Split Enzyme-Based Biosensors for Structural Characterization of Soluble and Insoluble β-Glucans

β-Glucan is widely distributed in various plants and microorganisms and is composed of β-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with β-1,6- or β-1,4-linkage. Numerous studies have investigated different β-glucans and revealed their bioactivities. To understand the structure-function relationship of β-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain β-1,6-branched β-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived β-1,3-glucan recognition protein and fungal endo-β-1,6-glucanase (Neg1)-derived β-1,6-glucan recognition protein, respectively. In this approach, two β-glucan recognition proteins bound to β-glucan molecules come into close proximity, resulting in the assembly of the full-length reporter enzyme and induction of transient luciferase activity, indicative of the structure of β-glucan. To test the applicability of this assay, β-glucan and two β-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a β-1,3-glucan with a long polymer of β-1,6-glucan in a dose-dependent manner. This simple test also allows the monitoring of real-time changes in the side chain structure and serves as a convenient method to distinguish between β-1,3-glucan and long-chain β-1,6-branched β-1,3-glucan in various soluble and insoluble β-glucans.

A Split Luciferase Complementation Assay for the Quantification of β-Arrestin2 Recruitment to Dopamine D2-Like Receptors

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of β-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify β-arrestin2 recruitment to D2long and D3 receptors and measure time-resolved β-arrestin2 recruitment to the D2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no β-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the β-arrestin recruitment to the D2longR and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/β-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.

Ralstonia solanacearum elicitor RipX Induces Defense Reaction by Suppressing the Mitochondrial atpA Gene in Host Plant

RipX of Ralstonia solanacearum is translocated into host cells by a type III secretion system and acts as a harpin-like protein to induce a hypersensitive response in tobacco plants. The molecular events in association with RipX-induced signaling transduction have not been fully elucidated. This work reports that transient expression of RipX induced a yellowing phenotype in Nicotiana benthamiana, coupled with activation of the defense reaction. Using yeast two-hybrid and split-luciferase complementation assays, mitochondrial ATP synthase F1 subunit α (ATPA) was identified as an interaction partner of RipX from N. benthamiana. Although a certain proportion was found in mitochondria, the YFP-ATPA fusion was able to localize to the cell membrane, cytoplasm, and nucleus. RFP-RipX fusion was found from the cell membrane and cytoplasm. Moreover, ATPA interacted with RipX at both the cell membrane and cytoplasm in vivo. Silencing of the atpA gene had no effect on the appearance of yellowing phenotype induced by RipX. However, the silenced plants improved the resistance to R. solanacearum. Moreover, qRT-PCR and promoter GUS fusion experiments revealed that the transcript levels of atpA were evidently reduced in response to expression of RipX. These data demonstrated that RipX exerts a suppressive effect on the transcription of atpA gene, to induce defense reaction in N. benthamiana.

Modular bioengineered kinase sensors via scaffold protein-mediated split-luciferase complementation

Phosphorylation is a key regulation event in cellular signaling. To sense the underlying kinase activity, we engineered modular and easy adaptable serine kinase sensors for the exemplary kinases PKA, PKB and CHK1.