The Anticancer Potential of the Bacterial Protein Azurin and Its Derived Peptide p28
Ananda M. Chakrabarty, Arsénio M. Fialho in Microbial Infections and Cancer Therapy, 2019
There are many reasons that support the theory that azurin has the potential to act as an anticancer agent. Besides its preferential entry into cancer cells, no adverse side effects were observed in in vivo studies [11, 51, 52]. As mentioned above, this protein also can mediate specific high-affinity interactions with various unrelated mammalian proteins relevant in cancer, conferring on it the property of a natural scaffold protein, which is probably the most important characteristic of this protein [17]. This ability to act on multiple targets is important since it might be harder to trigger resistance by the cells. Another advantage of this bacterial protein is that azurin is a water-soluble molecule with a hydrophobic patch and this might help in its tissue penetration and clearance from the bloodstream [9]. In addition to all this, azurin can be easily hyperexpressed in Escherichia coli, which makes the process of production very cheap [15]. All these reasons make azurin an attractive molecule to be used in cancer therapy.
mTOR Targeting Agents for the Treatment of Lymphoma and Leukemia
Gertjan J. L. Kaspers, Bertrand Coiffier, Michael C. Heinrich, Elihu Estey in Innovative Leukemia and Lymphoma Therapy, 2019
In addition, TORC1 enhances the translation of a different set of RNAs by phosphorylating 4E-BP1 (2,6). eIF4E is a component of a helicase complex that binds to the 7-methylguanine cap at the 5′ end of mRNAs and enhances the ability of ribosome-eIF complexes to scan the mRNA for initiation sites. 4E-BP1, in its unphosphorylated state, binds to eIF4E and inhibits the eIF4E-containing helicase complex. Activation of TORC1 signaling causes hyperphosphorylation of 4E-BP1, diminishing the stability of the 4E-BP1/eIF4E complex, and causing its dissociation. Free eIF4E then binds to the scaffold protein eIF4G and the RNA helicase eIF4A, forming an active helicase that facilitates translation of mRNAs containing long, highly folded 5′ untranslated regions. Included in this class of transcripts are messages encoding cyclin D1, c-Myc, hypoxia inducible factor-lα (HIF-lα), vascular endothelial growth factor and fibroblast growth factor as well as ribosomal proteins themselves (2,3,6). These molecules are not only critical for cell survival and proliferation, but also have the potential to be used to monitor therapy. Because HIF-lα regulates the glycolytic pathway and fluorodeoxyglucose positron emission tomography (FDG-PET) detects tumors by their elevated rates of glycolysis, FDG-PET can potentially be used to assess inhibition of this pathway after treatment with mTOR inhibitors (2,3).
Order Caudovirales
Paul Pumpens, Peter Pushko, Philippe Le Mercier in Virus-Like Particles, 2022
The canonic version of the P22-driven cargo experiments was based on the fact that the phage P22 assembled from 420 copies of the coat-protein gp5 subunit into an icosahedral capsid with the assistance of 100–330 copies of a scaffold protein, the C-terminus of which interacted with the coat protein and was necessary for self-assembly, whereas the N-terminus could be severely truncated or mutated with little to no effect on assembly (O’Neil et al. 2011). Therefore, the system genetically fused the cargo proteins to the truncated form of the P22 scaffold protein, which acted further as a template for capsid assembly as well as a specific encapsulation signal for the cargo. In this way, the additional space within the capsid from the truncation of the SP enabled the cargo to be assembled in vivo with high packaging efficiency. This method did not alter the coat protein in any way; eliminated the need for affinity tags, chemical linkers, or bridging ligands; and perhaps avoided the misassembly of VLPs. Instead, the fusion of the scaffold protein to the cargo naturally provided the necessary affinity to the interior of the capsid, and assembly was templated around the cargo. Thus, a vector containing the gene for P22 coat protein and a truncated variant of the wild-type scaffold protein encoding for amino acids 141–303 (SP141) was designed. This vector enabled the insertion of a series of fluorescent-protein genes, including eGFP and mCherry, for the creation of N-terminal fluorescent protein—SP141 fusions. In these protein fusions, the thrombin-cleavage site was located between SP141 and the fluorescent protein and acted as both a flexible linker sequence and an accessible cleavage site for the release of the fluorescent protein from the SP141 as needed. The fluorescent procapsids were indistinguishable from the wild-type P22 procapsids during the expression and purification process except for their brilliant color, which indicated the expression, encapsulation, and proper folding of the fluorescent protein (Figure 1.7b). On average, the expression yields were 150 mg of P22 with encapsulated fluorescent cargo per liter of E. coli culture after a single purification step by sucrose-cushion ultracentrifugation (O’Neil et al. 2011).
