Mechanisms for cellular copper import via secreted cuproproteins

项目概要： 铜是一种必需的微量营养素，也是真核生物所必需的酶的氧化还原活性辅因子。 呼吸、抗氧化应激和功能化细胞信号分子的产生。非常 最近，据报道新型隐球菌Bim 1代表一类新的分泌型和细胞表面- 相关的铜蛋白，通过高亲和力CTR Cu-转运蛋白促进真菌在宿主期间的Cu-吸收 殖民化与C.新变型Bim 1在几种真菌的基因组中高度代表 病原体影响人类，我们确定这个新的家庭铜清除蛋白作为Bim 1样蛋白 （BLP）。令人惊讶的是，在BLP活性位点和C-末端GPI处存在显著的序列多样性 锚定域事实上，没有什么是已知的，如何这样的序列变化影响铜运输功能。 我们的中心假设是，BLP活性位点的变化是用来调节铜结合亲和力和氧化 状态特异性，而C-末端结构域在细胞表面分配BLP蛋白。的总目标 本研究项目有两个方面：1.为了了解BLP家族中的活性位点多样性如何影响Cu- 结合性能2.了解BLP细胞外定位模式如何改变细胞铜稳态。 我们建议使用机会性真菌病原体Pseudogymnopathy中编码的三种BLP destructans（Pd）作为原型的自然多样性，这个新的家庭的细胞外铜清除剂。我们 将在以下（2）具体研究目标中检验我们的假设：目标1。确定BLP的影响 活性部位的变化。目标二。为了明确Bim 1样蛋白（BLP）亚型在细胞外Cu 贩卖人口在第一个目标下，我们将（i）开发重组表达平台以产生野生型， 变体PdBLP。我们将在（ii）中确定活性位点的变化如何改变金属结合性质， 铜配位环境最后，在（iii）中，我们将确定活性位点的变化如何改变Cu-氧化还原 特性.在目标2中，我们定义了BLP亚型在细胞外铜运输中的作用。我们将测试 创新假设BLP可以在细胞表面分配，将Cu中继到细胞表面并促进Cu- 进口效率。为了验证这一假设，我们将利用酿酒酵母（Sc） 遗传学来构建BLP/CTR摄取途径的模型。在（i-ii）中，我们将优化重组表达 PdCTR转运蛋白和PdBLP在S.酿酒。这将涉及蛋白质的严格表征 在质膜和细胞壁的表达和定位模式。在（iii）中，我们将评估 PdBLP表达水平和细胞外定位促进Cu从扩散性和固体中输入 支持Cu源。这项工作的预期成果是对这种新型BLP如何 Cu吸收途径的功能是确保在极端环境下将Cu原子充分递送到真菌病原体。 铜的生物利用度，类似于宿主感染期间发现的。

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.