Protein-protein interactions involving congenital cataract T5P gammaC-crystallin mutant: a confocal fluorescence microscopy study.

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein-protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy. The method detects protein-protein interactions in the natural environment of living cells. Crystallin genes (CRYGC T5P, CRYGC, and CRYAA) were fused to either the green fluorescence protein (GFP) or red fluorescence protein (DsRED or RFP) vector. Each of the following GFP-RFP (donor-acceptor) plasmid pairs was cotransfected into HeLa cells: gammaC-gammaC, gammaC-gammaCT5P, gammaCT5P-gammaCT5P, alphaA-gammaC, and alphaA-gammaCT5P. After culture, confocal fluorescence cell images were taken. Protein-protein interactions in the form of net FRET were evaluated. The confocal fluorescence images show that cells expressing T5P gammaC-crystallin contain many protein aggregates, but cells co-expressing with either gammaC- or alphaA-crystallin reduce the aggregation considerably. FRET determination indicates that gammaCT5P-gammaCT5P shows less protein-protein interaction than either gammaC-gammaC or gammaC-gammaCT5P. Cotransfection with alphaA-crystallin (alphaA-gammaC or alphaA-T5PgammaC) increases nFRET compared with gammaC-gammaC or gammaC-T5PgammaC. Our results demonstrate that T5P gammaC-crystallin shows more protein aggregates and less protein-protein interaction than WT gammaC-crystallin. Chaperone alphaA-crystallin can rescue T5P gammaC-crystallin from aggregation through increased protein interaction. The formation of congenital cataract may be due to reduced protein-protein interactions and increased aggregation from an insufficient amount of alpha-crystallin for protection.

Methylglyoxal induces prostaglandin E2 production in rat mesangial cells.

The formation of methylglyoxal (MG), a reactive dicarbonyl compound, is accelerated under hyperglycemia, presumably contributing to tissue injury in diabetes. On the other hand, prostaglandin E2 (PGE2) has been implicated in glomerular hyperfiltration, a characteristic change in the early stage of diabetic nephropathy. We therefore examined whether MG was capable of inducing PGE2 production in rat mesangial cells (RMC) to address a possible mechanism by which hyperglycemia-derived dicarbonyls accelerated the development of diabetic nephropathy. RMC were incubated with 0 - 200 microM of MG, followed by determination of secreted PGE2 by enzyme immunoassay (EIA). We further investigated the intracellular mechanisms mediating the MG-induced PGE2 synthesis, focusing particularly on cyclooxygenase-2 (COX-2) and the MAPK superfamily. Our results indicated that MG induced PGE2 production in a dose-dependent manner, accompanied by augmentation of COX-2 mRNA expression. This MG-induced PGE2 production was significantly suppressed by inhibiting either ERK1/2 or p38 MAPK, implicating involvement of the MAPK superfamily. Our results suggest a potential role of MG in the development of diabetic nephropathy through PGE2 production, and may serve as a novel insight into the therapeutic strategies for diabetic nephropathy.

Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching.

PURPOSE: The R120G mutation of alphaB-crystallin is known to cause desmin-related myopathy, but the mechanisms underlying the formation of cataract are not clearly established. We hypothesize that alteration of protein-protein interaction between R120G alphaB-crystallin and lens intermediate filament proteins is one of the mechanisms of congenital cataract. METHODS: Protein-protein interactions were determined by confocal fluorescence resonance energy transfer (FRET) microscopy using green fluorescence protein (GFP) as the donor and red fluorescence protein (RFP) as the acceptor. The lens vimentin gene was fused into a GFP vector and the alphaB-crystallin (WT or R120G mutant) gene was fused into the RFP vector. The donor-acceptor plasmid pairs of intermediate filament (IF)-GFP and alphaB-RFP were co-transfected into HeLa cells. After incubation, confocal fluorescence images of the transfected cells were taken. FRET was estimated by the acceptor photobleaching method. Protein-protein interaction was evaluated by FRET efficiency. RESULTS: The confocal fluorescence images showed that the cells expressing vimentin and R120G alphaB-crystallin contained large amounts of protein aggregates while few vimentin fibers were observed. FRET efficiency analyses indicated that vimentin had a significantly greater protein-protein interaction with R120G alphaB-crystallin than with WT alphaB-crystallin. CONCLUSIONS: Our results show that the R120G alphaB-crystallin mutant promoted vimentin aggregation through increased protein-protein interaction. This process may contribute to the formation of congenital cataract.