The Antibody Society’s antibody validation webinar series
Published in mAbs, 2020
Jan L.A. Voskuil, Anita Bandrowski, C. Glenn Begley, Andrew R.M. Bradbury, Andrew D. Chalmers, Aldrin V. Gomes, Travis Hardcastle, Fridtjof Lund-Johansen, Andreas Plückthun, Giovanna Roncador, Alejandra Solache, Michael J. Taussig, James S. Trimmer, Cecilia Williams, Simon L. Goodman
Several affinity binders based on scaffold proteins have been designed and used as alternatives to antibodies,33 including Adnectins, Affibodies, Affimers, Anticalins, Bicyclic peptides, DARPins, Fynomers, Kunitz domains, and Monobodies. The general concept for each is similar: a stable scaffold protein is used to display diversified amino acid sequences at exposed surface sites, and the affinity binders are selected using an appropriate display platform (phage, yeast or ribosome display). Because of the small size and their low-cost production, such alternatives to antibodies are being evaluated in clinical trials,33 and will likely soon enter the commercial tool affinity binder market. As with antibodies, validation for specific purposes and batch quality control remain necessary.
The Unc13A isoform is important for phasic release and olfactory memory formation at mushroom body synapses
Published in Journal of Neurogenetics, 2020
Jennifer Woitkuhn, Anatoli Ender, Christine B. Beuschel, Marta Maglione, Tanja Matkovic-Rachid, Sheng Huang, Martin Lehmann, Joerg R. P. Geiger, Stephan J. Sigrist
Our previous analysis of Unc13A and B in the adult Drosophila olfactory system demonstrated distinct “nanoscopic positioning” within individual active zones using stimulated emission depletion microscopy (STED). In detail, STED showed that Unc13A, a release factor whose synaptic localization is dependent on the active zone scaffold protein Bruchpilot (BRP), is located relatively close to the VGCCs clustering within the active zone centre (≈70 nm). By contrast, another active zone scaffold protein, Syd-1, clustered Unc13B further away (>100 nm) from VGCCs and the BRP-scaffold centres (Fulterer et al., 2018). We also found this spacing pattern across the synaptic active zones of three other principal neuron types: olfactory receptor neurons, antennal lobe inhibitory interneurons and projection neurons (Fulterer et al., 2018).
Selection of target-binding proteins from the information of weakly enriched phage display libraries by deep sequencing and machine learning
Published in mAbs, 2023
Tomoyuki Ito, Thuy Duong Nguyen, Yutaka Saito, Yoichi Kurumida, Hikaru Nakazawa, Sakiya Kawada, Hafumi Nishi, Koji Tsuda, Tomoshi Kameda, Mitsuo Umetsu
Here, we present a possibility of generating functional non-Ig scaffold proteins from the information of the “weakly enriched” libraries, i.e., where functional variants are weakly enriched, by deep sequencing and machine learning. We used a series of phage pools displaying a mutated non-Ig scaffold protein in which no prospective variants with target-specific affinity were experimentally identified (weakly enriched library). The phage pools have been obtained in the biopanning process against galectin-3, which can be a potential therapeutic target for cancer treatment and diagnostic biomarkers for several diseases, including heart failure and cancers.35,36 By using deep sequencing analysis to the weakly enriched library, we evaluated sequence frequencies at various timepoints in the biopanning, and appropriate data was chosen for clustering analysis. The clustering analysis revealed no distinct sequence patterns, but a Bayesian machine learning model trained with the selected deep sequencing data supplied several clusters with distinct sequence patterns. Selection from the phage libraries based on the patterns led to the discovery of improved variants with target-specific affinity. This study shows the possibility of deep sequencing and machine learning for designing a refined library with prospective variants in the surveyed sequence space.
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