Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles.

Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.

Mutant torsinA in the heterozygous DYT1 state compromises HSV propagation in infected neurons and fibroblasts.

Most cases of early onset torsion dystonia (DYT1) are caused by a 3-base pair deletion in one allele of the TOR1A gene causing loss of a glutamate in torsinA, a luminal protein in the nuclear envelope. This dominantly inherited neurologic disease has reduced penetrance and no other medical manifestations. It has been challenging to understand the neuronal abnormalities as cells and mouse models which are heterozygous (Het) for the mutant allele are quite similar to wild-type (WT) controls. Here we found that patient fibroblasts and mouse neurons Het for this mutation showed significant differences from WT cells in several parameters revealed by infection with herpes simplex virus type 1 (HSV) which replicates in the nucleus and egresses out through the nuclear envelope. Using a red fluorescent protein capsid to monitor HSV infection, patient fibroblasts showed decreased viral plaque formation as compared to controls. Mouse Het neurons had a decrease in cytoplasmic, but not nuclear HSV fluorescence, and reduced numbers of capsids entering axons as compared to infected WT neurons. These findings point to altered dynamics of the nuclear envelope in cells with the patient genotype, which can provide assays to screen for therapeutic agents that can normalize these cells.