Mechanisms for cellular copper import via secreted cuproproteins

项目总结： 铜是一种必需的微量营养素，也是真核生物必需的酶的氧化还原活性辅因子。 呼吸作用、抗氧化性和产生功能化的细胞信号分子。非常 最近，新生隐球菌Bim1被报道代表了一类新的分泌性和细胞表面- 寄主中通过高亲和力CTR铜转运蛋白促进真菌铜吸收的相关铜蛋白 殖民主义。新生芽孢杆菌Bim1的同源物在几种真菌的基因组中高度表达 影响人类的病原体，我们确定这个新的铜清除蛋白家族为Bim1样蛋白 (Bps)。令人惊讶的是，BLP活性位点和C-末端GPI存在显著的序列多样性 锚定域。关于这种序列变异如何影响铜的运输功能，我们几乎一无所知。 我们的中心假设是BLP活性位点的变化被用来调节铜的结合亲和力和氧化 状态特异性，而C-末端结构域在细胞表面分割BLP蛋白。的总目标是 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铜- 绑定属性。2.了解BLP胞外定位模式如何改变细胞内铜稳态。 我们建议使用机会性真菌病原菌假淋巴曲霉中编码的三个BLP 作为这一新的胞外铜清除剂家族自然多样性的原型。我们 我将在以下几个方面检验我们的假设：(2)具体的研究目标：目的1.确定BLP的影响 活性部位变异；目的2.确定Bim1样蛋白(BLP)亚型在胞外铜中的作用 贩卖人口。在第一个目标下，我们将(I)开发一个重组表达平台，以生产野生型和 变种PdBLP。我们将在(Ii)中确定活性中心的变化如何改变金属结合性质和 铜配合环境。最后，在(Iii)中，我们将确定活性中心的变化如何改变铜的氧化还原 属性。在目标2中，我们定义了BLP异构体在细胞外铜运输中的作用。我们将测试 创新的假设认为，BLPS可以在细胞表面进行分割，将铜传递到细胞表面，并促进铜- 进口效率。为了验证这一假设，我们将利用酿酒酵母(SC)的力量。 遗传学来建立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在酿酒酵母中的表达。这将涉及对蛋白质的严格表征。 质膜和细胞壁的表达和定位模式。在第(Iii)项中，我们会评估 PdBLP的表达水平和胞外定位促进铜从弥散性和固体输入 支持的铜源。这项工作的预期结果是对这一新颖的BLP如何 铜吸收途径的功能，以确保铜原子在极端情况下足够地输送到真菌病原体 铜的生物利用率，类似于在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.