Biomedical subjects

J Andrew Aquilina

Publications and source records attributed to J Andrew Aquilina.

16 recordsLinked to original sources

PubMed2007

Mimicking phosphorylation of alphaB-crystallin affects its chaperone activity.

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

Animals↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

Targeting C-reactive protein for the treatment of cardiovascular disease.

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Animals↗

PubMed2006

3-Hydroxykynurenine oxidizes alpha-crystallin: potential role in cataractogenesis.

The alpha-, beta-, and gamma-crystallins are the major structural proteins of mammalian lenses. The human lens also contains tryptophan-derived UV filters, which are known to spontaneously deaminate at physiological pH and covalently attach to lens proteins. 3-Hydroxykynurenine (3OHKyn) is the third most abundant of the kynurenine UV filters in the lens, and previous studies have shown this compound to be unstable and to be oxidized under physiological conditions, producing H2O2. In this study, we show that methionine and tryptophan amino acid residues are oxidized when bovine alpha-crystallin is incubated with 3-hydroxykynurenine. We observed almost complete oxidation of methionines 1 and 138 in alphaA-crystallin and a similar extent of oxidation of methionines 1 and 68 in alphaB-crystallin after 48 h. Tryptophans 9 and 60 in alphaB-crystallin were oxidized to a lesser extent. AlphaA-crystallin was also found to have 3OHKyn bound to its single cysteine residue. Examination of normal aged human lenses revealed no evidence of oxidation of alpha-crystallin; however, oxidation was detected at methionine 1 in both alphaA- and alphaB-crystallin from human cataractous lenses. Age-related nuclear cataract is associated with coloration and insolubilization of lens proteins and extensive oxidation of cysteine and methionine residues. Our findings demonstrate that 3-hydroxykynurenine can readily catalyze the oxidation of methionine residues in both alphaB- and alphaA-crystallin, and it has been reported that alpha-crystallin modified in this way is a poorer chaperone. Thus, 3-hydroxykynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens.

Animals↗

PubMed2006

Tandem mass spectrometry reveals the quaternary organization of macromolecular assemblies.

The application of mass spectrometry (MS) to the study of progressively larger and more complex macromolecular assemblies is proving increasingly useful for structural biologists. The scope of this approach has recently been widened through the application of a tandem MS procedure. This two-step technique involves the selection of specific assemblies in the gas phase and inducing their dissociation through collisions with argon atoms. Here, we investigate the mechanism of this process and show that dissociation of subunits from a macromolecular assembly follows a sequential pathway, with the partitioning of charge between the dissociation products governed primarily by their relative surface areas. Using this basis of understanding, we highlight differences in the dissociation pathways of three related macromolecular assemblies and show how these are a direct consequence of changes in both local and global oligomeric organization.

Animals↗

PubMed2005

Subunit exchange of polydisperse proteins: mass spectrometry reveals consequences of alphaA-crystallin truncation.

The small heat shock protein, alpha-crystallin, plays a key role in maintaining lens transparency by chaperoning structurally compromised proteins. This is of particular importance in the human lens, where proteins are exposed to post-translational modifications over the life-time of an individual. Here, we examine the structural and functional consequences of one particular modification of alphaA-crystallin involving the truncation of 5 C-terminal residues (alphaA(1-168)). Using novel mass spectrometry approaches and established biophysical techniques, we show that alphaA(1-168) forms oligomeric assemblies with a lower average molecular mass than wild-type alphaA-crystallin (alphaA(WT)). Also apparent from the mass spectra of both alphaA(WT) and alphaA(1-168) assemblies is the predominance of oligomers containing even numbers of subunits; interestingly, this preference is more marked for alphaA(1-168). To examine the rate of exchange of subunits between assemblies, we mixed alphaB crystallin with either alphaA(WT) or alphaA(1-168) and monitored in a real-time mass spectrometry experiment the formation of heteroligomers. The results show that there is a significant decrease in the rate of exchange when alphaA(1-168) is involved. These reduced exchange kinetics, however, have no effect upon chaperone efficiency, which is found to be closely similar for both alphaA(WT) and alphaA(1-168). Overall, therefore, our results allow us to conclude that, in contrast to mechanisms established for analogous proteins from plants, yeast, and bacteria, the rate of subunit exchange is not the critical parameter in determining efficient chaperone behavior for mammalian alphaA-crystallin.

Chromatography↗

PubMed2005

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.

Animals↗

PubMed2004

Tsp36, a tapeworm small heat-shock protein with a duplicated alpha-crystallin domain, forms dimers and tetramers with good chaperone-like activity.

