Judge, P. J. | bioRxiv Science

bioRxiv2020

Physicochemical characterization, toxicity and in vivo biodistribution studies of a discoidal, lipid-based drug delivery vehicle: Lipodisq nanoparticles containing doxorubicin

Many promising pharmaceutically active compounds have low solubility in aqueous environments and their encapsulation into efficient drug delivery vehicles is crucial to increase their bioavailability. Lipodisq nanoparticles are approximately 10 nm in diameter and consist of a circular phospholipid bilayer, stabilized by an annulus of SMA (a hydrolysed copolymer of styrene and maleic anhydride). SMA is used extensively in structural biology to extract and stabilize integral membrane proteins for biophysical studies. Here, we assess the potential of these nanoparticles as drug delivery vehicles, determining their cytotoxicity and the in vivo excretion pathways of their polymer and lipid components. Doxorubicin-loaded Lipodisqs were cytotoxic across a panel of cancer cell lines, whereas nanoparticles without the drug had no effect on cell proliferation. Intracellular doxorubicin release from Lipodisqs in HeLa cells occurred in the low-pH environment of the endolysosomal system, consistent with the breakdown of the discoidal structure as the carboxylate groups of the SMA polymer become protonated. Biodistribution studies in mice showed that, unlike other nanoparticles injected intravenously, most of the Lipodisq components were recovered in the colon, consistent with rapid uptake by hepatocytes and excretion into bile. These data suggest that Lipodisqs have the potential to act as delivery vehicles for drugs and contrast agents.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsDLPE1,2-dilauroyl-sn-glycero-3-phosphoethanolamineDLPE-Cy5Cy5 dye covalently conjugated to 1,2-dilauroyl-sn-glycero-3-phosphoethanolamineDLSdynamic light scatteringDMPC1,2-dimyristoyl-sn-glycero-3-phosphocholineDMPG1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol)DOXDoxorubicinEPRenhanced permeability and retention effectLipodisqLQNMRnuclear magnetic resonanceNPnanoparticleRTroom temperatureSMAhydrolysed co-polymer of styrene and maleic anhydrideSMA-Cy5Cy5 dye covalently conjugated to a thiol derivative of SMASMAnhnon-hydrolysed co-polymer of styrene and maleic anhydrideSMAnh-SHthiol derivative of SMAnhSMA-SHthiol derivative of SMAView Full Text

pharmacology and toxicology↗

bioRxiv2019

Conformational flexibility of GRASP protein and its constituent PDZ subdomains reveals structural basis of its promiscuous interactome

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix, which includes the Golgi Reassembly and Stacking Proteins (GRASPs), which participate in cisternae stacking and lateral linkage in vertebrates. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different protein binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content, however PDZ1 alone mediates nearly all the interactions between GRASPs and their binding partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual {beta}31{beta}4{beta}52{beta}6{beta}1{beta}2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable {beta}2-strand and a more flexible and unstable 2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.\n\nSignificance StatementGolgi Reassembly and Stacking Proteins (GRASPs) play pivotal roles in the maintenance of Golgi structure as well as in unconventional protein secretion. Their broad network of interactions is mainly sustained by the two-PDZ domains located in the N-terminal portion of the protein. The asymmetry of the PDZ domains in terms of number and diversity of interacting partners has been long recognized, but the molecular determinants of that asymmetry remains largely unknown. The biophysical data presented here provide a firm basis for understanding why PDZ1 behaves differently to PDZ2 in solution, despite their similar 3D structures. Furthermore, we propose that PDZ2 assist ligand binding to PDZ1, by means of conformational stabilization.

biophysics↗