Date of Award

1-1-2018

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xv, 245 pages) : color illustrations.

Dissertation/Thesis Chair

Marlene Belfort

Committee Members

Kathleen McDonough, Janice Pata, Nicholas Mantis, Cara Pager

Keywords

conditional protein splicing, cysteine chemistry, intein, mycobacteria, mycobacteriophages, ROS, Endonucleases, Mycobacteria, Proteins, Mycobacterial diseases, Mycobacterium tuberculosis, Amino acid sequence

Subject Categories

Biochemistry | Bioinformatics | Biology

Abstract

Inteins are intervening protein elements, capable of coordinating escape from a host protein through a self-catalyzed mechanism, called protein splicing. This results in free intein and a mature host protein product. Inteins are also mobile elements and many contain homing endonucleases that enable the targeting to ectopic sites and invasion of novel niches. Inteins have been found across all three domains of life and are often present in replication, recombination, and repair proteins. However, it is unclear if the observed distribution is simply a factor of endonuclease preference or if inteins have been selectively maintained due to an adaptation that is of advantage to the host. My dissertation explores intein evolution, dissemination, and regulatory potential, focusing on inteins in mycobacteria and their phages. Taking a bioinformatic approach, I investigated the role of mycobacteriophages in intein dissemination and evolution, finding that these phages have contributed to the movement of inteins among mycobacteria. Mycobacteriophages also appear to facilitate intein evolution through allowing alternative catalytic residue sampling. To delve into mechanisms of conditional splicing, inteins from two distinct mycobacterial hosts and proteins, the Mycobacterium tuberculosis SufB intein and the two Mycobacterium smegmatis DnaB inteins, were studied after exposure to reactive oxygen species (ROS) and reactive nitrogen species (RNS). These studies revealed the important role of cysteines as modulatory residues. The SufB work showed multiple intein invasions in various pathogenic mycobacterial species. Biochemical characterization revealed the catalytic Cys+1 residue is extraordinarily sensitive to modification in response to ROS and RNS stress. This cysteine forms an intramolecular disulfide bond with Cys1, effectively trapping the intein in an inactive state. My DnaB work further explored the modulatory capacity of two distinct inteins within a single host protein, the replicative helicase DnaB. These inteins display different splicing behavior and sensitivities to ROS and RNS stress. The sensitive intein, an atypical class 3 mini-intein, was again found to undergo cysteine dependent inhibition. Biochemical and structural insight, provided by the first class 3 intein structure, showed that a disulfide bond forms between the initiating cysteine nucleophile and a secondary cysteine in response to ROS. These collective studies support the hypothesis that inteins can serve as biosensors and provide benefit to the host under specific conditions through an exciting form of posttranslational control. Future work is needed to demonstrate such mechanisms in a native host, although preliminary studies in mycobacterial biofilms may suggest a path forward.

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