NMR spectroscopy of α-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of α-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved ¹H NMR spectrum is observed for α-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, αA- and αB-crystallin. These extensions are not involved in interactions with other proteins (e.g. β- and γ-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of αA-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic α-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the αA-crystallin subunit maintains its flexibility whereas the αB-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the ‘substrate’ protein. The conformation of ‘substrate’ proteins when they interact with α-crystallin has been probed by ¹H NMR spectroscopy and it is concluded that α-crystallin interacts with ‘substrate’ proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the ¹H and ³¹P NMR spectra of α-crystallin in the presence of increasing concentations of urea, it is proposed that α-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of α-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with ‘substrate’ proteins during the chaperone action.     

Thermodynamic stability of bovine α-crystallin in its interactions with other bovine crystallins

Light scattering measurements were performed on dilute solutions of α-crystallin mixed with different combinations of β_H, β_L and γ-fractions of bovine lens crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration and temperature. The temperature dependence of the second virial coefficients was used to obtain partial molar enthalpy and end entropy of solutions. The difference between the thermodynamic parameters of the crystallin mixtures and those of the weighted averages of the individual components yielded the excess enthalpy and entropy functions of the solutions. Both the excess enthalpy and entropy functions indicated that thermodynamic stability of α-crystallin is progressively enhanced by its interactions with γ(β_H+γ)(β_H+β_L+γ) crystallins. The last two combinations showed negative values both for excess enthalpy as well for excess entropy of solutions. Other combinations demonstrated increasing positive values. This implies that the combination of all four crystallins in the vertebrate lens enables the best solvation property as well as the best packing as opposed to any other single or combinatorial arrangements of crystallins. Similar conclusions have been obtained in the past from water and other vapor sorption studies.     

Mutations and modifications support a ‘pitted-flexiball’ model for α-crystallin

α-Crystallin is renown for resisting crystallization and electron microscopic image analysis. The spatial conformation thus remaining elusive, the authors explored the structure and chaperone functioning by analyzing the effects of site-directed mutagenesis, the properties of naturally occurring aberrant forms of α-crystallin and the influence of chemical modifications. The authors observed that the globular multimeric structure, as well as the chaperoning capacity are remarkably tolerant towards changes and modifications in the primary structure. The essential features of the quaternary structure—globular shape, flexibility, highly polar exterior and accessible hydrophobic surface pockets—support a ‘pitted-flexiball’ model, which combines tetrameric subunit building blocks in an open micelle-like arrangement.     

Quaternary structure of bovine α-crystallin: influence of temperature

The tertiary and quaternary structure of α-crystallin is still a matter of controversy. We have characterized the native α-crystallin quaternary structure by isolating it at the in vivo temperature and solvent conditions. It can be represented by a distribution of expanded particles with a weight average molar mass of 550 000 g/mol. On decreasing (to 4°C) or increasing (up to 50°C) the temperature, the size distribution increases to larger particles. Only at lower temperatures (4°C), a stable population of particles is obtained with weight average molar mass of 700 000 g/mol. In all conditions, α-crystallin behaves as a very expanded particle with a maximum hydrodynamic volume of 3.15 ml/g. The transitions in quaternary structure are rather slow: it takes several hours to evolve from a population of aggregates, characteristic for given solvent conditions, to another distribution in size and quaternary structure on changing the environment. The quaternary structure of α-crystallin is an uncharacteristic parameter of the particle: a broad distribution of values can be obtained on changing the environment. Any realistic model should include this property. Our studies favor an open loose structure, where peptides can be added or removed without drastic changes of secondary and tertiary structure of the peptides.     

Interaction of DNA with bovine lens α-crystallin: its functional implications

Under normal conditions, lens aggregates of α-crystallin subunits, αA and αB, are found in the cytoplasm. However, during stress in nonlenticular tissues, αB translocates to the nucleus. A sequence study revealed that both subunits share a consensus sequence with other DNA binding proteins. These observations prompted us to investigate DNA binding with α-crystallin by UV-mediated photo-crosslinking. The data show that both single and double stranded DNA crosslink mainly with tetramers of α-crystallin subunits. The formation of tetramers appears to modify α-crystallin interactive properties and, therefore, its induction may have functional significance. These observations suggest that α-crystallin may have a nuclear function which includes DNA binding.     

The possible role of α-crystallins in human senile cataractogenesis

α-Crystallins possess molecular chaperone properties and are one of the most abundant of the lenticular proteins. Posttranslational modifications of these proteins have been implicated as a possible etiology of human cataracts. This article will review current knowledge concerning the effects of known posttranslational modifications upon the molecular chaperone properties and aggregation behavior of α-A and α-B crystallin. Based upon these effects, experimental approaches will be discussed that may be useful in the development of reagents that may selectively inhibit the cataractogenic process in the aging human lens.     

