Mechanisms for cellular copper import via secreted cuproproteins

通过分泌铜蛋白输入细胞铜的机制

• 批准号: 10797773
• 负责人: Ryan Loren Peterson
• 金额: $ 10万
• 依托单位: TEXAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-21 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797773
• 关键词: Active Sites; Affect; Affinity; Amino Acid Sequence; Antifungal Agents; Aspergillus niger; Binding; Biological Availability; Biological Process; C-terminal; Cell Wall; Cell membrane; Cell surface; Coccidioides immitis; Coenzymes; Copper; Cryptococcus neoformans; Development; Ensure; Environment; Extracellular Protein; Family; Future; Gene Proteins; Genetic; Genome; Goals; Histoplasma capsulatum; Homeostasis; Homologous Gene; Human; Infection; Link; Meningitis; Metals; Micronutrients; Modeling; Mus; Outcome; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Pattern; Plasma Cells; Production; Property; Protein Family; Protein Isoforms; Proteins; Public Health; Recombinants; Reporting; Research; Research Project Grants; Resistance; Respiration; Role; Saccharomyces cerevisiae; Signal Transduction; Signaling Molecule; Solid; Source; Specificity; Talaromyces; Testing; Variant; Virulence; Work; design; extracellular; fungus; host colonization; innovation; novel; oxidation; pathogenic fungus; protein expression; protein transport; prototype; trafficking; treatment strategy; uptake; virtual

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.

项目摘要： 铜是一种必需的微量营养素，是真核酶的必需氧化还原活性辅助因子 呼吸，氧化应激抗性以及功能化细胞信号分子的产生。非常 最近，据报道，加密型新面球BIM1代表了一类新的分泌和细胞表面 相关的蛋白蛋白在主机期间通过高亲和力CTR Cu-Transsterster促进真菌Cu摄取 殖民化。 Neoformans bim1的同源物在几种真菌的基因组中高度表示 影响人类的病原体，我们将这种新的Cu扫除蛋白识别为BIM1样蛋白 （blps）。令人惊讶的是，BLP活性位点和C末端GPI具有显着的序列多样性 锚定域。几乎没有关于这种序列变化如何影响cu贩运函数的知之甚少。 我们的中心假设是BLP活动位点变化用于调节Cu结合亲和力和氧化 状态特异性，而C末端结构域分配BLP蛋白在细胞表面。总体目标 该研究项目是两个方面：1。了解BLP家族中的主动地点多样性如何影响Cu- 结合特性。 2。了解BLP细胞外定位模式如何改变细胞Cu Homeostasis。 我们建议使用在机会性真菌病原体pseudogymnoascus中编码的三个BLP destructans（PD）作为这个新的细胞外铜扫描剂家族的自然多样性的原型。我们 将在以下（2）特定研究目的中检验我们的假设：目的1。确定BLP的影响 主动站点变化。目标2。定义BIM1样蛋白（BLP）同工型在细胞外Cu中的作用 贩运。在第一个目标下，我们将（i）开发一个重组表达平台，以生产野生类型和 变体PDBLP。我们将在（ii）确定主动位点变化如何改变金属结合特性和 铜协调环境。最后，在（iii）中，我们将确定主动位点变化如何改变cu-redox 特性。在AIM 2中，我们定义了BLP同工型在细胞外Cu贩运中的作用。我们将测试 创新的假设，即BLP可以在细胞表面分配以将Cu转移到细胞表面并增强Cu- 进口效率。为了检验这一假设，我们将利用酿酒酵母（SC）的力量 遗传学以建立BLP/CTR摄取途径的模型。在（I-II）中，我们将优化重组表达式 酿酒酵母中的PDCTR转运蛋白和PDBLP。这将涉及蛋白质的严格特征 质膜和细胞壁上的表达和定位模式。在（iii）中，我们将评估 PDBLP表达水平和细胞外定位，可促进从扩散和实心 支持CU来源。这项工作的预期结果是对这种新颖BLP的基本理解 Cu摄取途径的功能，以确保在极端的真菌病原体中充分递送Cu原子 铜生物利用度，类似于宿主感染期间发现的铜。