Confocal fluorescence microscopy study of interaction between lens MIP26/AQP0 and crystallins in living cells.

MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only alpha-crystallin or some specific gamma-crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (alphaA-, alphaB-, betaB2-, or gammaC-crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed-through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from alphaA- and alphaB-crystallin to betaB2- and gammaC-crystallin. Competitive interaction study using untagged alphaA-crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and alphaA-crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm.

Protein-protein interactions among human lens acidic and basic beta-crystallins.

Human lens beta-crystallin contains four acidic (betaA1-->betaA4) and three basic (betaB1-->betaB3) subunits. They oligomerize in the lens, but it is uncertain which subunits are involved in the oligomerization. We used a two-hybrid system to detect protein-protein interactions systematically. Proteins were also expressed for some physicochemical studies. The results indicate that all acidic-basic pairs (betaA-betaB) except betaA4-betaBs pairs show strong hetero-molecular interactions. For acidic or basic pairs, only two pairs (betaA1-betaA1 and betaA3-betaA3) show strong self-association. betaA2 and betaA4 show very weak self-association, which arises from their low solubility. Confocal fluorescence microscopy shows enormous protein aggregates in betaA2- or betaA4-crystallin transfected cells. However, coexpression with betaB2-crystallin decreased both the number and size of aggregates. Circular dichroism indicates subtle differences in conformation among beta-crystallins that may have contributed to the differences in interactions.

Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells.

PURPOSE: To determine protein-protein interactions among lens crystallins in living cells. METHODS: Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (alphaA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: alphaB-, betaB2-, gammaC-crystallin, and R120G alphaB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells. After culture, confocal microscopy images were taken and FRET values were calculated. RESULTS: FRET occurs when the two proteins interact. The data show strong interactions between alphaA- and alphaB-crystallin and weak interactions between alphaA- and betaB2- or gammaC-crystallin, which is consistent with our previous two-hybrid system study. The R120G alphaB-crystallin mutant, however, showed significantly less FRET than wild-type alphaB-crystallin. There are also more R120G alphaB-crystallin transfected cells with protein aggregates than wild-type alphaB-crystallin transfected cells. Cotransfection with alphaA-crystallin could not rescue R120G alphaB-crystallin from aggregation. CONCLUSIONS: FRET microscopy gave excellent results on the protein-protein interactions among crystallins. It supports many previous studies and provides a novel technique for further study of protein-protein interactions among lens proteins including membrane and cytoskeletal proteins.

Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants.

Lens alpha-crystallin is an oligomeric protein with a molecular mass of 500-1000 kDa and a polydispersed assembly. It consists of two types of subunits, alphaA and alphaB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F alphaA-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C alphaA, wild-type and R120G alphaB, wild-type and Q155* betaB2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C alphaA and R120G alphaB mutations despite conformational changes.

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27.

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.

Domain interaction sites of human lens betaB2-crystallin.

betaB2-crystallin, the major component of beta-crystallin, is a dimer at low concentrations but can form oligomers under physiological conditions. The interaction domains have been speculated to be the beta-sheets, each of which is formed by two or more beta-strands. betaB2-crystallin consists of 16 beta-strands, 8 in the N-terminal domain and 8 in the C-terminal domain. Domain interaction sites may be removed by destroying the beta-strands, which can be done by site-specific mutations, substituting the beta-formers (Val, Phe, Leu) with Glu or Asn, strong beta-breakers. We have cloned the following beta-strand-deleted mutants, I20E, L34E, V54E, V60E, V73E, L97E, I109E, I124E, V144E, V152E, L162E, L165E, and V187E and their corresponding X --> Asn mutants. We also made two mutants, V46E and V129E, that were not on the beta-strand as controls. Disruption of protein-protein interactions was screened by a mammalian two-hybrid system assay. Protein-protein interactions decreased for all beta-strand-deleted mutants except I20E, L34E, and L162E mutants; this effect was not seen in the two mutant controls, V46E and V129E. The sequences around Val-54, Val-60, Val-73, and Leu-97 in the N-terminal region and Ile-109, Ile-124, Val-144, Val-152, Leu-165, and Val-187 in the C-terminal region that formed beta-strands appear to be important in dimerization. Some selected mutant proteins that showed strong (V46E and V129E) and reduced (V60E, V144E, V60N, and V144N) interactions were expressed in bacterial culture and were studied with spectroscopy and chromatography. The V60E and V144E mutants were found to be partially unfolded and incapable of forming a complete dimer.

