Mechanisms for cellular copper import via secreted cuproproteins

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Project Summary: Copper is an essential micronutrient and a required redox-active cofactor for enzymes necessary for eukaryotic respiration, oxidative stress resistance, and the production of functionalized cell signaling molecules. Very recently, Cryptococcus neoformans Bim1 was reported to represent a new class of secreted and cell surface- associated cuproproteins that promote fungal Cu-uptake via high-affinity CTR Cu-transporters during host colonization. Homologs to C. neoformans Bim1 are highly represented in the genome of several fungal pathogens affecting humans, and we identify this new family of Cu-scavenging proteins as Bim1-like proteins (BLPs). Surprisingly, there is significant sequence diversity at the BLP active site and C-terminal GPI anchoring domain. Virtually nothing is known on how such sequence variations affect Cu-trafficking function. Our central hypothesis is that BLP active site variation is used to modulate Cu-binding affinity and oxidation state specificity, whereas the C-terminal domain partitions BLP proteins at the cell surface. The overall goal of this research project is two-fold: 1. To understand how the active site diversity within the BLP family affects Cu- binding properties. 2. To understand how BLP extracellular localization patterns alter cellular Cu-homeostasis. We propose to use the three BLPs encoded in the opportunistic fungal pathogen Pseudogymnoascus destructans (Pd) as prototypes for the natural diversity of this new family of extracellular Cu-scavengers. We will test our hypothesis in the following (2) specific research aims: Aim 1. To determine the impact of BLP active site variation.; Aim 2. To define the role of Bim1-like protein (BLP) isoforms in extracellular Cu trafficking. Under the first aim, we will (i) develop a recombinant expression platform to produce wild type and variant PdBLPs. We will in (ii) determine how active site variation alters the metal-binding properties and the copper coordination environment. Finally, in (iii) we will determine how active site variation alters Cu-redox properties. In aim 2, we define the role of BLP isoforms in extracellular Cu trafficking. We will test the innovative hypothesis that BLPs can partition at the cell surface to relay Cu to the cell surface and boost Cu- import efficiency. To test this hypothesis, we will leverage the power of Saccharomyces cerevisiae (Sc) genetics to build a model of the BLP/CTR uptake pathway. In (i-ii) we will optimize the recombinant expression of PdCTR transporters and PdBLPs in S. cerevisiae. This will involve the rigorous characterization of protein expression and localization patterns at the plasma membrane and cell wall. In (iii) we will assess the impact of PdBLP expression levels and extracellular localization in facilitating Cu-import from diffusible and solid supported Cu sources. The expected outcomes of this work are a basic understanding of how this novel BLP Cu-uptake pathway functions to ensure adequate delivery of Cu-atoms to fungal pathogens under extremes in copper bioavailability, akin to that found during host infection.