Deciphering iron/heme homeostasis using the fission yeast model.

这项提议的长期目标是破译真核生物中获得血红素的分子基础。由于其作为几种关键细胞酶的辅因子的重要生理作用，生物体通过两种不同的方式来获得血红素：血红素的生物合成和从外部来源吸收血红素。与负责血红素生物合成的特性良好的酶相反，对参与获得外源血红素的参与者和机制的了解非常有限，而且仍然知之甚少。拉贝小组的研究旨在填补我们知识的这一空白。我们的研究使用了模式生物S.pombe。与酿酒酵母不同，这种酵母可以吸收外源血红素，从而允许利用遗传学的力量选择性地阻止血红素的生物合成，从而为研究细胞吸收外部血红素的机制创造了条件。我们的研究揭示了编码在血红素同化过程中具有新功能的蛋白质的基因的存在，但对它们的了解很少，包括它们的作用机制以及它们如何对血红素水平的变化做出调节。在接下来的5年里，我们的研究计划将重点放在以下具体目标上。1.阐明细胞表面锚定的血红素受体Shu1在血红素转运中与其相互作用伙伴合作的分子基础。Shu1经历了血红素刺激的内化，这涉及到与Nbr1的相互作用，Nbr1作为ESCRT蛋白的货物。我们将：a)调查Shu1-Nbr1复合体的异构性；b)确定促进Shu1-Nbr1关联的系留因子(S)。2.阐明血红素转运蛋白Str3和伴侣蛋白Tpx1在血红素的获得和运输中的分子参与。在微需氧条件下，Tpx1的破坏导致细胞无法使用外源氯化血红素。我们将：a)表征Tpx1的血红素结合特性；b)研究Tpx1和Str3相互作用的机制。3.确定低铁条件下抑制Fep1所需的血红素途径。Frp1是一种血红蛋白。在没有血红素的情况下，抑制子Fep1结构性地与Frp1启动子结合。相反，当加入外源氯化血红素时，Fep1从这个启动子上解离，导致Frp1表达。我们将：a)识别向Fep1发出血红素存在信号所需的蛋白质；b)使用RNA-和CHIP-Seq方法来识别真菌细胞中血红素调节子的全基因组图像。根据该计划培训的学生将获得多学科方法的教育/培训，包括酵母遗传学、分子和细胞生物学、生物化学、下一代测序和质谱学领域。不区分少数族裔的学生将发展关键技能，使他们能够获得加拿大生物技术部门和/或学术研究社区训练有素的科学家的地位。

The long-term goal of this proposal is to decipher the molecular basis of heme acquisition in eukaryotes. Due to its essential physiological role as a cofactor for several key cellular enzymes, organisms have evolved with two different ways to obtain heme: heme biosynthesis and heme uptake from external sources. As opposed to the well-characterized enzymes responsible for heme biosynthesis, knowledge of the players and mechanisms involved in acquisition of exogenous heme is very limited and remains poorly understood. Studies in the Labbe's group are aimed at filling this gap in our knowledge. Our studies use the model organism S. pombe. This yeast, unlike S. cerevisiae, can assimilate exogenous heme, allowing the use of the power of genetics to selectively block heme biosynthesis, setting conditions to investigate the mechanisms by which external heme is taken up by the cells. Our studies have revealed the existence of genes encoding proteins that have novel functions in heme assimilation, yet little is known about them, including their mechanism of action and how they are regulated in response to changes in heme levels. During the next 5 years, we will focus our research program on the following specific aims. 1. Elucidate the molecular basis whereby Shu1, a cell surface-anchored heme receptor, cooperates with its interacting partners in heme transport. Shu1 undergoes heme-stimulated internalization that involves an interaction with Nbr1, which functions as a cargo for ESCRT proteins. We will: a) investigate the heteromeric nature of the Shu1-Nbr1 complex; b) identify a tethering factor(s) that fosters Shu1-Nbr1 association. 2. Elucidate the molecular participation of the heme transporter Str3 and the chaperone Tpx1 in the acquisition and trafficking of heme. Under microaerophilic conditions, disruption of Tpx1 results in cells being unable to use exogenous hemin. We will: a) characterize the heme-binding properties of Tpx1; b) investigate the mechanism whereby Tpx1 and Str3 interact with one another. 3. Determine the pathway whereby heme is required for inhibition of Fep1 under low Fe conditions. Frp1 is a hemeprotein. The repressor Fep1 is constitutively bound to the Frp1 promoter in the absence of heme. In contrast, when exogenous hemin is added, Fep1 dissociates from this promoter, resulting in Frp1 expression. We will: a) identify proteins that are required to signal the presence of heme to Fep1; b) use RNA- & ChIP-Seq approaches to identify a genome-wide picture of the heme regulon in fungal cells. Students trained under this program will acquire education/training in multidisciplinary approaches, including areas of yeast genetics, molecular & cellular biology, biochemistry, next-generation sequencing & mass spectrometry. Students without distinction as to minorities will develop critical skills, allowing them to gain the status of highly trained scientists for the Canadian biotech sector &/or academic research community.