Characterization of Transmembrane Metal Transporters in Metal Homeostasis, Detoxification and Drug Resistance

Because of their unique chemistry, transition metals are central to the function of one-third of all proteins identified in the human proteome, thus, play pivotal roles in biological processes. Yet, abnormal metal distribution underlies cellular toxicity. Every cell, as well as whole organisms, is therefore challenged to tightly control and maintain appropriate concentrations of metals while excluding nonessential, toxic metals. Cellular metal import and export often require transmembrane transporter proteins embedded in the membrane. In this dissertation, we investigate the chemical roles underlying selectivity and translocation mechanism of transmembrane proteins involved in metal transport with the aim of shedding light on how homeostasis, detoxification and metal-based drug resistance is achieved. Atomic resolution understanding of metal selectivity by membrane proteins has remained largely elusive. The P1B-type ATPase pumps represent transporters with wide and overlapping substrate specificities. A Zn2+-transporting P-type ATPase (ZntA) was utilized as a model system to understand how metal promiscuity against Zn2+, Cd2+, Hg2+, and Pb2+ is obtained at a molecular level. We dissected the coordination chemistry of metal-bound forms of ZntA through biochemical tools and X-ray absorption spectroscopy analysis. We revealed that coordination plasticity is required to guarantee substrate promiscuity of ZntA. Moreover, our results indicated that divergence from ideal ligand sets and geometries is required for efficient metal translocation by ZntA during its catalytic cycle. The pathology and entrance into the cells of therapeutic metals must also be based on similar coordination chemistry principles of trafficking that govern physiological trace metals. Platinumcoordination complexes are among the most effective chemotherapeutic drugs used in clinic for the treatment of cancer. Despite their efficacy, cancer cells can develop drug resistance leading to treatment failure and relapse. Cellular uptake and extrusion of Pt(II)-complexes mediated by transmembrane proteins are critical in controlling the intracellular concentrations of Pt(II)-drugs and in developing pre-target resistance. TMEM205 is a human transmembrane protein (hTMEM205) overexpressed in cancer cells resistant to cisplatin, but its molecular function underlying resistance remains elusive. We developed a low-cost and high-throughput recombinant expression platform coupled to in vivo functional resistance assays to address the molecular mechanism by which the orphan hTMEM205 protects against Pt(II)-complex toxicity. We demonstrate that hTMEM205 mediates Pt(II)-drug export selectively towards cisplatin and oxaliplatin but not carboplatin. Thus, hTMEM205 represents a new potential target that can be exploited to reduce cellular resistance towards Pt(II)-drugs. We subsequently investigated the nature of the physiological role of the “orphan” hTMEM205 and addressed whether it could exert its function as a novel Cu(I) transporter. The rationale behind this hypothesis is two-pronged: 1) Cu homeostatic pathways have been proposed to act as cisplatin transport systems and 2) Cu(I) and Pt(II) interact with similar biological ligands due to their comparable chemical coordination chemistry preferences according to the Pearson acid-base theory. In this work, through a combination of in vivo and in vitro activity assays, we defined that hTMEM205 is a novel human Cu(I) transporter. First, we developed a bacterial-based approach to show the directionality of hTMEM205-mediated copper transport. Second, successful purification of recombinant hTMEM205 allowed the establishment of a hTMEM205-reconstituted proteoliposome platform to dissect its enzymatic transport activity. By mutagenesis, we revealed that hTMEM205 recognize and mediate Cu(I) extrusion by a putative sulfur-based translocation mechanism. This work sheds light on the physiological relevance of hTMEM205 in cellular metal metabolism and establishes a new transporter-based copper pathway.