Cardona-Correa, Albin A.

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    Detection and characterization of 𝘉𝘢𝘤𝘪𝘭𝘭𝘶𝘴 𝘢𝘯𝘵𝘩𝘳𝘢𝘤𝘪𝘴 lethal factor interacting proteins using human stomach T7 Phage Display cDNA libraries
    (2015) Cardona-Correa, Albin A.; Ríos Velázquez, Carlos; College of Arts and Sciencies - Sciences; Martínez Cruzado, Juan C.; Rodríguez Minguela, Carlos M.; Department of Biology; Zapata Serrano, Mildred
    The 2001 bioterrorism attacks to the USA using envelopes containing 𝘉. 𝘢𝘯𝘵𝘩𝘳𝘢𝘤𝘪𝘴 spores increased the concern of scientists to fully understand the mode of action of the anthrax disease. 𝘉. 𝘢𝘯𝘵𝘩𝘳𝘢𝘤𝘪𝘴 produces an exotoxin composed of three proteins. The first protein is the protective antigen (PA), which interacts with the human cell receptor TEM8 & CMG2 to allow the entrance of the two other subunits, edema factor (EF) and lethal factor (LF), to the cell by endocytosis. The EF interacts with the ATP molecules, causing an excess of cAMP which results in edema. On the other hand, the LF was found to cleavage most mitogen activated protein kinase (MAPK) kinases (MKKs) and then the NOD-like receptor proteins (NLRPs), resulting in cells apoptosis. If additional substrates suffer a LF-mediated cleavage, resulting in cell apoptosis, then we suggest novel LF-interacting partners to promote this biological process in gastrointestinal (GI) anthrax. T7 Phage Display (T7PD) is an 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 technique of combinatorial chemistry that allows the quick selection, amplification, and identification of potential ligands of specific biomolecules. The technique consists on the expression of cloned DNA sequences as fusion proteins on the T7 phage surface, allowing the study of protein-protein interactions. In this work, the technique was used to identify a profile of proteins that interact with LF, as additional substrates, in order to understand GI anthrax 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰. Four T7PD screenings, consisting of three biopanning rounds each one, were performed using a human stomach cDNA library in order to identify proteins that binds LF. A lethal factor wild-type (LF-WT) with metalloprotease activity and a mutant type (LF-MT) without protease activity were used as target to identify different sites for interaction. The T7PD LF- interacting partners were tittered; eventually, 192 were isolated for extract their DNA for a PCR reaction using specific primers to the cloning site. The DNA fragment sizes varied from 300- 900bp. After sequencing 124 clones a total of 33 proteins were identified 𝘪𝘯 𝘴𝘪𝘭𝘪𝘤𝘰, being mostly from protein families such as peptidase A1, lipase, and kruppel C2H2-type zinc-finger protein motifs. From these proteins, 18 were selected for a specificity test consisting of allowing the interaction of individual candidates with each type of LF and the blocking agent (5% casein). A total of 10 T7PD candidates were found to bind at least one type of LF tested. The candidate peptides belong to proteins from families such as peptidase A1, cytochrome, and Cation transport ATPase. The PAP46, a candidate from the peptidase A1 family, was found to interact with both types of LF tested with a target minimal concentration for interaction (MCI) of 1μg/mL. These findings could represent the identification of additional substrates for LF, besides the MKKs and NLRPs. In short, these putative novel peptides that bind LF 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 can be used to better understand anthrax disease at the molecular level, through the characterization of these substrates, and to develop new therapeutic agents.