Controlling Protein Activity and Degradation Using Blue Light

Abstract

Regulation of protein stability is a fundamental process in eukaryotic cells and pivotal to, e.g., cell cycle progression, faithful chromosome segregation, or protein quality control. Synthetic regulation of protein stability requires conditional degradation sequences (degrons) that induce a stability switch upon a specific signal. Fusion to a selected target protein permits to influence virtually every process in a cell. Light as signal is advantageous due to its precise applicability in time, space, quality, and quantity. Light control of protein stability was achieved by fusing the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin1 with a synthetic degron (cODC1) derived from the carboxy-terminal degron of ornithine decarboxylase to obtain the photosensitive degron (psd) module. The psd module can be attached to the carboxy terminus of target proteins that are localized to the cytosol or nucleus to obtain light control over their stability. Blue light induces structural changes in the LOV2 domain, which in turn lead to activation of the degron and thus proteasomal degradation of the whole fusion protein. Variants of the psd module with diverse characteristics are useful to fine-tune the stability of a selected target at permissive (darkness) and restrictive conditions (blue light).

Engineering Critical Enzymes and Pathways for Improved Triterpenoid Biosynthesis in Yeast

“…With the development of protein engineering, many mature strategies have been applied to control protein activity. For example, several conditional degrons have been designed to control the abundance of targeted protein by adapting experimental conditions, which is a promising strategy to repress the Erg7 expression [98,99]. Active-site mutagenesis of triterpenoid-related P450s might increase the catalytic efficiency and decrease the generation of by-products, which has been utilized to improve the selectivity and catalytic activity of the bacterial P450s [100].…”

An Optogenetic Toolbox for Synergistic Regulation of Protein Abundance

“…Altogether, this demonstrates that the novel psd3 V416L module increases the flexibility in creating lightsensitive mutants, in addition to the previously published modules. 12,17,25 The novel variants psd3 V416L and psTF (tetR-AsLOV2 V416L / Zdk1-psd3) will be of importance under circumstances, in which only low amounts of blue light are deliverable toward the construct-containing cells. These constructs are therefore very well-suited for bioreactor applications in which delivery of high light doses is not always possible due to high cell densities.…”

Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum–associated degradation pathway

“…Low-fluorescence medium was used to grow yeast cells in liquid cultures exposed to blue light using standard plastic cell culture flasks (Usherenko et al , 2014). Modification of yeast genes with the psd module at chromosomal level was performed as described using plasmid pDS96 as template (Lutz et al , 2016). Thus, all psd-modified variants of SEC61 , SEC62 , and SEC66 are the sole copies of these genes.…”