Functional Elements in Molecular Chaperone α-Crystallin: Identification of Binding Sites in αB-Crystallin

α-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in α-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′ dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine α-crystallin at 48°C. The complex was photolyzed and reduced with DTT and the subunits of α-crystallin, αA- and αB-, were separated. Fluorescence analysis showed that both αA- and αB-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of αB-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.     

Hydration Properties of the Molecular Chaperone α-Crystallin in the Bovine Lens

Topographic studies of crystalline fractions from different morphological layers of the young adult bovine lens were conducted. Crystallin profiles were obtained for each lens layer, using thin-layer isoelectric focusing in polyacrylamide gel (IEF). Water soluble (WS) crystallins from the lens equator revealed a separation into HM (high molecular weight) _L-, _H-, _L-, _S-, and -crystallins. The nature of the water insoluble (WI) protein fraction in the separated lens layers reflected the aggregated state of _L-, _L-, _S-, and -crystallins in different regions of the lens, concealed in the central cavity of the -crystallin chaperone model. The IEF data demonstrate a possible chaperone-like function for -crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. The water binding properties of bovine lens -crystallin, calf skin collagen, and bovine serum albumin (BSA) were investigated with various techniques. The water adsorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The NMR spin–-echo technique was used to study the hydration of protein samples and to determine the spin–-spin relaxation times (T₂) from the protons of water adsorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During water vapor uptake at relative humidity P/P₀ = 0.75, -crystallin did not adsorb water suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At relative humidity P/P₀ = 1.0, the adsorption of water by -crystallin was 17% with a single component decay character of spin echo (T₂ = 3 msec). Addition of water to -crystallin to about 50% of its weight/weight in the protein sample showed T₂ = 8 msec with only one single component decay of the spin–-echo signal. The single component decay character of the spin echo indicates water tightly bound by -crystallin. Under a relative humidity P/P₀ = 1.0, collagen and BSA adsorbed, correspondingly, 19.3 and 28% of water and showed a two-component decay curve with T₂ about 5 and 40 msec. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with -crystallin with similar distribution patterns in the lens layers. To conclude, it was found that -crystallin can immobilize water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of -crystallin and its superhydration properties and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.     

Functional characterization of amphipathic α-helix in the osmoregulatory ABC transporter OpuA

The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic α-helix is located close to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic α-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodisks. We show that the amphipathic α-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport.     

Chaperone–Protein Interactions That Mediate Assembly of the Bacteriophage Lambda Tail to the Correct Length

Bacteriophage λ makes two proteins with overlapping amino acid sequences that are essential for tail assembly. These two proteins, gpG and gpGT, are related by a programmed translational frameshift that is conserved among diverse phages and functions in λ to ensure that gpG and the frameshift product gpGT are made in a molar ratio of approximately 30:1. Although both proteins are required and must be present in the correct ratio for assembly of functional tails, neither is present in mature tails. During λ tail assembly, major tail protein gpV polymerizes to form a long tube whose length is controlled by the tape measure protein gpH. We show that the “G” domains of gpG and gpGT bind to all or parts of tail length tape measure protein gpH and that the “T” domain of gpGT binds to major tail shaft subunit gpV, and present a model for how gpG and gpGT chaperone gpH and direct the polymerization of gpV to form a tail of the correct length.     

Functional expression of the α7 and α4-containing nicotinic acetylcholine receptors on the neonatal rat carotid body

The carotid bodies (CBs) are chemosensory organs that respond to hypoxemia with transmitter neurosecretion, leading to a respiratory reflex response. It has been proposed that acetylcholine is a key regulator of transmitter release through activation of presynaptic nicotinic acetylcholine receptors (nAChRs). In the present work, we studied the identity of such nAChRs and their contribution to catecholamine release from CBs. Neonatal rat CBs were placed in a recording chamber for electrochemical recordings or disassociated for voltage-clamp studies on isolated cells. Fast nicotine superfusion increases catecholamine release from intact CBs. This response was diminished reversibly by the non-selective nAChR blocker hexamethonium, by the selective α7 blocker α-bungarotoxin and by the α4-containing nAChR blocker erysodine. In isolated CB cells the nAChR agonists nicotine, acetylcholine and cytisine all evoke inward currents with similar potencies. The nicotine-evoked current was fully blocked by mecamylamine and partially inhibited by α-bungarotoxin or erysodine. However, the combination of both α-bungarotoxin an erysodine failed to suppress this response. Immunodetection studies confirm the presence of α7 and α4 subunits in isolated dopaminergic CB cells. Our results show that activation of α7 and/or α4-containing nAChR subtypes have the ability to regulate catecholamine release from intact CB due to activation of fast inward currents expressed in chemoreceptor cells. Therefore, our results suggest that both nAChR subtypes contribute to the cholinergic nicotinic regulation of catecholamine signaling in the carotid body system.     

