Mechanisms for cellular copper import via secreted cuproproteins

通过分泌铜蛋白输入细胞铜的机制

基本信息

批准号：
10794575
负责人：
Ryan Loren Peterson
金额：
$ 5.62万
依托单位：
TEXAS STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10794575
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

项目摘要

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.

项目概要：铜是一种必需的微量营养素，也是真核生物必需的酶所需的氧化还原活性辅因子呼吸、氧化应激抵抗以及功能化细胞信号分子的产生。非常最近，据报道，新型隐球菌 Bim1 代表了一类新的分泌和细胞表面- 相关铜蛋白在宿主期间通过高亲和力 CTR 铜转运蛋白促进真菌对铜的吸收殖民化。新型隐球菌 Bim1 的同源物在多种真菌的基因组中高度存在影响人类的病原体，我们将这个新的铜清除蛋白家族鉴定为 Bim1 样蛋白（BLP）。令人惊讶的是，BLP 活性位点和 C 端 GPI 存在显着的序列多样性锚定域。事实上，我们对这种序列变异如何影响铜转运功能一无所知。我们的中心假设是 BLP 活性位点变化用于调节 Cu 结合亲和力和氧化状态特异性，而 C 端结构域在细胞表面划分 BLP 蛋白。总体目标为该研究项目有两个方面： 1. 了解 BLP 家族内的活性位点多样性如何影响 Cu- 绑定属性。 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假裸子囊菌中编码的三种 BLP 破坏蛋白（Pd）作为这种新的细胞外铜清除剂家族的自然多样性的原型。我们将在以下 (2) 个具体研究目标中检验我们的假设：目标 1. 确定 BLP 的影响活性位点变异。目标 2. 确定 Bim1 样蛋白 (BLP) 亚型在细胞外 Cu 中的作用贩运。在第一个目标下，我们将（i）开发重组表达平台来生产野生型和变体 PdBLP。我们将在 (ii) 中确定活性位点变化如何改变金属结合特性和铜配位环境。最后，在 (iii) 中，我们将确定活性位点变化如何改变铜氧化还原特性。在目标 2 中，我们定义了 BLP 同工型在细胞外铜运输中的作用。我们将测试创新的假设是，BLP 可以在细胞表面进行分配，将 Cu 传递到细胞表面并增强 Cu- 进口效率。为了检验这一假设，我们将利用酿酒酵母 (Sc) 的力量遗传学建立 BLP/CTR 摄取途径模型。在(i-ii)中，我们将优化重组表达酿酒酵母中 PdCTR 转运蛋白和 PdBLP 的影响。这将涉及蛋白质的严格表征质膜和细胞壁的表达和定位模式。在(iii)中，我们将评估以下因素的影响： PdBLP 表达水平和细胞外定位促进扩散和固体中的铜输入支持铜源。这项工作的预期成果是对这种新颖的 BLP 如何进行基本理解铜吸收途径的作用是确保在极端条件下将铜原子充分输送给真菌病原体铜的生物利用度，类似于宿主感染期间发现的。