Kinetics of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin

Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-Crystallin performs such a function in the ocular lens. Dynamic light scattering (DLS) measurements were performed to gain insight into the kinetics and mechanism of alpha-crystallin chaperoning. Experiments were conducted as a function of alpha-lactalbumin concentration as well as the alpha-crystallin/alpha-lactalbumin ratio over a 24 h period. In the particle distribution patterns the lactalbumin concentration was partitioned into three compartments: (a) monomeric free lactalbumin; (b) lactalbumin in the chaperoning complex; and (c) lactalbumin aggregates. DLS intensities were converted to molar concentrations by assuming a model of a spherical chaperoning complex. In the model, alpha-crystallin is the central core and alpha-lactalbumin molecules occupy a ring surrounding the core. The kinetics of chaperoning was studied by proposing a simple scheme with four rate constants. The reversible reaction of the formation of the chaperoning complex is characterized by rate constants k(1) and k(2). The rate constants k(3) and k(4) govern the irreversible aggregation of lactalbumin: the former from the free monomeric lactalbumin pool and the latter describing the aggregation of the denatured lactalbumin released from the chaperoning complex. The rate constants, k(3) and k(4) are four magnitudes larger than k(1) and k(2). The equilibrium constant of chaperoning complex formation lies in favor of the reactants. k(4) is somewhat faster than k(3) and it is three times faster than k(s) governing the self-aggregation of lactalbumin in the absence of alpha-crystallin.     

The mode of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin.

Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-crystallin performs such a function in the ocular lens. To gain insight into the mechanism of the anti-aggregation activity of alpha-crystallin, we performed dynamic light scattering (DLS) measurements investigating its interaction with partially denatured alpha-lactalbumin over a 24 hr period. Analyses were conducted as a function of the concentration of alpha-lactalbumin as well as the bovine alpha-crystallin/alpha-lactalbumin ratio. Additional studies of the systems were performed by HPLC and SDS gel electrophoresis. The particle distribution patterns derived from the DLS data indicated that the chaperoned complex (lactalbumin plus crystallin) is a loose fluffy globular entity. After the complex becomes saturated with lactalbumin, it appears to release the partially denatured lactalbumin which may aggregate into high molecular weight moieties. These eventually may precipitate out of solution. On longer standing, 24hr and over, the chaperoned complex as well as the lactalbumin aggregates become more compact. The chaperoned complex (alpha-crystallin plus alpha-lactalbumin) is in dynamic equilibrium both with the monomeric and the aggregated alpha-lactalbumin population.     

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.     

Identification of a region in alcohol dehydrogenase that binds to alpha-crystallin during chaperone action

alpha-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with alpha-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of alpha-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to alpha-crystallin during chaperone-like action. The first method involved the incubation of alpha-crystallin with ADH peptides at 48 degrees C for 1 h followed by separation and analysis of bound peptides. In the second method, alpha-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 degrees C for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH(40-60)) as the alpha-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between alpha-crystallin and a synthetic peptide representing the binding site of ADH.     

Zn2+ enhances the molecular chaperone function and stability of alpha-crystallin

Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a micronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation.     

Native quaternary structure of bovine alpha-crystallin

Alpha-crystallin is the most important soluble protein in the eye lens. It is responsible for creating a high refractive index and is known to be a small heat-shock protein. We have used static and dynamic light scattering to study its quaternary structure as a function of isolation conditions, temperature, time, and concentration. We have used tryptophan fluorescence to study the temperature dependence of the tertiary structure and its reversibility. Gel filtration, analytical ultracentrifugation, polyacrylamide gel electrophoretic analysis, and absorption measurements were used to study the chaperone-like activity of alpha-crystallin in the presence of destabilized lysozyme. We have demonstrated that the molecular mass of the in vivo alpha-crystallin oligomer is about 700 kDa (alpha(native)) while the 550 kDa molecule (alpha(37 degrees C),diluted), which is often found in vitro, is a product of prolonged storage at 37 degrees C of low concentrated alpha-crystallin solutions. We have proven that the molecular mass of the alpha-crystallin oligomer is concentration dependent at 37 degrees C. We have found strong indications that, during chaperoning, the alpha-crystallin oligomer undergoes a drastic rearrangement of its peptides during the process of complex formation with destabilized lysozyme. We propose the hypothesis that all these processes are governed by the phenomenon of subunit exchange, which is well-known to be strongly temperature-dependent.     