Small heat shock proteins (sHSPs), which range in monomer size between 12 and 42 kDa, are characterized by a conserved C-terminal alpha-crystallin domain of 80-100 residues. They generally form large homo- or heteromeric complexes, and typically have in vitro chaperone-like activity, keeping unfolding proteins in solution. A special type of sHSP, with a duplicated alpha-crystallin domain, is present in parasitic flatworms (Platyhelminthes). Considering that an alpha-crystallin domain is essential for the oligomerization and chaperone-like properties of sHSPs, we characterized Tsp36 from the tapeworm Taenia saginata. Both wild-type Tsp36 and a mutant (Tsp36C-->R) in which the single cysteine has been replaced by arginine were expressed and purified. Far-UV CD measurements of Tsp36 were in agreement with secondary structure predictions, which indicated alpha-helical structure in the N-terminal region and the expected beta-sandwich structure for the two alpha-crystallin domains. Gel permeation chromatography and nano-ESI-MS showed that wild type Tsp36 forms dimers in a reducing environment, and tetramers in a non-reducing environment. The tetramers are stabilized by disulfide bridges involving a large proportion of the Tsp36 monomers. Tsp36C-->R exclusively occurs as dimers according to gel permeation chromatography, while the nondisulfide bonded fraction of wild type Tsp36 dissociates from tetramers into dimers under nonreducing conditions at increased temperature (43 degrees C). The tetrameric form of Tsp36 has a greater chaperone-like activity than the dimeric form.

Amino Acid Sequence↗

PubMed2004

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity.

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.

Amyloid↗

PubMed2004

Phosphorylation of alphaB-crystallin alters chaperone function through loss of dimeric substructure.

Phosphorylation is the most common posttranslational modification of the alpha-crystallins in the human lens. These phosphorylated forms are not only important because of their abundance in aging lenses and the implications for cataract but also because they have been identified in patients with degenerative brain disease. By using mimics corresponding to the reported in vivo phosphorylation sites in the human lens, we have examined the effects of phosphorylation upon the chaperone-like properties and structure of alphaB-crystallin. Here we show that phosphorylation of alphaB-crystallin at Ser-45 results in uncontrolled aggregation. By using an innovative tandem mass spectrometry approach, we demonstrate how this alteration in behavior stems from disruption of dimeric substructure within the polydisperse alphaB-crystallin assembly. This structural perturbation appears to disturb the housekeeping role of alphaB-crystallin and consequently has important implications for the disease states caused by protein aggregation in the lens and deposition in non-lenticular tissue.

Binding Sites↗

PubMed2004

Temperature and concentration-controlled dynamics of rhizobial small heat shock proteins.

A hallmark of alpha-crystallin-type small heat shock proteins (sHsps) is their highly dynamic oligomeric structure which promotes intermolecular interactions involved in subunit exchange and substrate binding (chaperone-like activity). We studied the oligomeric features of two classes of bacterial sHsps by size exclusion chromatography and nanoelectrospray mass spectrometry. Proteins of both classes formed large complexes that rapidly dissociated upon dilution and at physiologically relevant heat shock temperatures. As the secondary structure was not perturbed, temperature- and concentration-dependent dissociations were fully reversible. Complexes formed between sHsps and the model substrate citrate synthase were stable and exceeded the size of sHsp oligomers. Small Hsps, mutated in a highly conserved glycine residue at the C-terminal end of the alpha-crystallin domain, formed labile complexes that disassembled more readily than the corresponding wild-type proteins. Reduced complex stability coincided with reduced chaperone activity.

Chromatography↗

PubMed2003

Investigating interactions of the pentraxins serum amyloid P component and C-reactive protein by mass spectrometry.

The oligomeric state of human SAP (serum amyloid P component) in the absence and presence of known ligands has been investigated using nanoelectrospray ionization MS. At pH 8.0, in the absence of Ca2+, SAP has been shown to consist of pentameric and decameric forms. In the presence of physiological levels of Ca2+, SAP was observed to exist primarily as a pentamer, reflecting its in vivo state. dAMP was shown not only to promote decamerization, but also to lead to decamer stacking involving up to 30 monomers. A mechanism for this finding is proposed. CRP (C-reactive protein), a pentraxin closely related to SAP, exists as a pentamer in the presence or absence of Ca2+. Pentamers of CRP and SAP were shown to form mixed decamers in Ca2+-free buffer; however, in the presence of Ca2+, this interaction was not observed. Furthermore, no exchange of monomeric subunits was observed between the SAP and CRP oligomers, suggesting a remarkable stability of the individual pentameric complexes.

C-Reactive Protein↗

PubMed2003

Polydispersity of a mammalian chaperone: mass spectrometry reveals the population of oligomers in alphaB-crystallin.

