Curated Optogenetic Publication Database

Abstract: Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC), by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.

Abstract: Apoptosis is a cell death program involved in the development of multicellular organisms, immunity, and pathologies ranging from cancer to HIV/AIDS. We present an engineered protein that causes rapid apoptosis of targeted cells in monolayer culture after stimulation with blue light. Cells transfected with the protein switch L57V, a tandem fusion of the light-sensing LOV2 domain and the apoptosis-executing domain from caspase-7, rapidly undergo apoptosis within 60 min after light stimulation. Constant illumination of under 5 min or oscillating with 1 min exposure had no effect, suggesting that cells have natural tolerance to a short duration of caspase-7 activity. Furthermore, the overexpression of Bcl-2 prevented L57V-mediated apoptosis, suggesting that although caspase-7 activation is sufficient to start apoptosis, it requires mitochondrial contribution to fully commit.