Evidence of Disrupted-in Schizophrenia 1 (DISC1) as an arsenic binding protein and implications regarding its role as a translational activator.

Disrupted-in-schizophrenia-1 (DISC1) is a scaffold protein that plays a pivotal role in orchestrating signaling pathways involved in neurodevelopment, neural migration, and synaptogenesis. Among those, it has recently been reported that the role DISC1 in the Akt/mTOR pathway can shift from a global translational repressor to a translational activator in response to oxidative stress induced by arsenic. In this study we are providing evidence that DISC1 can directly bind arsenic via a C-terminal cysteine motif (C-X-C-X-C). A series of fluorescence-based binding assays were conducted with a truncated C-terminal domain construct of DISC1 and a of series of single, double, and triple cysteine mutants. We found that arsenous acid, a trivalent arsenic derivative, specifically binds to the C-terminal cysteine motif of DISC1 with low micromolar affinity. All three cysteines of the motif are required for high-affinity binding. Electron microscopy experiments combined with in silico structural predictions revealed that that the C-terminal of DISC1 forms an elongated tetrameric complex. The cysteine motif is consistently predicted to be located within a loop, fully exposed to solvent, providing a simple molecular framework to explain the high-affinity of DISC1 toward arsenous acid. This study sheds light on a novel functional facet of DISC1 as an arsenic binding protein and highlights its potential role as both a sensor and translational modulator within the Akt/mTOR pathway.

Evidence of DISC1 as an arsenic binding protein and implications regarding its role as a translational activator.

Disrupted-in-schizophrenia-1 (DISC1) is a scaffolding protein that plays a pivotal role in orchestrating signaling pathways involved in neurodevelopment, neural migration, and synaptogenesis. Among those, it has recently been reported that the role of DISC1 in the Akt/mTOR pathway can shift from a global translational repressor to a translational activator in response to oxidative stress induced by arsenic. In this study we provide evidence that DISC1 can directly bind arsenic via a C-terminal cysteine motif (C-X-C-X-C). A series of fluorescence-based binding assays were conducted with a truncated C-terminal domain construct of DISC1 and a series of single, double, and triple cysteine mutants. We found that arsenous acid, a trivalent arsenic derivative, specifically binds to the C-terminal cysteine motif of DISC1 with low micromolar affinity. All three cysteines of the motif are required for high-affinity binding. Electron microscopy experiments combined with in silico structural predictions reveal that the C-terminal of DISC1 forms an elongated tetrameric complex. The cysteine motif is consistently predicted to be located within a loop, fully exposed to solvent, providing a simple molecular framework to explain the high-affinity of DISC1 toward arsenous acid. This study sheds light on a novel functional facet of DISC1 as an arsenic binding protein and highlights its potential role as both a sensor and translational modulator within Akt/mTOR pathway.

Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore.

SNARE-dependent membrane fusion is essential for neurotransmitter release at the synapse. Recently, α-synuclein has emerged as an important regulator for membrane fusion. Misfolded α-synuclein oligomers are potent fusion inhibitors. However, the function of normal α-synuclein has been elusive. Here, we use the single vesicle-to-supported bilayer fusion assay to dissect the role of α-synuclein in membrane fusion. The assay employs 10 kD Rhodamine B-dextran as the content probe that can detect fusion pores larger than ∼6 nm. We find that the SNARE complex alone is inefficient at dilating fusion pores. However, α-synuclein dramatically increases the probability as well as the duration of large pores. When the SNARE-interacting C-terminal region of α-synuclein was truncated, the mutant behaves the same as the wild-type. However, the double proline mutants compromising membrane-binding show significantly reduced effects on fusion pore expansion. Thus, our results suggest that α-synuclein stimulates fusion pore expansion specifically through its membrane binding.