The quaternary structure of the polydisperse mammalian chaperone alphaB-crystallin, a member of the small heat-shock protein family, has been investigated by using electrospray mass spectrometry. The intact assemblies give rise to mass spectra that are complicated by the overlapping of charge states from the different constituent oligomers. Therefore, to determine which oligomers are formed by this protein, tandem mass spectrometry experiments were performed. The spectra reveal a distribution, primarily of oligomers containing 24-33 subunits, the relative populations of which were quantified, to reveal a dominant species being composed of 28 subunits. Additionally, low levels of oligomers as small as 10-mers and as large as 40-mers were observed. Interpretation of the tandem mass spectral data was confirmed by simulating and summing spectra arising from the major individual oligomers. The ability of mass spectrometry to quantify the relative populations of particular oligomeric states also revealed that, contrary to the dimeric associations observed in other small heat-shock proteins, there is no evidence for any stable substructures of bovine alphaB-crystallin isolated from the lens.

Animals↗

PubMed2002

Glutathione and NADH, but not ascorbate, protect lens proteins from modification by UV filters.

Age-dependent human lens colouration and fluorescence may stem primarily from the covalent binding of UV filters to crystallins. The tendency of the kynurenine (Kyn) UV filters to deaminate at neutral pH, with the generation of reactive alpha,beta-ketoalkenes, underlies this phenomenon. In this study the authors examined the ability of small molecular weight antioxidants, which are known to be present in the lens, to inhibit this process. Crystallins were incubated with Kyn at pH 7 in the presence of glutathione (GSH), ascorbate or NADH. Ascorbate, even at high (15 m M) levels, was not found to significantly retard the time-dependent covalent binding of Kyn to the proteins. GSH, and to a lesser extent NADH, however, had a major impact in preventing this modification. The increase in protein UV absorbance and fluorescence was inhibited by GSH intercepting the reactive ketone intermediate, to form a GSH-Kyn adduct. NADH seemed to protect by both reduction of the reactive ketone intermediate and by competing with Kyn for presumably hydrophobic sites on the crystallins. This may indicate that the covalent attachment of aromatic Kyn molecules could be facilitated by initial hydrophobic interactions. Since GSH is present at far greater concentrations than NADH, these results show that in primate lenses, GSH is the key agent responsible for protecting the crystallins from covalent modification.

Animals↗

PubMed2002

UV filter instability: consequences for the human lens.

Human lenses appear to become coloured with age primarily due to the covalent binding of UV filter compounds to lens proteins. These crystallin modifications result from the inherent instability of the kynurenine UV filters. Here we investigate this decomposition, the role this may have in the formation of other primate UV filters, and the interaction of the intermediates (alpha,beta-ketoalkenes) with lens components. The UV filters kynurenine, 3-hydroxykynurenine and 3-hydroxykynurenine glucoside were incubated at neutral pH in the presence or absence of NADH or NADPH. The three UV filters underwent spontaneous deamination, such that at pH 7 less than half of the starting materials (kynurenine (42%), 3-hydroxykynurenine glucoside (30%) and 3-hydroxykynurenine (21%)) remained after 7 days. In the presence of NAD(P)H, the double bond of the UV filter-derived deamination compounds, were reduced. Deamination of 3-hydroxykynurenine glucoside, followed by reduction with NAD(P)H, could thus account for the formation of the major lens UV filter 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside. beta-Benzoylacrylic acid, which possesses the same alpha,beta-ketoalkene sidechain as the deaminated kynurenine UV filters, underwent Michael addition with glutathione, was reduced (hydrogenated) by NAD(P)H, however was unreactive with ascorbate. Surprisingly, at pH 7 the UV filter-derived alpha,beta-ketoalkene intermediates, do not readily undergo intramolecular cyclization. This feature makes the double bond more available for reaction with protein nucleophilic residues and other lens components such as glutathione. On the basis of these data it is likely that glutathione and NAD(P)H, but not ascorbate, protect proteins in the lens from modification by UV filters.

Acrylates↗

PubMed2001

Novel protein modification by kynurenine in human lenses.

It is known that human lenses increase in color and fluorescence with age, but the molecular basis for this is not well understood. We demonstrate here that proteins isolated from human lenses contain significant levels of the UV filter kynurenine covalently bound to histidine and lysine residues. Identification was confirmed by synthesis of the kynurenine amino acid adducts and comparison of the chromatographic retention times and mass spectra of these authentic standards with those of corresponding adducts isolated from human lenses following acid hydrolysis. Using calf lens proteins as a model, covalent binding of kynurenine to lens proteins has been shown to proceed via side chain deamination in a manner analogous to that observed for the related UV filter, 3-hydroxykynurenine O-beta-D-glucoside. Levels of histidylkynurenine and lysylkynurenine were low in human lenses in subjects younger than 30, but thereafter increased in concentration with the age of the individual. Post-translational modification of lens proteins by tryptophan metabolites therefore appears to be responsible, at least in part, for the age-dependent increase in coloration and fluorescence of the human lens, and this process may also be important in other tissues in which up-regulation of tryptophan catabolism occurs.

Adult↗