alpha-Synuclein enhances bioluminescent activity of firefly luciferase by facilitating luciferin localization.

alpha-Synuclein, the pathological component of Parkinson's disease, has been demonstrated to be highly interactive with various protein partners. alpha-Synuclein has been shown to exert a novel effect on the bioluminescence of firefly luciferase by stimulating the oxyluciferin formation from its substrate of luciferin, which results in a significant enhancement of the spike of flashing light via concomitant augmentation for both rapid rise and quick decay of the luminescence. Binding affinity between alpha-synuclein and luciferase was evaluated with K(d) of 8.1 microM based on a dose-dependent enhancement of the luciferase activity by alpha-synuclein. Kinetic analyses indicated that alpha-synuclein has facilitated luciferin localization to the luciferase by decreasing apparent K(m), which makes the maximum rate of bioluminescence no longer dependent upon ATP concentration. Catalytic consequences of the alpha-synuclein binding to luciferase have led to a delayed onset of the coenzyme A-mediated retardation of the quick decay of flashing light as well as a shift in the emission spectra of bioluminescence. Taken together, the novel effects of alpha-synuclein toward the bioluminescence of luciferase have been demonstrated to be initiated by the specific molecular interaction between the proteins which has influenced the substrate (luciferin) localization to the enzyme.

Arabidopsis proteins important for modulating defense responses to Pseudomonas syringae that secrete HopW1-1.

Plant infection responses result from the interaction of pathogen-derived molecules with host components. For the bacterial pathogen Pseudomonas syringae, these molecules are often effector proteins (Hops) that are injected into plant cells. P. syringae carrying hopW1-1 have restricted host range on some Arabidopsis thaliana accessions. At least two Arabidopsis genomic regions are important for the natural variation that conditions resistance to P. syringae/hopW1-1. HopW1-1 elicits a resistance response, and consequently the accumulation of the signal molecule salicylic acid (SA) and transcripts of HWI1 (HopW1-1-Induced Gene1). This work identified three HopW1-1-interacting (WIN) plant proteins: a putative acetylornithine transaminase (WIN1), a protein phosphatase (WIN2) and a firefly luciferase superfamily protein (WIN3). Importantly, WIN2 and WIN3 are partially required for HopW1-1-induced disease resistance, SA production and HWI1 expression. The requirement for WIN2 is specific for HopW1-1-induced resistance, whereas WIN3 is important for responses to several effectors. Overexpression of WIN2 or WIN3 confers resistance to virulent P. syringae, which is consistent with these proteins being defense components. Several known genes important for SA production or signaling are also partially (EDS1, NIM1/NPR1, ACD6 and ALD1) or strongly (PAD4) required for the robust resistance induced by HopW1-1, suggesting a key role for SA in the HopW1-1-induced resistance response. Finally, WIN1 is an essential protein, the overexpression of which over-rides the resistance response to HopW1-1 (and several other defense-inducing effectors), and delays SA and HWI1 induction. Thus, the WIN proteins have different roles in modulating plant defense.

Stable RNA interference in Spodoptera frugiperda cells by a DNA vector-based method.

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study.