Oxidative dimer formation is the critical rate-limiting step for Parkinson's disease alpha-synuclein fibrillogenesis

Intraneuronal deposition of alpha-synuclein as fibrils and oxidative stress are both implicated in the pathogenesis of Parkinson's disease. We found that the critical rate-limiting step in nucleation of alpha-synuclein fibrils under physiological conditions is the oxidative formation and accumulation of a dimeric, dityrosine cross-linked prenucleus. Dimer formation is accelerated for the pathogenic A30P and A53T mutant alpha-synucleins, because of their greater propensity to self-interact, which is reflected in the smaller values of the osmotic second virial coefficient compared to that of wild-type synuclein. Our finding that oxidation is an essential step in alpha-synuclein aggregation supports a mechanism of Parkinson's disease pathogenesis in which the separately studied pathogenic factors of oxidative stress and alpha-synuclein aggregation converge at the critical step of alpha-synuclein dimer formation.     

Parkinson's disease-associated alpha-synuclein is more fibrillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.     

Mechanical design of hedgehog spines and porcupine quills

The spines or quills of hedgehogs and porcupines are morphologically and mechanically different. In simple terms, it seems that the quills of porcupines are proportioned to be as long as possible without bending too easily when loaded axially. By contrast, the spines of the hedgehog are as short as possible while still able to bend when loaded axially. In addition, the hedgehog spines have an internal morphology which delays the onset of local buckling under these loads, thus enabling the spines to absorb larger amounts of mechanical energy. By contrast, the quills of Hystrix are designed to break at the tip. Thus, whilst the quills of the porcupine seem to be well adapted for keeping an enemy as far away as possible, the spines of the hedgehog probably have this as an incidental function. Their main function is much more likely to be that of a shock absorber or storage of impact energy when the animal falls from a height, a behavioural attribute which is reportedly common.     

Circular dichroism of reduced and oxidized recombinant human epidermal growth factor.

To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r-HuEGF) we studied the structure of reduced and oxidized r-HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r-HuEGF in 10 mM sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of alpha-helix, beta-sheet, and unordered structure. The addition of the alpha-helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of alpha-helix, at the apparent expense of beta-sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r-HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding.     

Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (M_r 120 000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843–850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450_BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450_BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its M_r of 38 000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH₂-terminal amino acid is proline; the penultimate NH₂-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450_BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450_BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450_BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450_BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.     

Changes in Conformation and Stability upon Formation of Complexes of Erythropoietin (EPO) and Soluble EPO Receptor

Erythropoietin (EPO) is a glycoprotein hormone which belongs to the four-helical-bundle cytokine family and regulates the level of circulating red blood cells. The EPO receptor (EPOR) belongs to the cytokine-receptor family of proteins. While many of the downstream events following receptor/ligand interaction have been defined, both ligand-induced receptor dimerization and conformational changes induced by binding have been implicated as the initial step in signal transduction. In a recent paper [Philo et al. (1996), Biochemistry 38, 1681–1691] we described the formation of both 1:1 and 2:1 EPOR/EPO complexes. In this paper, we examine changes in protein conformation and stability resulting from the formation of both 1:1 and 2:1 complexes of the soluble extracellular domain of EPOR and the recombinant EPO derived from either Chinese hamster ovary cells or from Escherichia coli cells. Occupation of the first binding site results in a slight conformational change that is apparent in both the far- and near-UV circular dichroism spectra. Formation of the 2:1 complex results in an even greater change in conformation which involves the local environment of one or more aromatic amino acids, accompanied perhaps by a small increase in helical content of the complex. This change in local conformation could occur in the EPO molecule, in the EPOR, in both EPOR molecules due to dimerization, or in all molecules in the trimer. The 1:1 complex exhibits increased stability to thermal-induced denaturation relative to the individual protein component; indeed, the E. coli-derived (nonglycosylated) EPO stays folded in the complex at temperatures where the EPO alone would have unfolded and precipitated. Glycosylation of the receptor increases the reversibility of thermal denaturation, but does not affect the temperature at which this unfolding reaction occurs.     

Solvent modulation in hydrophobic interaction chromatography

Solvents play a critical role in hydrophobic interaction chromatography (HIC), since the separation of proteins by HIC is based on the hydrophobicity of the proteins presented to the solvents. This review first describes the solvent properties which determine the effect of cosolvents on the binding and elution of proteins in HIC; i.e., the protein solvent interactions and the surface tension of water/cosolvent mixture. Second are presented the various cosolvents which have been tested as facilitating binding or elution of the proteins. Last, some examples of solvent manipulation which resolved complex mixtures of proteins by HIC are reviewed.