Conversion of human alpha-lactalbumin to an apo-like state in the complexes with basic poly-amino acids: toward understanding of the molecular mechanism of antitumor action of HAMLET

It was recently shown that alpha-lactalbumin associated with oleic acid (HAMLET) interacts with core histones thereby triggering apoptosis of tumor cells (J. Biol. Chem. 2003, 278, 42131). In previous work, we revealed that monomeric alpha-lactalbumin in the absence of fatty acids can also interact with histones and, moreover, with basic poly-amino acids (poly-Lys and poly-Arg) that represent simple models of histone proteins (Biochemistry 2004, 43, 5575). Association of alpha-lactalbumin with histone or poly-Lys(Arg) essentially changes its properties. In the present work, the character of the changes in structural properties and conformational stability of alpha-lactalbumin in the complex with poly-Lys(Arg) has been studied in detail by steady-state fluorescence, circular dichroism, and differential scanning calorimetry. Complex formation strongly depends on ionic strength, confirming its electrostatic nature. Experiments with the poly-amino acids of various molecular masses demonstrated a direct proportionality between the number of alpha-lactalbumin molecules bound per poly-Lys(Arg) and the surface area of the poly-amino acid random coil. The binding of the poly-amino acids to Ca2+-saturated human alpha-lactalbumin decreases its thermal stability down to the level of its free apo-form and decreases Ca2+-affinity by 4 orders of magnitude. The conformational state of alpha-lactalbumin in a complex with poly-Lys(Arg), named alpha-LActalbumin Modified by Poly-Amino acid (LAMPA), differs from all other alpha-lactalbumin states characterized to date, representing an apo-like (molten globule-like) state with substantially decreased affinity for calcium ion. The requirement for efficient conversion of alpha-lactalbumin to the LAMPA state is a poly-Lys(Arg) chain consisting of several tens of amino acid residues.     

The distribution and relative immunogenicity of calf alpha-crystallin antigenic determinants on different subunits.

In the native alpha-crystallin molecule, 45.9% of all reactive antigenic determinants were found to be located on SH-containing subunits. Of these, the majority (35.3%) were reaggregation dependent, and 10.6% were reactive on monomeric subunits. By contrast, only 10.9% of all antigenic determinants were located on SH-free subunits, and the ratio of aggregation-dependent determinants (4.4%) to those of monomeric subunits (6.5%) was reversed compared to SH-containing subunits. Among all antigenic determinants reactive in native alpha-crystallin, 44.1% were dependent on the presence of both types of subunits. These data indicate that the antigenic determinants requiring subunit interaction were formed from SH-containing and SH-free subunits in a ratio of 1:1. Direct analysis showed that in the alpha-crystallin molecule, the ratio of these subunits is 2:1. The experiments indicate that some conformations of subunits in the native molecule persist in separated subunits. The relative immunogenicity of each type of antigenic determinant expressed as the ratio of the percentage of the determinant reactive in the native calf lens alpha-crystallin to the percentage of corresponding antibodies induced by native alpha-crystallin was found to be close to 1.     

The N-terminal domain of alphaB-crystallin is protected from proteolysis by bound substrate

Alpha-crystallin, a major structural protein of the lens can also function as a molecular chaperone by binding to unfolding substrate proteins. We have used a combination of limited proteolysis at low temperature, and mass spectrometry to identify the regions of alpha-crystallin directly involved in binding to the structurally compromised substrate, reduced alpha-lactalbumin. In the presence of trypsin, alpha-crystallin which had been pre-incubated with substrate showed markedly reduced proteolysis at the C-terminus compared with a control, indicating that the bound substrate restricted access of trypsin to R157, the main cleavage site. Chymotrypsin was able to cleave at residues in both the N- and C-terminal domains. In the presence of substrate, alpha-crystallin showed markedly reduced proteolysis at four sites in the N-terminal domain when compared with the control. Minor differences in cleavage were observed within the C-terminal domain suggesting that the N-terminal region of alpha-crystallin contains the major substrate interaction sites.     