The Duplicated α7 Subunits Assemble and Form Functional Nicotinic Receptors with the Full-length α7

The α7 nicotinic acetylcholine receptor gene (CHRNA7) is linked to schizophrenia. A partial duplication of CHRNA7 (CHRFAM7A) is found in humans on 15q13–14. Exon 6 of CHRFAM7A harbors a 2-bp deletion polymorphism, CHRFAM7AΔ2bp, which is also associated with schizophrenia. To understand the effects of the duplicated subunits on α7 receptors, we fused α7, dupα7, and dupΔα7 subunits with various fluorescent proteins. The duplicated subunits co-localized with full-length α7 subunits in mouse neuroblastoma cells (Neuro2a) as well as rat hippocampal neurons. We investigated the interaction between the duplicated subunits and full-length α7 by measuring Förster resonance energy transfer using donor recovery after photobleaching and fluorescence lifetime imaging microscopy. The results revealed that the duplicated proteins co-assemble with α7. In electrophysiological studies, Leu at the 9′-position in the M2 membrane-spanning segment was replaced with Cys in dupα7 or dupΔα7, and constructs were co-transfected with full-length α7 in Neuro2a cells. Exposure to ethylammonium methanethiosulfonate inhibited acetylcholine-induced currents, showing that the assembled functional nicotinic acetylcholine receptors (nAChRs) included the duplicated subunit. Incorporation of dupα7 and dupΔα7 subunits modestly changes the sensitivity of receptors to choline and varenicline. Thus, the duplicated proteins are assembled and transported to the cell membrane together with full-length α7 subunits and alter the function of the nAChRs. The characterization of dupα7 and dupΔα7 as well as their influence on α7 nAChRs may help explain the pathophysiology of schizophrenia and may suggest therapeutic strategies.     

A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor Clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the α7β1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, α7A and α7B, and the extracellular spliced forms, α7X1 and α7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the α7β1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-α7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-α7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the α7A and α7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the α7X2 extracellular domain were active. These results demonstrate that the α7β1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the α7 chain, and that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

The Molecular Chaperone Hsp90 Modulates Intermediate Steps of Amyloid Assembly of the Parkinson-related Protein ��-Synuclein

α-Synuclein is an intrinsically unstructured protein that binds to membranes, forms fibrils, and is involved in neurodegeneration. We used a reconstituted in vitro system to show that the molecular chaperone Hsp90 influenced α-synuclein vesicle binding and amyloid fibril formation, two processes that are tightly coupled to α-synuclein folding. Binding of Hsp90 to monomeric α-synuclein occurred in the low micromolar range, involving regions of α-synuclein that are critical for vesicle binding and amyloidogenesis. As a consequence, both processes were affected. In the absence of ATP, the accumulation of non-amyloid α-synuclein oligomers prevailed over fibril formation, whereas ATP favored fibril growth. This suggests that Hsp90 modulates the assembly of α-synuclein in an ATP-dependent manner. We propose that Hsp90 affects these folding processes by restricting conformational fluctuations of α-synuclein.     

Efficient Expression of Functional (α6β2)2β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)₂β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)₂β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.     

In Vivo Function of the Chaperonin TRiC in α-Actin Folding during Sarcomere Assembly

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Effects of Calcium Ion on the Catalytic Activity of α-Chymotrypsin in Organic Solvents

Addition of small amounts of calcium ion markedly accelerated the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester by the catalysis of α-chymotrypsin in organic solvents. Maximum increase of the reaction rate was about 12-fold in the presence of 25 μm of calcium ion in ethanol. The rate increase was strongly dependent on calcium ion concentration and nature of organic solvents. Esterification of N-acetyl-l-tyrosine and hydrolysis of N-acetyl-l-tyrosine ethyl ester by α-chymotrypsin in organic solvents were also accelerated by calcium ion. The reactions obeyed Michaelis–Menten kinetics, and the acceleration of the reactions was due to the increase in k_cat.     

