OptoREACT: Optogenetic Receptor Activation on Nonengineered Human T Cells.

Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.

Reversible Shielding and Immobilization of Liposomes and Viral Vectors by Tailored Antibody-Ligand Interactions.

Controlling the time and dose of nanoparticulate drug delivery by administration of small molecule drugs holds promise for efficient and safer therapies. This study describes a versatile approach of exploiting antibody-ligand interactions for the design of small molecule-responsive nanocarrier and nanocomposite systems. For this purpose, antibody fragments (scFvs) specific for two distinct small molecule ligands are designed. Subsequently, the surface of nanoparticles (liposomes or adeno-associated viral vectors, AAVs) is modified with these ligands, serving as anchor points for scFv binding. By modifying the scFvs with polymer tails, they can act as a non-covalently bound shielding layer, which is recruited to the anchor points on the nanoparticle surface and prevents interactions with cultured mammalian cells. Administration of an excess of the respective ligand triggers competitive displacement of the shielding layer from the nanoparticle surface and restores nanoparticle-cell interactions. The same principle is applied for developing hydrogel depots that can release integrated AAVs or liposomes in response to small molecule ligands. The liberated nanoparticles subsequently deliver their cargoes to cells. In summary, the utilization of different antibody-ligand interactions, different nanoparticles, and different release systems validates the versatility of the design concept described herein.

Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC.

Thiolatocobalamins are a class of cobalamins comprised of naturally occurring and synthetic ligands. Glutathionylcobalamin (GSCbl) occurs naturally in mammalian cells, and also as an intermediate in the glutathione-dependent dealkylation of methylcobalamin (MeCbl) to form cob(I)alamin by pure recombinant CblC from C. elegans. Glutathione-driven deglutathionylation of GSCbl was demonstrated both in mammalian as well as in C. elegans CblC. Dethiolation is orders of magnitude faster than dealkylation of Co-C bonded cobalamins, which motivated us to investigate two synthetic thiolatocobalamins as substrates to repair the enzymatic activity of pathogenic CblC variants in humans. We report the synthesis and kinetic characterization of cysteaminylcobalamin (CyaCbl) and 2-mercaptopropionylglycinocobalamin (MpgCbl). Both CyaCbl and MpgCbl were obtained in high purity (90-95%) and yield (78-85%). UV-visible spectral properties agreed with those reported for other thiolatocobalamins with absorbance maxima observed at 372 nm and 532 nm. Both CyaCbl and MpgCbl bound to wild type human recombinant CblC inducing spectral blue-shifts characteristic of the respective base-on to base-off transitions. Addition of excess glutathione (GSH) resulted in rapid elimination of the ß-ligand to give aquacobalamin (H2OCbl) as the reaction product under aerobic conditions. Further, CyaCbl and MpgCbl underwent spontaneous dethiolation thereby repairing the loss of activity of pathogenic variants of human CblC, namely R161G and R161Q. We posit that thiolatocobalamins could be exploited therapeutically for the treatment of inborn errors of metabolism that impair processing of dietary and supplemental cobalamin forms. While these disorders are targets for newborn screening in some countries, there is currently no effective treatment available to patients.