Sarkis, J. | bioRxiv Science

bioRxiv2025

Experimental galactose-1 phosphate uridylyltransferase (GALT) mRNA therapy improves motor-related phenotypes in a mouse model of Classic Galactosemia

Despite life-saving newborn screening programs and a life-long galactose-restricted diet, many patients with Classic Galactosemia continue to develop long-term debilitating neurological deficits, speech dyspraxia, and primary ovarian insufficiency (POI). Earlier, we showed that administration of an experimental human GALT mRNA predominantly expressed in the liver of the GalT gene-trapped mouse model augmented the expression of hepatic GALT activity, which reduced build-up of galactose and its toxic metabolites not only in the liver, but also in the peripheral tissues. Moreover, we showed that administration of GALT mRNA in the mutant mice restored whole-body galactose oxidation (WBGO), a functional biomarker. In this study, we extended our proof-of-concept (POC) efficacy studies to a disease-relevant phenotype, motor impairment. We found that a biweekly dosing regimen at 2mg/kg for 2 months could improve the motor performance of the animals in Rotarod and Composite Phenotype Scoring tests.

genetics↗

bioRxiv2022

Probing weak lipid-lipid binding domain interactions with a mechanically transduced immunosorbent assay (METRIS)

Protein-lipid interactions constitute a very important class of biological interactions critical for multiple cell and tissue functions. It is believed that most lipid-protein interactions are very weak, with affinities in the 1mM-1M range. Here we study the interactions of multiple protein lipid binding domains with lipid membranes containing signaling lipids known as phosphatidylinositol phosphates (PIPs) using a new mechanically transduced immunosorbent assay (METRIS). We demonstrate that this assay can measure extremely weak interactions at PIP bilayer concentrations below 1%, which is close to the biological lipid concentration regime. In particular, we have studied the interaction of DrrAWT, DrrAK568A, PH-{delta}, and 2XFYVE as well as previously unexplored lipid binding domains such as Auxilin 1 (PTEN) and Auxilin 2 (GAK) against a wide palette of PIPs. Our results confirm that each of these domains interacts specifically with a PIP partner. In the case of Auxilin 1 and Auxilin 2, both proteins in the Clathrin endocytotic pathway, we find that their PTEN-like domain interacts specifically yet with ultra low affinity with PI3P and PI4P respectively. We have also found a new unknown medium-high affinity interaction between GAK with PI34P2. Our work, thus, provides a direct and robust method to measure and catalog protein lipid interactions which are important in many processes such as signaling and membrane sculpting. Furthermore, this assay can be extended in a straightforward manner to study other interactions such as ligand-receptor or antibody-antigen.

biophysics↗