1. "SNAP" - Versatile intracellular cargo delivery system based on the Shiga toxin subunit B
Kresik Leanid
Shiga toxin is a bacterial exotoxin possessing an AB5 molecular configuration. An enzymatically active monomeric A subunit (Shiga toxin subunit A; StxA) is non-covalently associated with a homopentamer consisting of five identical B fragments that form the subunit B (Shiga toxin subunit B; StxB). B subunit is responsible for binding to the cell surface receptors and the toxin internalisation. StxB binding is possible through the interaction with the specific receptor the neutral glycosphingolipid globotriaosylceramide (Gb3) present on the surface of cells. Gb3 was found to be restrictedly expressed in epithelial cells and overexpressed in various primary human cancers and cancer cell lines. Notably, in the absence of the enzymatically active StxA, StxB still adopts its pentameric structure and the ability for receptor binding, which makes it a potential candidate for designing the intracellular delivery system. This work aimed to develop a versatile StxB-based intracellular delivery system called "SNAP" using a modified StxB and a chemical conjugation method requiring a maleimide-polyethylene glycol-(2)-succinimidyl ester (Mal-PEG2-NHS) linker. EGFP and mCherry fluorescence proteins were used as example cargo proteins. The success of the StxB conjugates formation was assessed by SDS-PAGE electrophoresis and size-exclusion chromatography, followed by the internalisation studies using the VeroE6 cell line and confocal microscopy imaging. "SNAP" method developed in these studies could be implemented as an StxB-mediated intracellular cargo delivery technique and could overturn the protein-protein conjugation in general.
2. The use of glass substrates with bi-functional silanes for designing micropatterned cell-secreted cytokine immunoassays
Biomaterials, Volume 32, Issue 23, August 2011, 5478-5488. doi: https://doi.org/10.1016/j.biomaterials.2011.04.026 Jeong Hyun Seo, Li-Jung Chen, Stanislav V. Verkhoturov, Emile A. Schweikert, Alexander Revzin
It is often desirable to sequester cells in specific locations on the surface and to integrate sensing elements next to the cells. In the present study, surfaces were fabricated so as to position cytokine sensing domains inside non-fouling poly(ethylene glycol) (PEG) hydrogel microwells. Our aim was to increase sensitivity of micropatterned cytokine immunoassays through covalent attachment of biorecognition molecules. To achieve this, glass substrates were functionalized with a binary mixture of acrylate- and thiol-terminated methoxysilanes. During subsequent hydrogel photopatterning steps, acrylate moieties served to anchor hydrogel microwells to glass substrates. Importantly, glass attachment sites within the microwells contained thiol groups that could be activated with a hetero-bifunctional cross-linker for covalent immobilization of proteins. After incubation with fluorescently-labeled avidin, microwells fabricated on a mixed acryl/thiol silane layer emitted ∼ 6 times more fluorescence compared to microwells fabricated on an acryl silane alone. This result highlighted the advantages of covalent attachment of avidin inside the microwells.
3. Anisotropy in mechanical unfolding of protein upon partner-assisted pulling and handle-assisted pulling
Communications Biology, Communications Biology volume 4, Article number: 925 (2021). doi: https://doi.org/10.1038/s42003-021-02445-y Nisha Arora, Jagadish Prasad Hazra & Sabyasachi Rakshit
Proteins as force-sensors respond to mechanical cues and regulate signaling in physiology. Proteins commonly connect the source and response points of mechanical cues in two conformations, independent proteins in end-to-end geometry and protein complexes in handshake geometry. The force-responsive property of independent proteins in end-to-end geometry is studied extensively using single-molecule force spectroscopy (SMFS). The physiological significance of the complex conformations in force-sensing is often disregarded as mere surge protectors. However, with the potential of force-steering, protein complexes possess a distinct mechano-responsive property over individual force-sensors. To decipher, we choose a force-sensing protein, cadherin-23, from tip-link complex and perform SMFS using end-to-end geometry and handshake complex geometry.
