Self-splicing introns and inteins attract attention for their molecular mechanisms, phylogentic diversity, role in genome evolution, and application in research, biotechnology and medicine. Interest in these elements stems from their self-splicing properties at the RNA level for introns and protein level for inteins, and from their ability to ac as mobile genetic elements at the DNA level. In the past funding period, we made considerable progress in structural and functional characterization of these self-splicing introns and inteins. For the next research phase, we will focus on the relatively understudied inteins. Inteins exist at the crossroads of the disparate disciplines of protein chemistry, biotechnology and molecular evolution. Their autocatalytic peptide cleavage and ligation reactions make them useful tools in modern chemical biology, whereas their existence within proteins critical to vital cellular processes raises provocative questions about their function in nature. We propose the following three specific aims, based on discoveries made in the past funding period: In the first aim, we will analyze the role of the flanking host sequences, the exteins, on intein structure, splicing an evolution. This work is enabled by our collaborations with physicists and structural biologists. We will also address a bold hypothesis, that inteins persist in specific exteins because they confer a selective advantage on their host, through adaptive interactions with flanking extein residues. In the second aim, we will study intein inhibitors as mechanistic probes and antimicrobials. Thus we will exploit the existence of inteins in critical genes of microbial pathogens, to probe inteins as novel targets for bacterial and fungal antibiotics. We will further characterize cisplatin, the chemotherapeutic agent, which we identified as a protein splicing inhibitor. We will investigate cisplatin's efficacy against infection by Mycobacterium tuberculosis and also test its ability to curtail activity of cryptococcal inteins. Additionally, we aim to isolte small-molecule and peptide inhibitors, with a view to comparing their properties with each other and with cisplatin. In the third aim, we will use molecular methodologies previously developed in our lab (redox traps, gain-of-fluorescence protease sensors, and phage display selections), to fashion tools for biotechnology and medicine. Thus, we will exploit our ability to isolate wild-typ intein precursors for biological and chemical applications, and construct sensors for proteases in a botulism toxin diagnostic and to detect tuberculosis (TB) biomarkers as a TB diagnostic. Once again we are taking collaborative, interdisciplinary approaches, which combine genetics, biochemistry and microbiology with physics and structural biology. In this way, we will enhance our understanding of the structure, function and evolution of inteins, as a means to exploit them as potential targets for drug development and as novel reagents in biotechnology and medical diagnostics.
The overall goal of this application is to build upon progress made in the previous funding period, using interdisciplinary approaches to study intein structure, function, evolution and application. The applied aspects of the proposal relate to isolation and characterization of inhibitors of microbial inteins, as a means to discover novel antibiotics against tuberculosis and mycoses. We will also exploit intein technology to develop a diagnostic sensor for botulinum toxin and for tuberculosis biomarkers.
|Novikova, Olga; Jayachandran, Pradeepa; Kelley, Danielle S et al. (2016) Intein Clustering Suggests Functional Importance in Different Domains of Life. Mol Biol Evol 33:783-99|
|Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia et al. (2016) Structure of a group II intron in complex with its reverse transcriptase. Nat Struct Mol Biol 23:549-57|
|Chan, Hon; Pearson, C Seth; Green, Cathleen M et al. (2016) Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy. J Biol Chem 291:22661-22670|
|Lambowitz, Alan M; Belfort, Marlene (2015) Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution. Microbiol Spectr 3:MDNA3-0050-2014|
|Lambowitz, Alan M; Belfort, Marlene (2015) Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution. Microbiol Spectr 3:|
|Owen, Timothy S; Ngoje, George; Lageman, Travis J et al. (2015) FÃ¶rster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor. Anal Biochem 488:1-5|
|Topilina, Natalya I; Green, Cathleen M; Jayachandran, Pradeepa et al. (2015) SufB intein of Mycobacterium tuberculosis as a sensor for oxidative and nitrosative stresses. Proc Natl Acad Sci U S A 112:10348-53|
|Owen, Timothy S; Xie, Xie Jian; Laraway, Benjamin et al. (2015) Active site targeting of hedgehog precursor protein with phenylarsine oxide. Chembiochem 16:55-8|
|Pearson, C Seth; Belfort, Georges; Belfort, Marlene et al. (2015) Backbone assignments of mini-RecA intein with short native exteins and an active N-terminal catalytic cysteine. Biomol NMR Assign 9:235-8|
|Novikova, Olga; Smith, Dorie; Hahn, Ingrid et al. (2014) Interaction between conjugative and retrotransposable elements in horizontal gene transfer. PLoS Genet 10:e1004853|
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