Biliverdin Reductase-A Deficiency Brighten and Sensitize Biliverdin-binding Chromoproteins

Abstract

Tissue absorbance, light scattering, and autofluorescence are significantly lower in the nearinfrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/-mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/-mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/-mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.

Near-infrared imaging in fission yeast by genetically encoded biosynthesis of phycocyanobilin

“…The copyright holder for this preprint (which this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.444883 doi: bioRxiv preprint cells as reported previously (Kobachi et al, 2020), but did not show any change in iRFP fluorescence with BV or PCB treatment (Fig. S6B), probably because all iRFP molecules were occupied by BV.…”

“…The copyright holder for this preprint (which this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.444883 doi: bioRxiv preprint generate BV, and the knocked out of biliverdin reductase A (BVRA), which degrades BV to generate bilirubin, improves the brightness of iRFP through the additional accumulation of BV (Kobachi et al, 2020;Shemetov et al, 2017). On the other hand, because Caenorhabditis elegans can hardly produce BV (Ding et al, 2017), it is incapable of imaging iRFP by simply introducing only the iRFP gene in nematodes.…”

Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging

“…For these reasons, it remains challenging to use NIR-GECO2 and NIR-GECO2G to image Ca 2+ dynamics with single-cell resolution in rodents where neuronal BV concentrations are low and cannot be substantially increased by the strategy of co-expressing HO1 [8]. A promising approach to overcome this challenge was recently described by Kobachi and colleagues, who demonstrated that knocking out the gene for BV reductase increases the BV concentration in mice and improves the brightness of NIR--GECO1 by 4.3-fold [22]. While the off-kinetics of NIR-GECOs are very similar to that of GCaMP6s or jGCaMP7s, the on-kinetics of NIR-GECO series are slower than that of GCaMP series.…”

Improvement of Phycocyanobilin Synthesis for Genetically Encoded Phytochrome-Based Optogenetics

“…Importantly, the improved SynPCB systems could overcome the requirement of BVRA depletion for the PhyB-PIF optogenetics (Figure ), which could alleviate the concerns caused by toxicity through BVRA KO or KD. It has been reported that BVRA KO might cause modest abnormalities such as sensitization of oxidative stress. − Meanwhile, all reports using BVRA KO mice indicate that BVRA deficiency does not affect the reproductive rate, weight, growth, and lifespans . We had not observed any detectable changes in proliferation rate in BVRA KO HeLa cells and BVRA KD mESCs .…”

“…53−55 Meanwhile, all reports using BVRA KO mice indicate that BVRA deficiency does not affect the reproductive rate, weight, growth, and lifespans. 56 We had not observed any detectable changes in proliferation rate in BVRA KO HeLa cells and BVRA KD mESCs. 35 On the basis of the evidence, BVRA depletion with RNAi or CRISPR/Cas9-mediated knockout is still the best way to readily utilize the PhyB-PIF system.…”