Effects of water content on the enantioselectivity of α-chymotrypsin catalysis in organic media

Summary The -chymotrypsin-catalyzed transesterification between N-trifluoroacetyl-DL-phenylalanine trifluoroethyl ester and 1-propanol was carried out in a variety of organic solvents. The addition of small quantities of water enhanced both the rate of reaction and enantioselectivity. A high enantioselectivity was achieved in ethyl acetate (E = 120), diethyl ether (86), or acetonitrile (60). The competing hydrolysis became significant at water content higher than 0.5% (w/w).     

Effects of phospholipids on the antioxidant activity of α-tocopherol in the singlet oxygen oxidation of canola oil

This study evaluated the effects of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the antioxidative activity of α-tocopherol during oxidation of canola oil by singlet oxygen at 10°C for seven hours. Singlet oxygen was produced by chlorophyll b (4 ppm) under 1,700 lux. The oxidation of oil was evaluated by headspace oxygen consumption by gas chromatography and peroxide values (POVs). Concentrations of PC, PE, chlorophyll, and α-tocopherol were determined by HPLC. PC and PE protected chlorophyll from degradation, but they accelerated the degradation of α-tocopherol under singlet oxygen. Contents of PC and PE did not change for seven hours under singlet oxygen. α-Tocopherol significantly lowered POV and headspace oxygen consumption of canola oil under singlet oxygen, and its antioxidant activity was increased by the co-presence of PC and PE. PC and PE increased chemical quenching of singlet oxygen by tocopherol in decreasing the oil oxidation.     

Synthesis and Functional Identification of Oligopeptides Derived from the α3/5-Conotoxins

Conotoxins are small peptides comprising of 12–50 residues. The α3/5-conotoxins have the primary sequence motif -CCX3CX5C- and are paralytic, presumably targeted to the muscle nicotinic acetylcholine receptors. Some oligopeptide motifs chosen from the two loops of the α3/5-conotoxins, were synthesized and evaluated for biological activities on the rat nerve-skeletal muscle contractility, muscle cell contraction, electrophysiology and cytotoxicity. Three peptides (HPA, NPA and WNPA) showed high inhibitory effects and low cytotoxicities. They had the similar bioactivities as the anti-wrinkle product SYN-AKE did. The results in the present study indicated that these oligopeptides may be potential ingredients for anti-wrinkle product. However, further studies using in vivo animal models and human clinical trials are necessary before consumer use.     

Unfolding and Refolding of Bovine α-Crystallin in Urea and Its Chaperone Activity

We undertook an unfolding and refolding study of α_L-crystallin in presence of urea to explore the breakdown and formation of various levels of structure and to find out whether the breakdown of various levels of structure occurs simultaneously or in a hierarchal manner. We used various techniques such as circular dichroism, fluorescence spectroscopy, light scattering, polarization to determine the changes in secondary, tertiary, and quaternary structure. Unfolding and refolding occurred through a number of intermediates. The results showed that all levels of structure in α_L-crystallin collapsed or reformed simultaneously. The intermediates that occurred in the 2–4 M urea concentration range during unfolding and refolding differed from each other in terms of the polarity of the tryptophan environment. The ANS binding experiments revealed that refolded α_L-crystallin had higher number of hydrophobic pockets compared to native one. On the other hand, polarity of these pockets remained same as that of the native protein. Both light scattering and polarization measurements showed smaller oligomeric size of refolded α_L-crystallin. Thus, although the secondary structural changes were almost reversible, the tertiary and quaternary structural changes were not. The refolded α_L-crystallin had more exposed hydrophobic sites with increased binding affinity. The refolded form also showed higher chaperone activity than native one. Since the refolded form was smaller in oligomeric size, some buried hydrophobic sites were available. The higher chaperone activity of lower sized oligomer of α_L-crystallin again revealed that chaperone activity was dependent on hydrophobicity and not on oligomeric size.     

Expression of a Soluble Functional Form of the Integrin α4β1 in Mammalian Cells

The integrin α₄β₁(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human α₄ and β₁ subunits were fused to the genomic DNA encoding the human γ1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote α₄β₁ heterodimer formation. The soluble α₄β₁ Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent K_d for VCAM-1 binding were similar for both the soluble and native forms of α₄β₁. In addition, the integrin–Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble α₄β₁ should be generally applicable to a range of integrins.