-crystallin fragments with cleavage at the D136-G137 bond in the WS proteins from normal lenses. MALDI-TOF mass spectrometric analysis showed that all TCA soluble polypeptides from cataractous lenses, except one from normal lenses, contained residue number 130 to 175 from alphaB-crystallin. No further truncation occurred at the C-terminal region of the alphaB-crystallin polypeptides. Following comparison of the isotopic distribution in MALDI-TOF profiles of a tryptic fragment having a mass of 2,014 among the alphaB-crystallin polypeptides, a gain of one single Dalton was observed. This suggested deamidation of the N146 residue in alphaB-crystallin fragments. CONCLUSIONS: The results show that the N146 residue in human alphaB-crystallin undergoes in vivo deamidation and several fragments containing this modification exist in both WS and WI protein fractions of normal and cataractous human lenses.