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Reconstituted Membrane Systems for Assaying Membrane Proteins in Controlled Lipid Environments
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Small proteoliposomes (say less than 100 nm in diameter) have intrinsic curvature which may affect the protein function. Nanodiscs offer a solution to insert proteins into a small patch of planar membrane (Figure 6.1). A nanodisc is composed of a patch of phospholipid bilayer with its hydrophobic edge shielded by two copied of an amphipathic scaffold protein. The nanodiscs are a particularly attractive option for studying membrane proteins in the context of ligand-receptor interactions and are very useful for controlling both sides of the transmembrane proteins in a bilayer. Sligar’s group developed the method for the reconstitution of membrane proteins into nanodiscs.39–41 They took advantage of the special property of naturally occurring amphipathic apolipoprotein A-1 of high-density lipoproteins. Detergent-solubilized phospholipids, membrane scaffold protein (MSP) and proteins of interest are mixed in an empirical ratio. After incubation for 1–2 hours, detergents are removed using BioBeads or dialysis. The MSPs shield the edge of the bilayer formed around the transmembrane protein. The nanodiscs may vary in diameter from 9.8 to 17 nm, depending on the MSPs (Figure 6.1), and can accommodate different sizes of transmembrane proteins.39,41–45 The nanodiscs have advantage over reconstitution vesicles by keeping the proteins in a more native-like environment. Nanodiscs are widely used for the structural studies using cryoEM. There are more than 100 structures of membrane proteins in nanodiscs. A detailed procedure for the preparation of nanodiscs has been described by Sligar’s Lab.42,43 We have adapted the Sligar’s procedure with the detergent removal methods described in the last section and have been successful in making nanodiscs.
Cytochrome P450 Enzymes for the Synthesis of Novel and Known Drugs and Drug Metabolites
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Sanjana Haque, Yuqing Gong, Sunitha Kodidela, Mohammad A. Rahman, Sabina Ranjit, Santosh Kumar
To apply CYP enzymes in broad scale commercial manufacturing, further investigations need to be conducted on screening and protein engineering of CYPs. Mutagenesis of CYPs along with enzymatic coupling may improvise the already known pharmacophores, moreover, can generate libraries of novel compounds. CYP enzymes are being modified to produce active metabolites, natural lead products, and drug targets, which may reduce the difficulties to manufacture these by means of chemical synthesis (Kandel et al., 2014). Several groups are currently working on generating rich libraries of engineered CYPs from bacteria, plant, and mammals, continuously revealing newer and improved CYP enzyme systems. As most of the CYPs require an electron transfer system for the enzymatic reactions, improving this by introducing fusion proteins and CYP reductase will vastly enhance the enzymatic efficiency (Sakaki et al., 1990; Fisher et al., 1992). Another way to overcome this hurdle is to develop an efficient ‘shunt pathway’ for the CYP catalytic reactions, which will deliver active oxygen molecule. Using carboxylic acids as decoy molecule in this case may provide necessary power to the enzymatic reaction (Shoji et al., 2010). Future investigations on laboratory formulated strains of bacteria, transgenic yeasts, or plants as host system and optimizing the reaction process might open the door to identify newer CYP isoforms as well as enable higher expression of CYPs (Martinez and Rupashinghe, 2013; Grunwald, 2014). Another obstacle in the way of industrial CYP usage is the structural instability. Use of immobilized CYPs on gold electrodes is showing promising effect (Lamb et al., 1998; Nicoli et al., 2008). A nanodisc system comprising CYP and CYP reductase has demonstrated potential in enhancing CYP activity and stability, which may also be explored for further possibilities (Denisov and Sligar, 2011; Yoshida, 2012). To conclude, by engineering wild-type CYPs, improvising the host system and catalytic reaction pathways, CYP enzymes can be efficiently employed in industrial drug and drug metabolite synthesis.
Biomaterials-assisted construction of neoantigen vaccines for personalized cancer immunotherapy
Published in Expert Opinion on Drug Delivery, 2023
It has been reported that sufficient internalization of neoantigens and activation by APCs are prerequisites to prime profound antitumor immune responses [60]. Motivated by the high potency of diverse nanocarriers in promoting the delivery of both antigen and immunostimulatory agents to secondary lymphoid organs, diverse nano-biomaterials have recently been extensively explored in the construction of neoantigen nanovaccines to enable personalized cancer immunotherapy [64]. In 2016, James J. Moon and coworkers demonstrated a simple yet scalable strategy to prepare high-density lipoprotein-mimicking nanodiscs with synthetic 22-amino-acid apolipoprotein A1-mimetic peptides and phospholipids via a hydrophobic interaction driven self-assembly process [52]. After being coupled with neoantigen peptides through the cysteine-serine-serine linker-mediated covalent conjugation and cholesterol-modified CpG adjuvants, the yielded nanovaccines could markedly improve the delivery of both antigen and adjuvant to lymphoid organs and thus promote the presentation of antigens on DCs. As the result, such nanovaccines elicited potent neoantigen-specific CD8+ T cells at 47-fold and 31-fold greater frequencies compared to those elicited by soluble vaccines and vaccines formulated with clinically used adjuvant of Montanide, respectively. Furthermore, upon being loaded with multiple tumor-specific neoantigens, the yield nanodisc-based nanovaccines could prime a broad-spectrum adaptive anti-tumor cellular immunity to suppress the growth of different tumors in mice, particularly in synergizing with ICB immunotherapies.
