Alice Vrielink

Cell membrane-derived vesicles play essential roles in intercellular communication, material transport, and waste disposal. Despite their biomedical and industrial potential, isolating extracellular vesicles from natural sources remains technically challenging, limiting purification efficiency and scalability. This study introduces cell membrane extrusion as an alternative approach to optimize the production of cell membrane-derived vesicles (CSMs), from eukaryotic and prokaryotic cells. CSMs, generated from HeLa and SH-SY5Y cells exhibited a distinctive cup-shaped morphology and sizes of 151.36 ± 72.36 nm, and 416.86 ± 108.49 nm at 20 °C by DLS respectively, showing remarkable thermal stability at 4-70 °C range. Furthermore, loaded vesicles interacted with mammalian cells and achieved successful cargo internalization. CSMs were also produced from E. coli membranes, forming unilamellar vesicles of approximately 100 nm, as observed by Cryo-TEM. These vesicles displayed an inverse correlation between vesicle size and thermal stability and efficient cargo incorporation detected in 85% ± 3% of CSMs. However, under tested conditions, no interaction with prokaryotic cells occurred, and consequently, no delivery of the loaded molecule was observed. Overall, thesefindings highlight the potential of generating cell membrane-derived nanovesicles through extrusion, offering a promising strategy to mimic extracellular vesicles for innovative biomedical and industrial applications, including targeted drug delivery system.

Mitochondria are membrane bound organelles of endosymbiotic origin with limited protein coding capacity. As a consequence, the continual import of nuclear-encoded protein and nucleic acids such as DNA and small non-coding RNA is required and essential for maintaining organelle mass, number and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNA ^ala via conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with 6 SAM domains per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of Tric1 SAM domain was essential for protein function whereby mutation of Gly241 resulted in the disruption of the oligomer and the loss of RNA binding capability in Tric1. Furthermore, complementation of Arabidopsis thaliana Tric1/2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype suggesting the oligomerization is essential for function in planta . AlphaFold2 structure prediction of the SAM domain and Tric1 support a cyclic hexamer generating a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane. Our results highlight the importance of oligomerization of Tric1 for protein function.

A new high-pressure single-crystal diffraction setup has been designed and implemented at the Australian Synchrotron for collecting molecular and protein crystal structures. The setup incorporates a modified micro-Merrill–Bassett cell and holder designed specifically to fit onto the horizontal air-bearing goniometer, allowing high-pressure diffraction measurements to be collected with little to no modification of the beamline setup compared with ambient data collections. Compression data for the amino acid, L-threonine, and the protein, hen egg-white lysozyme, were collected, showcasing the capabilities of the setup.

Many pathogenic gram-negative bacteria have developed mechanisms to increase resistance to cationic antimicrobial peptides by modifying the lipid A moiety. One modification is the addition of phosphoethanolamine to lipid A by the enzyme phosphoethanolamine transferase (EptA). Previously we reported the structure of EptA from Neisseria, revealing a two-domain architecture consisting of a periplasmic facing soluble domain and a transmembrane domain, linked together by a bridging helix. Here, the conformational flexibility of EptA in different detergent environments is probed by solution scattering and intrinsic fluorescence-quenching studies. The solution scattering studies reveal the enzyme in a more compact state with the two domains positioned close together in an n-dodecyl-β-D-maltoside micelle environment and an open extended structure in an n-dodecyl-phosphocholine micelle environment. Intrinsic fluorescence quenching studies localize the domain movements to the bridging helix. These results provide important insights into substrate binding and the molecular mechanism of endotoxin modification by EptA.

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesize a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomycesantibioticus termed TNYR SaPLD was developed capable of synthesizing 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

The tumor necrosis factor (TNF)‐like core domain of receptor activator of nuclear factor‐κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast‐poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF‐like core domain. However, the structure–function relationship of this mutation is not clear. Sequence‐based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site‐directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast‐related signal transduction. However, no antagonistic effect was found in M200s against wild‐type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor–ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199‐induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.