Binding of destabilized betaB2-crystallin mutants to alpha-crystallin: the role of a folding intermediate

Age-related changes in protein-protein interactions in the lens play a critical role in the temporal evolution of its optical properties. In the relatively non-regenerating environment of the fiber cells, a critical determinant of these interactions is partial or global unfolding as a consequence of post-translational modifications or chemical damage to individual crystallins. One type of attractive force involves the recognition by alpha-crystallins of modified proteins prone to unfolding and aggregation. In this paper, we explore the energetic threshold and the structural determinants for the formation of a stable complex between alpha-crystallin and betaB2-crystallin as a consequence of destabilizing mutations in the latter. The mutations were designed in the framework of a folding model that proposes the equilibrium population of a monomeric intermediate. Binding to alpha-crystallin is detected through changes in the emission properties of a bimane fluorescent probe site-specifically introduced at a solvent exposed site in betaB2-crystallin. alpha-Crystallin binds the various betaB2-crystallin mutants, although with a significantly lower affinity relative to destabilized T4 lysozyme mutants. The extent of binding, while reflective of the overall destabilization, is determined by the dynamic population of a folding intermediate. The existence of the intermediate is inferred from the biphasic bimane emission unfolding curve of a mutant designed to disrupt interactions at the dimer interface. The results of this paper are consistent with a model in which the interaction of alpha-crystallins with substrates is not solely triggered by an energetic threshold but also by the population of excited states even under favorable folding conditions. The ability of alpha-crystallin to detect subtle changes in the population of betaB2-crystallin excited states supports a central role for this chaperone in delaying aggregation and scattering in the lens.     

Modification of alpha-crystallin subunit chains and formation of alpha-neoprotein molecules: role of SH groups

Immunoadsorbents with bound antibodies restricted to determinants dependent on alpha-crystallin's quaternary structure permitted the fractionation of the population of 125I-labeled alpha-crystallin molecules, treated by iodoacetic acid, into molecules in which the native structure was still preserved and molecules with a completely different quaternary structure than the native protein. Parallel experiments with [14C]iodoacetic acid yielded information on the percentage of blocked SH groups in each of the above two fractions. The presence of molecules formed by A with B-chain association was established by sequential binding first to an immunoadsorbent with antibodies restricted to determinants located on alpha-crystallin's A-subunit chains as ligand and second, after desorption, to an immunoadsorbent with antibodies to B chains as ligand. With the aid of these techniques, it was established that (i) The modified alpha-crystallin molecules with quaternary determinants of the native protein contained a maximum of 23% blocked SH groups, indicating that the carboxymethylation involved only the fast-reacting surface SH groups. (ii) The modified alpha-crystallin molecules without the native protein's quaternary structure were built by a different association between A and B subunits than in alpha-crystallin, indicating formation of alpha-neoprotein molecules. (iii) Monomeric A chains with all SH groups carboxymethylated, and monomeric B chains in a ratio of 1A:5B, 2A:1B, and 5A:1B in urea solution, associate on dialysis, forming alpha-neoprotein molecules.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.     

The selective inhibition of serpin aggregation by the molecular chaperone, alpha-crystallin, indicates a nucleation-dependent specificity

