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An Analysis of Protein Interaction and Its Methods, Metabolite Pathway and Drug Discovery
Published in Ayodeji Olalekan Salau, Shruti Jain, Meenakshi Sood, Computational Intelligence and Data Sciences, 2022
The two-hybrid system is the widely used technique in screening and protein interaction prediction. In yeast, various transcription factors of eukaryotic domain interfere to properly activate the process of transcription and binding of DNA. To map the interaction of the genome, Y2H has been improved and modified with the required information. [81] Y2H is reasonably priced to use, and it is an in vivo method and necessitates small optimization for protein interaction prediction [82]. By using the Y2H method belongs to the nucleus for protein interaction prediction is complex, when compared to the cytoplasm-based protein interaction prediction [83,84].
Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
Monobodies are the protein scaffolds based on Fibronectin type III domain. Fibronectin is a large protein that interacts with the extracellular matrix proteins and regulates cell-cell communication. Structurally, it is a large protein containing two 250 kD subunits connected by disulfide bridges. These subunits contain the repeats of three domains (type I, II, III). The first monobody was engineered using the tenth repeat of type III domain of Fibronectin (FNfn10) against ubiquitin using phage display system. FNfn10 is devoid of disulfide bonds, and also contains 7 β-strands and 3 surface loops that can be used to add diversity to the monobodies. Their structural simplicity makes them compatible with any screening method such as phage display, yeast two-hybrid system, and peptide-ribonucleic acid. They can be expressed in large quantities in bacteria and are highly thermo stable (Koide and Koide, 2007). Such properties of monobodies make them ideal candidates for therapeutic protein scaffolds. Monobodies have been engineered for the numerous proteins including EGFR (Hackel et al., 2012), IL-23 (Tang et al., 2012), Ableson (Abl) kinase SH2 domain (Wojcik et al., 2010), etc.
Imaging Cellular Networks and Protein-Protein Interactions In Vivo
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Snehal Naik, Britney L. Moss, David Piwnica-Worms, Andrea Pichler-Wallace
Compared with studies of protein interactions in cultured cells, strategies to interrogate protein-protein interactions in living organisms impose even further constraints on reporter systems and mechanisms of detection. Most strategies for detecting protein-protein interactions in intact cells are based on fusion of the pair of interacting molecules to defined protein elements to reconstitute a biological or biochemical function. Examples of reconstituted activities include activation of transcription, repression of transcription, activation of signal transduction pathways, or reconstitution of a disrupted enzymatic activity (4). A variety of these techniques have been developed to investigate protein-protein interactions in cultured cells. The two-hybrid system is the most widely applied method to identify and characterize protein interactions.
New cyclopentaquinoline and 3,5-dichlorobenzoic acid hybrids with neuroprotection against oxidative stress for the treatment of Alzheimer’s disease
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Kamila Czarnecka, Małgorzata Girek, Paweł Kręcisz, Robert Skibiński, Kamil Łątka, Jakub Jończyk, Marek Bajda, Piotr Szymczyk, Grzegorz Galita, Jacek Kabziński, Ireneusz Majsterek, Alba Espargaró, Raimon Sabate, Paweł Szymański
To exclude the potential protein-protein interactions between bait (ACHE) and four prey (A4, BACE1A, MAO B, MAPT) a small scale mating procedure (5 ml) was performed according to Matchmaker Gold yeast two-hybrid system manual recommendations. Prepared cell suspension was plated on DDO agar plates containing aureobasidin A (200 ng/ml) and X-α-Gal (40 µg/ml) (DDO/X/A) to screen for potential protein-protein interactions. A small scale mating procedure (10 ml) was then performed in the presence of hybrid ligands (10 µM) to initiate the possible hybrid ligand-mediated protein interactions. A suspension of mated cells was plated on DDO agar plates containing 10 µM of hybrid -ligand, aureobasidin A (200 ng/ml) and X-α- Gal (40 µg/ml) (QDO/X/A). Blue colonies were transferred on the QDO/X/A agar plates containing hybrid ligand (10 µM) confirm the presence of interactions. Obtained blue colonies were transferred on QDO/X/A agar plates. No hybrid ligand was added to the QDO/X/A agar to confirm that the interaction is dependent on hybrid ligand.
Protein interactions study through proximity-labeling
Published in Expert Review of Proteomics, 2019
Benoît Béganton, Isabelle Solassol, Alain Mangé, Jérôme Solassol
While a large number of techniques of protein-protein interactions studies have been developed, we have seen that the use of affinity purification methods or of the two-hybrid system is limited to the identification of strong, essentially permanent, interactions at a given time and sometimes in a non-adapted cellular context. Moreover, these conventional methods are little or non-suitable for the analysis of membrane proteins, while it is a key location when the interactome at the cell level needs is to be characterized. Two recent techniques (BioID and APEX) for in vivo analysis of PPIs based on proximity tagging have been proposed to overcome these limitations. These methods are essentially based on the fusion of a protein of interest to an enzyme, BirA biotin ligase for BioID and ascorbate peroxidase for APEX. When active, these enzymes generate a reactive product that diffuses around the active site of the protein of interest and covalently tags all the proteins that are in an environment close to the protein of interest. Biotin tagging then makes it possible to purify and identify the interactors (Figure 2).
The application of gene silencing in proteomics: from laboratory to clinic
Published in Expert Review of Proteomics, 2018
Maura Brioschi, Cristina Banfi
The well-known relevance of protein–protein interactions in cell signaling has led to the development of several approaches to dissect them. Traditionally, interactions have been studied using the yeast two-hybrid system, which is based on the fusion of two proteins, a ‘bait’ and a ‘prey,’ with transcription activators that are able to activate the transcription of a reporter gene only when the two proteins interact one with each other. However, this method can give many false-positive and false-negative identifications, neglecting also the effects of subcellular localization or PTMs too, because it studies the interactions outside normal physiological conditions [82]. Alternatively, interaction partners could be identified using overexpressed proteins tagged to allow a specific purification of the target protein together with its interactors. However, also in this case, the properties of the protein of interest could be modified by the overexpression itself, which could also saturate the system and mask the dynamic changes of interactions in response to biological stimuli [83]. Thus, the method of choice is still the co-immunopurification of interactors with antibodies/resins against the protein of interest in a physiological context, even if issues on the specificity of the approach still exist.