Binding of alpha-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes.

Parkinson's disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein alpha-synuclein (alpha-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and alpha-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between alpha-syn and Fe(II) (5.8x10(3)M(-1)) has been measured, but studies on the binding between alpha-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between alpha-syn and Fe(II) and the redox property of the resultant alpha-syn-Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O(2)) to the putative alpha-syn-Fe(III) complex, with H(2)O(2) as a co-product. The reduction potential was estimated to be 0.025V vs. Ag/AgCl, which represents a shift by -0.550V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between alpha-syn and Fe(III), 1.2x10(13)M(-1), to be deduced. Despite the relatively high binding affinity, alpha-syn-Fe(III) generated from the oxidation of alpha-syn-Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)(3) and/or ferrihydrite gel. The roles of alpha-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed alpha-syn aggregation, and iron transfer processes.

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus.

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.