R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable.

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.

The interaction of the molecular chaperone alpha-crystallin with unfolding alpha-lactalbumin: a structural and kinetic spectroscopic study.

The unfolding of the apo and holo forms of bovine alpha-lactalbumin (alpha-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein alpha-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and (1)H NMR spectroscopy. From these data, a description and a time-course of the events that result from the unfolding of both forms of the protein, and the state of the protein that interacts with alpha-crystallin, have been obtained. alpha-LA contains four disulphide bonds and binds a calcium ion. In apo alpha-LA, the disulphide bonds are reduced completely over a period of approximately 1500 seconds. Fully reduced alpha-LA adopts a partly folded, molten globule conformation that aggregates and, ultimately, precipitates. In the presence of an equivalent mass of alpha-crystallin, this precipitation can be prevented via complexation with the chaperone. alpha-Crystallin does not interfere with the kinetics of the reduction of disulphide bonds in apo alpha-LA but does stabilise the molten globule state. In holo alpha-LA, the disulphide bonds are less accessible to DTT, because of the stabilisation of the protein by the bound calcium ion, and reduction occurs much more slowly. A two-disulphide intermediate aggregates and precipitates rapidly. Its precipitation can be prevented only in the presence of a 12-fold mass excess of alpha-crystallin. It is concluded that kinetic factors are important in determining the efficiency of the chaperone action of alpha-crystallin. It interacts efficiently with slowly aggregating, highly disordered intermediate (molten globule) states of alpha-LA. Real-time NMR spectroscopy shows that the kinetics of the refolding of apo alpha-LA following dilution from denaturant are not affected by the presence of alpha-crystallin. Thus, alpha-crystallin is not a chaperone that is involved in protein folding per se. Rather, its role is to stabilise compromised, partly folded, molten globule states of proteins that are destined for precipitation.

Direct detection and quantification of transition metal ions in human atherosclerotic plaques: evidence for the presence of elevated levels of iron and copper.

OBJECTIVE: The involvement of transition metals in atherosclerosis is controversial. Some epidemiological studies have reported a relationship between iron (Fe) and cardiovascular disease, whereas others have not. Experimental studies have reported elevated levels of iron and copper (Cu) in diseased human arteries but have often used methods that release metal ions from proteins. METHODS AND RESULTS: In this study, we have used the minimally invasive technique of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICPMS) to quantify iron and copper in ex vivo healthy human arteries and carotid lesions. The EPR spectra detected are characteristic of nonheme Fe(III) complexes. Statistically elevated levels of iron were detected in the intima of lesions compared with healthy controls (0.370 versus 0.022 nmol/mg tissue for EPR, 0.525 versus 0.168 nmol/mg tissue by ICPMS, P<0.05 in each cases). Elevated levels of copper were also detected (7.51 versus 2.01 pmol/mg tissue, lesion versus healthy control, respectively, P<0.05). Iron levels did not correlate with the gender or age of the donor, or tissue protein or calcium levels, but cholesterol levels correlated positively with iron accumulation, as measured by EPR. CONCLUSIONS: These data support the hypothesis that iron accumulates in human lesions and may contribute to disease progression.

Casein proteins as molecular chaperones.

Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.

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

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

The quaternary organization and dynamics of the molecular chaperone HSP26 are thermally regulated.

The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein.

Glycosylation of sputum mucins is altered in cystic fibrosis patients.

Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.

Proteomic analysis of sputum from adults and children with cystic fibrosis and from control subjects.