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that prevent the misfolding and aggregation of proteins. However, specific details about their substrate specificity and mechanism of chaperone action are lacking. alpha1-Antichymotrypsin (ACT) and alpha1-antitrypsin (alpha1-AT) are two closely related members of the serpin superfamily that aggregate through nucleation-dependent and nucleation-independent pathways, respectively. The sHsp alpha-crystallin was unable to prevent the nucleation-independent aggregation of alpha1-AT, whereas alpha-crystallin inhibited ACT aggregation in a dose-dependent manner. This selective inhibition of ACT aggregation coincided with the formation of a stable high molecular weight alpha-crystallin-ACT complex with a stoichiometry of 1 on a molar subunit basis. The kinetics of this interaction occur at the same rate as the loss of ACT monomer, suggesting that the monomeric species is bound by the chaperone. 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding and far-UV circular dichroism data suggest that alpha-crystallin interacts specifically with a non-native conformation of ACT. The finding that alpha-crystallin does not interact with alpha1-AT under these conditions suggests that alpha-crystallin displays a specificity for proteins that aggregate through a nucleation-dependent pathway, implying that the dynamic nature of both the chaperone and its substrate protein is a crucial factor in the chaperone action of alpha-crystallin and other sHsps.     

How α-crystallin prevents the aggregation of insulin

Using steady-state, polarized, and phase-modulation fluorometry, the dithiothreitol-induced denaturation of insulin and formation of its complex with alpha-crystallin in solution were studied. Prevention of the aggregation of insulin by alpha-crystallin is due to formation of chaperone complexes, i.e. interaction of chains of the denatured insulin with alpha-crystallin. The conformational changes in alpha-crystallin that occur during complex formation are rather small. It is unlikely that N-termini are directly involved in the complex formation. The 8-anilino-1-naphthalenesulfonate (ANS) is not sensitive to the complex formation. ANS emits mainly from alpha-crystallin monomers, dimers, and tetramers, but not from oligomers or aggregates. The possibility of highly sensitive detection of aggregates by light scattering using a spectrofluorometer with crossed monochromators is demonstrated.     

Molecular characterization of alpha-lactalbumin folding variants that induce apoptosis in tumor cells

This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk alpha-lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of alpha-lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule alpha-lactalbumin showed that the active fraction contains oligomers of alpha-lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve alpha-lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of alpha-lactalbumin.     

The molecular chaperone,alpha-crystallin,protects against loss of antigenicity and activity of esterase caused by sugars,sugar phosphate and a steroid

Previously we showed that glycation-induced inactivation and loss of antigenicity of enzymes occur simultaneously. Alpha-crystallin, a major structural protein of the mammalian lens, prevents the aggregation of other proteins and protects enzyme function against post-translational modification in vitro. However, it is not known whether alpha-crystallin can also protect against loss of antigenicity of enzymes. Esterase activity in the lens is decreased in senile cataract and diabetes. We investigated the loss of antigenicity of esterase caused by different insults and the ability of alpha-crystallin to protect. Inactivation of carboxylesterase by sugars, fructose 6-phosphate (F6P) and a steroid, prednisolone-21-hemisuccinate (P-21-H), was measured spectrophotometrically in the presence and absence of alpha-crystallin, while loss of antigenicity was monitored simultaneously using an immunoprecipitation method. The esterase was progressively inactivated by fructose, F6P, ribose, and P-21-H. Bovine alpha-crystallin fully protected against inactivation of esterase by all four compounds, and also protected against loss of antigenicity of the esterase by fructose, ribose and P-21-H at a molar ratio of 1:1. The results indicated that alpha-crystallin, under our experimental conditions, clearly exhibited the ability to prevent loss of antigenicity and inactivation of esterase. The protective effect of alpha-crystallin against loss of antigenicity indicates a novel aspect of its chaperoning function.     

CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity

A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSPα resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSPα results in massive neurodegeneration that impairs survival in mice and flies. In CSPα-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSPα may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSPα-Hsc70-SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSPα produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSPα overexpression, and enhanced by CSPα deletion. Thus, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSPα-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment.     

Eye lens alphaA- and alphaB-crystallin: complex stability versus chaperone-like activity.

The major lens protein alpha-crystallin is composed of two related types of subunits, alphaA- and alphaB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric alphaA-crystallin complexes perform better in preventing thermal aggregation of proteins, while alphaB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50 degrees C alphaA- performs better than alphaB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of alphaA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of alphaA-crystallin's secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of alphaA- and alphaB-crystallin, since regardless of the experimental temperature alphaB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of alphaA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of alphaB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric alpha-crystallin as a lens protein.