Interaction and biophysical properties of human lens Q155* betaB2-crystallin mutant.

PURPOSE: Missense mutations in crystallin genes have been identified in autosomal dominant congenital cataracts. A truncation in the CRYBB2 gene (Q155*) has been associated with cerulean cataract, however its effects on biophysical properties have not been reported. We sought to determine the changes in conformation and protein-protein interactions brought about by this mutation. METHODS: Site specific mutations were performed to obtain the Q155* betaB2-crystallin mutant. Protein-protein interactions were screened by a mammalian two-hybrid system assay. Conformational changes were studied with spectroscopy (circular dichroism and fluorescence) and FPLC chromatography. RESULTS: We detected a decrease in protein-protein interactions for the Q155* betaB2-crystallin mutant. The Q155* mutant shows decreased ordered structure and stability but the partially unfolded protein retains some dimer structure. CONCLUSIONS: The Q155* mutation in betaB2-crystallin causes changes in biophysical properties that might contribute to cataract formation.

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells.

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-L-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.

Relation between serum 3-deoxyglucosone and development of diabetic microangiopathy.

OBJECTIVE: 3-Deoxyglucosone (3-DG), a highly reactive intermediate of the glycation reaction, has been suggested to contribute to the development of diabetes complications. To verify this hypothesis, we assessed the relation between serum 3-DG concentrations and the severity of diabetic microangiopathy in diabetic patients. RESEARCH DESIGN AND METHODS: We conducted a high-performance liquid chromatography assay to determine the serum 3-DG concentrations of 110 diabetic patients with different degrees of severity of diabetic microangiopathy and 57 age-matched control subjects. RESULTS: The fasting serum 3-DG level in diabetic patients was significantly (P < 0.001) higher than that in control subjects (353 +/- 110 vs. 199 +/- 53 nmol/l). The 3-DG levels were significantly (P < 0.001) elevated even in the diabetic patients showing normoalbuminuria (n = 62, 322 +/- 79 nmol/l) compared with control subjects. The 3-DG levels were further elevated in the patients with microalbuminuria (n = 30, 383 +/- 146 nmol/l) and overt proteinuria (n = 18, 410 +/- 100 nmol/l) (P = 0.027 and P < 0.001 vs. normoalbuminuria group, respectively). This phenomenon was basically reproduced in a category of retinopathy. Furthermore, the diabetic patients with low nerve conduction velocity showed a tendency to display higher 3-DG levels. CONCLUSIONS: The present results show that the fasting serum 3-DG level is elevated in diabetic patients and that the patients with relatively higher 3-DG levels were prone to suffer from more severe complications, indicating a possible association of 3-DG with diabetic microangiopathy.

Methylglyoxal induces apoptosis through activation of p38 mitogen-activated protein kinase in rat mesangial cells.

BACKGROUND: The formation of methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated through several pathways, including the glycation reaction under diabetic conditions, presumably contributing to tissue injury in diabetes. On the other hand, apoptotic cell death of glomerular cells has been suggested to play a role in the development of glomerulosclerosis in various types of glomerular injuries. We therefore examined whether MG was capable of inducing apoptosis in rat mesangial cells to address the possible mechanism by which hyperglycemia-related products accelerated pathologic changes in diabetic glomerulosclerosis. METHODS: Rat mesangial cells were incubated with 0 to 400 micromol/L MG, followed by the detection of apoptosis by both TUNEL method and electrophoretic analysis for DNA fragmentation. In addition, we investigated intracellular mechanisms mediating MG-induced apoptosis, focusing especially on the p38 mitogen-activated protein kinase (MAPK) pathway. RESULTS: MG induced apoptosis in rat mesangial cells in a dose-dependent manner and was accompanied by the activation of p38alpha isoform. Aminoguanidine and N-acetyl-l-cysteine inhibited the MG-induced p38 MAPK activation, as well as apoptosis in rat mesangial cells, suggesting the involvement of oxidative stress in these phenomena. SB203580, a specific inhibitor of p38 MAPK also suppressed the MG-induced apoptosis in rat mesangial cells. CONCLUSIONS: These results suggest a potential role for MG in glomerular injury through p38 MAPK activation under diabetic conditions and may serve as a novel insight into the therapeutic strategies for diabetic nephropathy.