α-

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

Environmental factors influencing the chaperone-like activity of α-crystallin

The effects of mild environmental changes (e.g. the addition of divalent cations or EDTA, as well as variations of buffer pH) on the heat stability and chaperone-like activity of native α-crystallin, and denatured–renatured α-crystallin in the native molar isoform ratio, have been investigated using circular dichroism (CD) spectropolarimetry and functional assays. The presence or absence of divalent cations has little or no effect on the secondary structure of renatured samples, although chaperone-like activity levels can vary widely; the only relevant spectral difference observed is a loss of some α-helical content in all the renatured samples relative to the native protein, but this change has no impact on function. The range of concentration over which the inhibitory Mg²⁺ effect is observed is 10-fold higher for dialyzed fresh protein than for protein renatured into buffers containing Mg²⁺, but for both sets of samples, the full effect is established below physiological Mg²⁺ concentrations. Renaturing into various pH buffers, in contrast, affects both heat stability and chaperone-like activity below pH 7.0, with essentially no functionality observed at pH 6.0. CD spectra of these samples indicate that acidic conditions lead to some degree of unfolding, and that this unfolding correlates directly with functionality. Similar results are obtained for fresh protein dialyzed against these pH levels. Overall, these results suggest that heat stability is a function of the protein's secondary structure and folding state, while chaperone-like activity is primarily a function of factors at the tertiary and quaternary levels of organization.     

Genealogy of the α-crystallin—small heat-shock protein superfamily

Sequences of 40 very diverse representatives of the α-crystallin–small heat-shock protein (α-Hsp) superfamily are compared. Their characteristic C-terminal ‘α-crystallin domain' of 80–100 residues contains short consensus sequences that are highly conserved from prokaryotes to eukaryotes. There are, in addition, some positions that clearly distinguish animal from non-animal α-Hsps. The α-crystallin domain is predicted to consist of two hydrophobic β-sheet motifs, separated by a hydrophilic region which is variable in length. Combination of a conserved α-crystallin domain with a variable N-terminal domain and C-terminal extension probably modulates the properties of the various α-Hsps as stress-protective and structural oligomeric proteins. Phylogeny reconstruction indicates that multiple α-Hsps were already present in the last common ancestor of pro- and eukaryotes. It is suggested that during eukaryote evolution, animal and non-animal α-Hsps originated from different ancestral gene copies. Repeated gene duplications gave rise to the multiple α-Hsps present in most organisms.     

α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

Laminin-5 and α3β1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/α3β1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 α3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes, which is initiated by α3β1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3ζ and σ, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3ζ with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes is initiated by laminin-5 stimulation via the α3β1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.     

The absorption, fluorescence and phosphorescence of phalloidin and α-amanitin

The peptides, phalloidin and α-amanitin, contain two unusual indole-derived chromophores, whose absorption, fluorescence and phosphorescence in aqueous solutions are described. Fluorescence is very weak, but phosphorescence at 77 K is intense. Phalloidin seems to undergo adiabatic photodissociation in alkaline solutions and to fluorescence from the indole anion form. In contrast, neither the phenolic nor the indolic protons of α-amanitin undergo photodissociation. The pK_a value of the phenolic hydroxy group of α-amanitin is 9.71 at 23°C.     

Iodination of αs1-casein and its effect on the calcium-induced aggregation reaction of the modified protein

The iodination of the tryosyl residues of α_s1-casein has been studied and a spectrophotometric procedure for differentiating monoiodotyrosine, diiodotyrosine and tyrosine is described. The calcium-induced aggregation behaviour of the iodinated caseins has been investigated. The initial stages of the reaction are shown to be dominated by electrostatic charge effects. The iodinated caseins behave as native α_s1-casein when the extent of conversion to diiodotyrosine is taken into account. The later stages of the reaction are found to be influenced by effects of the iodination not related to the change in charge.     

Effects of 6-methoxy-2-benzoxazolinone on the germination and α-amylase activity in lettuce seeds

Germination of lettuce seeds was inhibited by 6-methoxy-2-benzoxazolinone (MBOA) at concentrations greater than 0.03 mmol/L. MBOA also inhibited the induction of α-amylase activity in the lettuce seeds at concentrations greater than 0.03 mmol/L. These two concentration–response curves for the germination and α-amylase indicate that the percentage of the germination was positively correlated with the activity of α-amylase in the seeds. Lettuce seeds germinated around 18 h after incubation and inhibition of α-amylase by MBOA occurred within 6 h after seed incubation. These results show that MBOA may inhibit the germination of lettuce seeds by inhibiting the induction of α-amylase activity.     

β-secretase (BACE1)-inhibiting stilbenoids from Smilax Rhizoma

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of Smilax Rhizoma (the dried rhizomes of Smilax china L.) showed potent inhibitory activity. The active compounds were identified as a trans/cis-resveratrol mixture, oxyresveratrol, veraphenol, and cis-scirpusin A. They were shown to non-competitively inhibit BACE1 with the Ki values of 5.4 x 10(-6), 5.4 x 10(-6), 3.4 x 10(-6), and 5.4 x 10(-6)M and IC(50) values of 1.5 x 10(-5), 7.6 x 10(-6), 4.2 x 10(-6), and 1.0 x 10(-5)M, respectively. The active compounds were less inhibitory to alpha-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

α1-Antitrypsin and α2-macroglobulin do not inhibit the kinin-releasing activity of kallikreins from human urine and saliva

α₁-Antitrypsin, α₂-macroglobulin and low-molecular weight kininogen were isolated from human serum and kallikreins from human urine and saliva.α₁-Antitrypsin and α₂-macroglobulin inhibited the activity of trypsin in releasing kinin from low-molecular weight kininogen, due to their binding with the enzyme, but did non inhibit or bind with urinary and salivary kallikreins.