Membrane proteins as drug targets
Mokry, David Zachary
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To date, half of all pharmaceutical drugs target membrane proteins, which are encoded by 30% of a typical genome. However, less than 0.3% of unique entries in protein structure databases are membrane proteins. Two protein groups pertinent to this dissertation that are promising drug targets are the CaaX proteases (Ste24 and Rce1), and the Hemoglobin Receptor (HmbR). The former are involved in the maturation of certain eukaryotic isoprenylated proteins bearing a C-terminal CaaX motif (C, cysteine; a, aliphatic amino acid; and X, one of several amino acids), and have been identified in several organisms, including the disease causing agent, Trypanosoma brucei. HmbR and related proteins are found in gram negative bacteria, and is involved in the acquisition of heme from the host. In the first study, the CaaX proteases from Trypanosoma brucei were heterologously expressed in Saccharomyces cerevisiae. The results suggest the conserved presence of two CaaX protease activities in trypanosomatids, dispelling a previous notion of only one. This study also identified a trypanosomal Hsp40 chaperone as a substrate of both Tb CaaX proteases; the first substrate outside of yeast that is known to be cleaved by both CaaX proteases, and provides evidence that the target specificity of the Tb CaaX proteases is influenced by protein context. Finally, our findings support the potential use of small molecule CaaX protease inhibitors as general tools for cell biological studies on the trafficking of CaaX proteins. The second study spectroscopically evaluated HmbR heterologously expressed and purified from Escherichia coli. The results indicate an overall β-fold structure, and the presence of a 5-coordinate high-spin ferric heme with an axial negatively charged oxygen ligand. We also identified four amino acid residues involved in maintaining heme binding properties. Our study is the first spectroscopic characterization of any heme transporter in Neisseria meningitidis, and expands the variety of ways outer membrane heme transporters can coordinate heme. In summary, these projects showcase the multitude of approaches that can be applied toward the understanding of membrane protein function, even in the absence of purified components, and provides a rational starting point for the development of novel pharmaceuticals.