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Hexacoordinate Globins: Characterization and Engineering for Development of Novel Therapeutic Agents

Amdahl, Matthew (2018) Hexacoordinate Globins: Characterization and Engineering for Development of Novel Therapeutic Agents. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

This work was intended to characterize the hexacoordinate heme globins, most notably cytoglobin (Cygb), in the hopes of learning more about the native function of these proteins, but also exploring the potential of these proteins to be developed into therapeutic molecules.
The first portion of the work examined the reduction of vertebrate globins by the cytochrome b5 (CYB5) reducing system and ascorbate and found that Cygb is reduced by CYB5 much faster than any other globin. This interaction provides some insight into potential physiologic functions of Cygb, which has been theorized to function as an NO dioxygenase in vivo, limiting NO diffusion. NO dioxygenation oxidizes Cygb, and so rapid reduction by CYB5 could support rapid Cygb turnover and catalytic NO consumption. The reduction of Cygb by CYB5 was also documented, and found to be very similar, for the zebrafish homologs of these proteins. This level of evolutionary conservation suggests a role of significant physiologic importance, and even suggests that CYB5 and Cygb may have originally evolved together as redox partners.
Subsequent work generated novel recombinant Cygb mutants as potential Cygb-based therapeutic molecules intended to function as either an oxygen carrier or an antidotal therapy capable of scavenging carbon monoxide (CO) and/or cyanide. Key functional characteristics of these mutants were then quantified, primarily their rates of nitrite reduction (production of nitric oxide from nitrite), autoxidation, and cyanide binding. This work revealed one mutant (V85I) with promise as an oxygen carrier, and a separate mutant (L46F + H81Q) that may function as a CO and cyanide antidote, as it was shown to bind cyanide very rapidly. The V85I mutant produced NO from nitrite more rapidly than the wild-type protein, which may offset the NO scavenging and vasoconstriction that has historically been caused by infusion of cell-free globins. In a small animal model designed to test vasoactivity, this mutant did not show a clear advantage over unmodified Hb. Future work will continue to search for a potential Cygb-based oxygen carrier, as well as further explore L46F + H81Q Cygb’s ability to scavenge carbon monoxide and cyanide.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Amdahl, Matthewmba27@pitt.edumba270000-0002-5169-3340
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairGladwin, Markgladwinmt@upmc.edu
Committee MemberTejero, Jesus
Committee MemberShroff, Sanjeev
Committee MemberSundd, Prithu
Committee MemberWagner, William
Date: 24 September 2018
Date Type: Publication
Defense Date: 6 July 2018
Approval Date: 24 September 2018
Submission Date: 25 July 2018
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 230
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Bioengineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Nitric Oxide; Cytoglobin; Heme Globins; Cytochrome b5; Cytochrome b5 Reductase; Protein Engineering; Nitrite
Date Deposited: 24 Sep 2018 14:14
Last Modified: 24 Sep 2018 14:14
URI: http://d-scholarship.pitt.edu/id/eprint/34979

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