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THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS

Moseng, Mitchell A.
http://orcid.org/0000-0003-4314-7283
http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463

Abstract Details

2019, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
HSPA9, the gene coding for the mitochondrial chaperone mortalin, is involved in various cellular roles such as mitochondrial protein import, folding, degradation, Fe-S cluster biogenesis, mitochondrial homeostasis, and regulation of the anti-apoptotic protein p53. Due to the crucial role in cell survivability mortalin is also an aim for drug design with a strong emphasis on anticancer treatments. Mutations in the HSPA9 gene, particularly within the region coding for the nucleotide-binding domain (NBD), cause the autosomal disorder identified as EVEN-PLUS syndrome. The resulting mutants R126W and Y128C are located on the exterior of the mortalin-NBD near the interface of the interdomain linker (IDL). We used differential scanning fluorimetry (DSF), biolayer interferometry, X-ray crystallography, ATP hydrolysis assays, and Rosetta docking simulations to study the structural and functional consequences of the EVEN-PLUS syndrome-associated R126W and Y128C mutations within the mortalin-NBD. In order to determine a potential mortalin inhibitor we screened adenosine-5’-diphosphate (ADP) analogs by isothermal titration calorimetry (ITC) and inhibition assays. As part of our initial efforts to determine a promising covalent inhibitor of mortalin we crystallized human mortalin nucleotide binding domain (NBD) with N6-propargyl ADP. The acquired structure highlighted the ability of the nucleotide binding pocket to accommodate modified ADP compounds and reveals two possible sites for modification on nucleotides to increase specificity for mortalin. A library of ADP analogues containing modifications at either the 2C or N6 positions of adenosine were screened against mortalin-NBD to determine binding affinities. These results of the surface mutation studies indicate that the R126W and Y128C mutations have a far-reaching effect and disrupt ATP hydrolysis, interdomain linker binding, and thermostability. The structural differences observed provide insight into how the conformations of mortalin vary from other heat shock protein 70 (Hsp70) homologs. Combined, our biophysical and structural studies contribute to the understanding of the molecular basis for how disease-associated mortalin mutations affect mortalin functionality and the pathogenesis of EVEN-PLUS syndrome. The results of competitive inhibition and binding assays of the analogs demonstrate that modifications at the 2C or N6 positions have potential to bind and inhibit mortalin uniquely compared to other Hsp70 homologs. In particular, this data indicates that modifications at the 2C position confer the greatest selectivity in binding and inhibition of mortalin-NBD compared to the cytosolic homologs, Hsc70 and Hsp70.
Rick Page (Advisor)
153 p.
Biochemistry
Mortalin

Recommended Citations

Citations

  • Moseng, M. A. (2019). THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463

    APA Style (7th edition)

  • Moseng, Mitchell. THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS . 2019. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463.

    MLA Style (8th edition)

  • Moseng, Mitchell. "THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS ." Doctoral dissertation, Miami University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=miami1563537216760463

    Chicago Manual of Style (17th edition)

miami1563537216760463
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© 2019, some rights reserved.Creative Commons License THE BIOPHYSICAL HINDRANCE ON MORTALIN FUNCTION FROM EVEN-PLUS SYNDROME MUTATIONS AND MODIFIED ADP ANALOG INHIBITORS by Mitchell A. Moseng is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Miami University and OhioLINK.