The phosphoinositide code is read by a plethora of protein domains
Published in Expert Review of Proteomics, 2021
Michael Overduin, Troy A. Kervin
The ongoing challenge now is to resolve the diverse lipidons and memteins at atomic resolution, including quantifying the dynamic interactions between lipid and protein molecules as they exist in vivo, including the regulated states. Such complexes are the best representations of many therapeutic targets and encompass not only the membrane readers described here but also integral membrane systems. The design of improved native nanodisc systems to extract, purify, and measure such assemblies with minimal perturbation or bias remains an ongoing goal of the SMALP network. Being able to extract and present intact assemblies of membrane readers bound to lipidons from cells and tissues will allow screening of drug-like molecules and antibodies that are more specific for biologically relevant states. Disruption of protein:PI interactions through PTMs or mutations of membrane readers reprograms cellular states and contributes to many diseases including cancer. Indeed, proteins bound to membranes and signaling lipids including PIs represent the most valuable drug targets, and their exploitation will be accelerated by understanding their signaling and regulatory mechanisms in greater predictive detail.
Encapsulation of propolis extracts in aqueous formulations by using nanovesicles of lipid and poly(styrene-alt-maleic acid)
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2023
Chatmani Buachi, Charothar Thammachai, Brian J. Tighe, Paul D. Topham, Robert Molloy, Patchara Punyamoonwongsa
Interaction of the nanodiscs with Vero cells was demonstrated through the MTT assay. Vero cells (epithelial kidney cell line) were chosen because of their resistance and ease of cultivation within 24 h. The Vero cell assay was considered as the gold standard to rapidly screen the cytotoxicity profiles of new therapeutic agents [45,46]. Different concentrations of the propolis-loaded and unloaded nanodiscs (1000 P/DMPC, and 2000 P/DMPC) were evaluated for their cell viability (%). The results provided a key for a future design of PSMA/lipid nanostructure with the greatest healthcare potency but minimal adverse effects. Data obtained from the MTT measurement suggests an increased cytotoxic effect with the increasing of 1000 P/DMPC and 2000 P/DMPC concentrations (Figure 11 and Figure 12). Between them, Vero cells are most sensitive to the 2000 P/DMPC formulations. At 313 µg/mL, their cell viability is below 10%, almost comparable to the positive control (Triton-X). The 1000 P/DMPC treatments however showed less cytotoxicity. The cell viability remains above 85%, implying a non-cytotoxicity for biomedical application. According to Figure 11 and Figure 12, within a low concentration region (<78 µg/mL), both 1000 P/DMPC and 2000 P/DMPC formulations exhibit ability to promote the Vero cell growth. All of the treated cells display the increased cell viability (>105%) comparing to the control group. The explanation may be related to the enhanced vesicle-cell interactions. To the best of our knowledge, interaction of nanodiscs with Vero cell line has never been reported. Due to a similar bilayer arrangement, the mechanisms of liposome-cell interactions [47] may be applied for the nanodisc scenario. Led by the accelerated cell growth effect, it is believed that the phospholipid of the nanodiscs can associate with the cellular membrane, resulting in the formation of a micro-pore defect [48,49], that enables passage of essential ingredients (e.g., hormones, growth factors and minerals in the foetal bovine serum) across the plasma membrane. When these interactions were excessively too strong, as in the case of the high nanodisc concentrations (>156 µg/mL), the merging (or fusion) of the Vero cell membrane with the phospholipid and/or PSMA of the nanodiscs can be occurred, causing the cells to lose their structural integrity and biological function. The chemical nature of the nanodiscs, such as hydrophobicity, size and surface charge, can influence the cellular interactions. Driven primarily by the hydrophobically-associative force, 2000 P/DMPC is more capable of interacting with the plasma membrane than the 1000 P/DMPC, thus explaining the enhanced cytotoxicity effects in Figure 12.