Showing 1 - 7 of 7 results
Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum-associated degradation pathway.
Protein quality mechanisms are fundamental for proteostasis of eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) is a well-studied pathway that ensures quality control of secretory and endoplasmic reticulum (ER)-resident proteins. Different branches of ERAD are involved in degradation of malfolded secretory proteins, depending on the localization of the misfolded part, the ER lumen (ERAD-L), the ER membrane (ERAD-M), and the cytosol (ERAD-C). Here we report that modification of several ER transmembrane proteins with the photosensitive degron (psd) module resulted in light-dependent degradation of the membrane proteins via the ERAD-C pathway. We found dependency on the ubiquitylation machinery including the ubiquitin-activating enzyme Uba1, the ubiquitin--conjugating enzymes Ubc6 and Ubc7, and the ubiquitin-protein ligase Doa10. Moreover, we found involvement of the Cdc48 AAA-ATPase complex members Ufd1 and Npl4, as well as the proteasome, in degradation of Sec62-myc-psd. Thus, our work shows that ERAD-C substrates can be systematically generated via synthetic degron constructs, which facilitates future investigations of the ERAD-C pathway.
Optogenetic downregulation of protein levels with an ultrasensitive switch.
Optogenetic control of protein activity is a versatile technique to gain control over cellular processes, e.g. for biomedical and biotechnological applications. Among other techniques, the regulation of protein abundance by controlling either transcription or protein stability found common use as this controls the activity of any type of target protein. Here, we report modules of an improved variant of the photosensitive degron module and a light-sensitive transcription factor, which we compared to doxycycline-dependent transcriptional control. Given their modularity the combined control of synthesis and stability of a given target protein resulted in the synergistic down regulation of its abundance by light. This combined module exhibits very high switching ratios, profound downregulation of protein abundance at low light-fluxes as well as fast protein depletion kinetics. Overall, this synergistic optogenetic multistep control (SOMCo) module is easy to implement and results in a regulation of protein abundance superior to each individual component.
Development of a Synthetic Switch to Control Protein Stability in Eukaryotic Cells with Light.
In eukaryotic cells, virtually all regulatory processes are influenced by proteolysis. Thus, synthetic control of protein stability is a powerful approach to influence cellular behavior. To achieve this, selected target proteins are modified with a conditional degradation sequence (degron) that responds to a distinct signal. For development of a synthetic degron, an appropriate sensor domain is fused with a degron such that activity of the degron is under control of the sensor. This chapter describes the development of a light-activated, synthetic degron in the model organism Saccharomyces cerevisiae. This photosensitive degron module is composed of the light-oxygen-voltage (LOV) 2 photoreceptor domain of Arabidopsis thaliana phototropin 1 and a degron derived from murine ornithine decarboxylase (ODC). Excitation of the photoreceptor with blue light induces a conformational change that leads to exposure and activation of the degron. Subsequently, the protein is targeted for degradation by the proteasome. Here, the strategy for degron module development and optimization is described in detail together with experimental aspects, which were pivotal for successful implementation of light-controlled proteolysis. The engineering of the photosensitive degron (psd) module may well serve as a blueprint for future development of sophisticated synthetic switches.
Controlling Protein Activity and Degradation Using Blue Light.
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).
Biophotography: concepts, applications and perspectives.
Synthetic biology aims at manipulating biological systems by rationally designed and genetically introduced components. Efforts in photoactuator engineering resulted in microorganisms reacting to extracellular light-cues with various cellular responses. Some of them lead to the formation of macroscopically observable outputs, which can be used to generate images made of living matter. Several methods have been developed to convert colorless compounds into visible pigments by an enzymatic conversion. This has been exploited as a showcase for successful creation of an optogenetic tool; examples for basic light-controlled biological processes that have been coupled to this biophotography comprise regulation of transcription, protein stability, and second messenger synthesis. Moreover, biological reproduction of images is used as means to facilitate quantitative characterization of optogenetic switches as well as a technique to investigate complex cellular signaling circuits. Here, we will compare the different techniques for biological image generation, introduce experimental approaches, and provide future-perspectives for biophotography.
Photo-sensitive degron variants for tuning protein stability by light.
Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus.
A LOV2 domain-based optogenetic tool to control protein degradation and cellular function.
Light perception is indispensable for plants to respond adequately to external cues and is linked to proteolysis of key transcriptional regulators. To provide synthetic light control of protein stability, we developed a generic photosensitive degron (psd) module combining the light-reactive LOV2 domain of Arabidopsis thaliana phot1 with the murine ornithine decarboxylase-like degradation sequence cODC1. Functionality of the psd module was demonstrated in the model organism Saccharomyces cerevisiae. Generation of conditional mutants, light regulation of cyclin-dependent kinase activity, light-based patterning of cell growth, and yeast photography exemplified its versatility. In silico modeling of psd module behavior increased understanding of its characteristics. This engineered degron module transfers the principle of light-regulated degradation to nonplant organisms. It will be highly beneficial to control protein levels in biotechnological or biomedical applications and offers the potential to render a plethora of biological processes light-switchable.