RATIONALE: Recurrent pulmonary exacerbations are associated with progressive lung disease in cystic fibrosis (CF). Current definitions of an exacerbation, although not precisely defined, include new/worsening symptoms, declining lung function, and/or changing radiologic appearance. Early diagnosis of exacerbations by rapid noninvasive means should expedite therapeutic intervention, thereby minimizing lung damage. OBJECTIVES: To identify biomarkers of lung exacerbation for point-of-care monitoring of CF lung disease progression. METHODS: Saline-induced sputum was collected from adults with CF with an exacerbation and requiring hospitalization (FEV(1) < 60%), a subset of these adults at hospital discharge, children with stable CF and preserved lung function (FEV(1) > 70%), and control subjects (FEV(1) > 80%). Sputum was arrayed by two-dimensional electrophoresis and differentially expressed proteins were identified by proteomic analysis. MEASUREMENTS AND MAIN RESULTS: Sputum profiles from adults with CF with an exacerbation were characterized by extensive proteolytic degradation and influx of inflammation-related proteins, with some adults with CF approaching a "healthy" protein profile after hospitalization. Two children with CF showed profiles and biomarker expression resembling those of adults with an exacerbation. Levels of differentially expressed myeloperoxidase, cleaved alpha(1)-antitrypsin, IgG degradation, interleukin-8, and total protein concentration, together with their correlation to FEV(1), were statistically significant. Statistical correlation analyses indicated that changes in myeloperoxidase expression and IgG degradation were the strongest predictors of FEV(1). CONCLUSIONS: We identified extensive protein degradation and differentially expressed proteins as biomarkers of inflammation relating to pulmonary exacerbations. Prediction of exacerbation onset and more precise evaluation of the extent of resolution with treatment could be achieved by including biomarkers in standard assessment.

An immunoproteomic approach for identification of clinical biomarkers for monitoring disease: application to cystic fibrosis.

Circulating antibodies can be used to probe protein arrays of body fluids, prepared by two-dimensional gel electrophoresis, for antigenic biomarker detection. However, detected proteins, particularly low abundance antigens, often remain unidentifiable due to proteome complexity and limiting sample amounts. Using a novel enrichment approach exploiting patient antibodies for isolation of antigenic biomarkers, we demonstrate how immunoproteomic strategies can accelerate biomarker discovery. Application of this approach as a means of identifying biomarkers was demonstrated for cystic fibrosis (CF) lung disease by isolation and identification of inflammatory-associated autoantigens, including myeloperoxidase and calgranulin B from sputum of subjects with CF. The approach was also exploited for isolation of proteins expressed by the Pseudomonas aeruginosa strain PA01. Capture of PA01 antigens using circulating antibodies from CF subjects implicated in vivo expression of Pseudomonas proteins. All CF subjects screened, but not controls, were immunoreactive against immunocaptured Pseudomonas proteins, representing stress (GroES and ferric iron-binding protein HitA), immunosuppressive (thioredoxin), and alginate synthetase pathway (nucleoside-diphosphate kinase) proteins, implicating their clinical relevance as biomarkers of infection.

The eye lens protein alphaA-crystallin of the blind mole rat Spalax ehrenbergi: effects of altered functional constraints.

The rudimentary eyes of the mole rat Spalax ehrenbergi have lost their visual function, but are still required for the control of circadian rhythms. It has previously been found that alphaA-crystallin, a major eye lens protein in other mammals, evolved much faster in the mole rat than in rodents with normal vision. Yet, although mole rat alphaA-crystallin seems superfluous as a lens protein, its rate of change is still much slower than that of pseudogenes, suggesting some remaining function. The authors therefore studied the structure and function of recombinant mole rat alphaA-crystallin. Circular dichroism (CD), tryptophan fluorescence and gel permeation analyses indicated that the overall structure and stability of mole rat alphaA-crystallin are comparable to that of rat alphaA-crystallin. However, the chaperone-like activity of mole rat alphaA-crystallin is considerably lower than that of its rat orthologue. Two-dimensional NMR spectroscopy of mole rat alphaA-crystallin suggests that this may be in part due to a diminished flexibility of the C-terminal extension, which is thought to be important for the chaperoning capacity. Overall, mole rat alphaA-crystallin appears to still be a viable protein, confirming that it has some as yet elusive role, despite the loss of its primary lens function.