<p>ZMPSTE24 is a seven transmembrane
domain zinc metalloprotease that resides in the ER and inner nuclear membranes
of mammalian cells. The crystal structures of both the mammalian and yeast
homologs, ZMPSTE24 and Ste24, respectively, were solved recently and revealed a
common novel structure. Both structures contain a large chamber of mixed
hydrophobicity that is capped on both sides. The canonical catalytic HExxH
zinc-binding motif lies inside the chamber. Defects in the enzymatic function
of human ZMPSTE24 have been shown to cause premature aging disorders. In
addition to the well-defined role ZMPSTE24 and Ste24 play in the maturation of prelamin
A in mammals and <b>a</b>-factor in yeast, both proteins have been proposed to
play protective roles in Type 2 diabetes and viral infections by interactions
with the cellular translocon. ZMPSTE24 can also be inhibited by several common HIV
aspartyl protease inhibitors, possibly causing the frequent and common
side-effects of these prescribed drugs. As
of now, no precise location for substrate binding has been identified in either
ZMPSTE24 or Ste24. Thus, the goal of this project is to localize residues in
the enzyme that are important for substrate binding. The yeast homolog Ste24
was used as a model system as it functionally complements the mammalian enzyme
and can be reliably cloned, overexpressed, and purified in an active form. </p>
<p>Three approaches were taken to
directly determine the <i>K<sub>D</sub> </i>values for substrates of
Ste24. The ability to perform a direct
analysis of <i>K<sub>D</sub></i> values of Ste24 mutations was successfully
optimized using microscale thermophoresis. Through <i>K<sub>D</sub></i>
analysis, the Ste24 mutation G255A, while completely inactive, does not prevent
substrate binding. Alternatively, L441A and L410A mutations showed both an
increase in thermal stability and a decrease in binding affinity, that could
explain their lower activity levels. A photoaffinity labeling-based proteomics
experiment was utilized to precisely locate the site of the prenyl group to a
hydrophobic patch lying just under a side portal of Ste24, near K234, during
the -aaXing cleavage of <b>a</b>-factor maturation. To assess the method of
inhibition of HIV protease inhibitors on Ste24 the conserved aspartate mutants
were explored. All mutations of these aspartate residues resulted in a severe
loss of Ste24 function and instability of the protein.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/16590338 |
| Date | 22 November 2021 |
| Creators | Chelsea C St. Germain (11409800) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/CHARACTERIZATION_OF_THE_BINDING_SITE_OF_STE24_DURING_THE_-AAXING_CLEAVAGE/16590338 |
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