Mendel, R. R. | Search bioRxiv

bioRxiv2020

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficient C. elegans

The molybdenum cofactor (Moco) is a 520 dalton prosthetic group that is synthesized in all domains of life. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco-biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy, however the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement, rescuing a C. elegans model of Moco deficiency. We demonstrate that diverse Moco:protein complexes are stable and bioavailable, suggesting a new strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

developmental biology↗

bioRxiv2020

Split-HaloTag Imaging Assay for in vivo 3D-Microscopy and Subdiffractional Analyses of Protein-Protein Interactions

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualisation of protein complex localisation. Due to its simple protocols, it has become one of the most frequently used methods. However, standard fluorescent proteins entail several drawbacks for sophisticated microscopy. With the HaloTag(R) system, these drawbacks can be overcome as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we established the Split-HaloTag(R) imaging assay in plants which is based on the reconstitution of a functional HaloTag(R) protein upon protein-protein interaction and subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using well-characterised interactions as an example for protein-protein interaction at cellular structures: the molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Additionally, a specific interaction was visualised with subdiffractional polarisation microscopy in a more distinctive manner as example for sophisticated imaging. Split-GFP and Split-HaloTag(R) can complement one another as Split-HaloTag(R) represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag(R) imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interaction with specific microscopic techniques such as 3D-imaging, single molecule tracking and super-resolution microscopy.

plant biology↗