ABSTRACT
Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein-protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
Subject(s)
Light , Optogenetics/methods , Protein Engineering/methods , Protein Interaction Domains and Motifs/radiation effects , Animals , Caspase 9/radiation effects , Gene Library , Genetic Engineering , HEK293 Cells , HumansABSTRACT
Selenoprotein H (SelH) is one of the 25 so far identified selenoproteins. Selenoproteins may function as antioxidants, heavy metal antidotes, and neural survival factors. Previous studies have shown that overexpression of SelH in HT22 cells protected the cells from UVB irradiation-induced death by reducing superoxide formation. The objective of this study was to determine the effects of SelH on cell signaling pathways after UVB irradiation. We exposed both human SelH- and vector-transfected HT22 cells to UVB irradiation and collected samples at 5 and 17 h of recovery. Cell viability was assessed, as well as protein levels of caspase-3, -8, -9, apoptosis-inducing factor (AIF), P53, nuclear respiratory factor-1 (NRF-1) and heat shock protein 40 (HSP40). Mitochondrial membrane potential was determined by flow cytometry. Overexpression of SelH protected cells against UVB-induced injury by blockade of the mitochondria-initiated cell death pathway, prevention of mitochondrial membrane depolarization, and suppression of the increase of p53. Furthermore, overexpression of SelH increased levels of NRF-1, an antioxidant, and HSP40, a protein chaperone that repairs denatured protein. We conclude that SelH protects neurons against UVB-induced damage by inhibiting apoptotic cell death pathways, by preventing mitochondrial depolarization, and by promoting cell survival pathways.
Subject(s)
DNA-Binding Proteins/physiology , Neurons/metabolism , Neurons/radiation effects , Selenoproteins/physiology , Signal Transduction/genetics , Signal Transduction/radiation effects , Ultraviolet Rays , Animals , Apoptosis/genetics , Apoptosis/physiology , Apoptosis/radiation effects , Blotting, Western , Caspase 3/metabolism , Caspase 3/radiation effects , Caspase 9/metabolism , Caspase 9/radiation effects , Cell Line , Cell Survival/genetics , Cell Survival/physiology , Cell Survival/radiation effects , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression/physiology , Genetic Vectors , HSP40 Heat-Shock Proteins/biosynthesis , HSP40 Heat-Shock Proteins/genetics , Humans , Membrane Potentials/physiology , Mice , Mitochondrial Membranes/physiology , Nuclear Respiratory Factor 1/biosynthesis , Nuclear Respiratory Factor 1/genetics , Selenoproteins/biosynthesis , Selenoproteins/genetics , Tumor Suppressor Protein p53ABSTRACT
BACKGROUND: The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis. METHODS: Tumour cells were treated with gemcitabine, radiation, or the combination. 0-72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT(2) Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment. RESULTS: Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment. CONCLUSION: A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.
