Collaborative Research: Signaling via Opsins and Opsin-Related Proteins in Fungi

合作研究：真菌中视蛋白和视蛋白相关蛋白的信号传导

• 批准号: 0078306
• 负责人: Donald Natvig
• 金额: $ 27万
• 依托单位: University of New Mexico
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0078306&HistoricalAwards=false
• 关键词: Collaborative; Research; Signaling; via; Opsins

Opsins are seven-transmembrane helix proteins that bind all-trans or 11-cis isomers of retinal to form light-absorbing pigments known as rhodopsins. Previously, opsin-encoding genes had only been cloned from animals and the archaea. The principal investigator recently identified the first opsin gene from eukaryotic microbes, Neurospora crassa nop-1. The NOP-1 protein sequence is most similar to that of archaeal opsins, with conservation of all 22 retinal binding pocket residues, including the lysine residue that forms a Schiff base linkage with retinal. NOP-1 is also similar to several predicted proteins from various fungal species, including Saccharomyces cerevisiae Hsp30p. With the exception of two predicted proteins from filamentous fungi, all other related fungal proteins lack the Schiff base lysine residue and we have referred to them as Opsin-Related Proteins (ORPs). NOP-1 has been overexpressed in Pichia pastoris and its spectral properties determined. Similar to archaeal opsins, NOP-1 binds all-trans retinal with a Schiff base linkage. The resultant pigment has an absorption maximum at 534 nm (green). The relatively long photocycle of the NOP-1 pigment is similar to that of archaeal sensory rhodopsins, suggesting NOP-1 functions as a sensory receptor in N. crassa. The nop-1 message is most abundant under conditions that favor asexual sporulation (conidiation) in N. crassa. Although ?nop-1 mutants do not have visible phenotypes at 30oC, these strains exhibit green light-dependent defects in asexual spore-forming structures and cell viability at elevated growth temperatures (37-42oC). Also, the principal investigator identified an EST encoding a N. crassa ORP (orp-1). The predicted ORP-1 protein sequence is most similar to HSP30 from Coriolus versicolor. In contrast to NOP-1, ORP-1 only shows conservation of 50% of the 22 retinal binding pocket residues with archaeal rhodopsins, including substitution of isoleucine for the Schiff base lysine residue. The sequence similarity between NOP-1 and archaeal rhodopsins; the evolutionary relationship between NOP-1 and S. cerevisiae Hsp30p; the light-dependent expression of nop-1; the sensory rhodopsin-like photocycle of NOP-1; the light-dependent conidiation defect of ?nop-1 mutants in the presence of oligomycin, and the light-dependent defects in cell growth at elevated temperatures lead to the following hypothesis: Opsins and ORPs regulate cell growth, viability and development during cellular stress responses in N. crassa, potentially via light-dependent and independent pathways, respectively. The Research Objectives of this project are 1) To determine the localization and native chromophore of NOP-1 in N. crassa and to further characterize the involvement of nop-1 in stress and developmental regulation; 2) to identify genes that regulate nop-1 gene expression and genes whose expression is regulated by nop-1; 3) to create and analyze a ?orp-1 mutant and to over-express and characterize the encoded ORP-1 protein, and 4) to identify other genes encoding opsins and ORPs, and other nop-1 pathway components. During this collaborative research project, one laboratory will focus on Objectives 1 and 3, while the other will focus on Objectives 2 and 4. This collaborative research project will strengthen and extend the existing collaboration, and exploit the complementary skills and resources available to the two principal investigators. One principal investigator has extensive expertise in genome analysis, molecular evolution, the genetics and biochemistry of stress responses and the genetics of filamentous fungi in general. The other has significant experience in protein biochemistry, mutational analysis of fungal genes, stress responses and signal transduction via both G proteins and histidine kinases. The former has access to the resources of the Neurospora Genome Project, including automated sequencing and microarray facilities. The latter is in close proximity to a leader in biophysical analysis of archaeal rhodopsins and who has been a past and is a current collaborator. The existence of seven-helix opsin receptors has been postulated in eukaryotic microbes for decades. Many of these organisms utilize light signals to regulate cell growth, reproduction, phototaxis or the circadian clock. Therefore, the identification of an opsin in N. crassa has enormous repercussions for filamentous fungi and other eukaryotes. Furthermore, the lack of an opsin in the yeast Saccharomyces cerevisiae points to the importance of studying this class of proteins in the model filamentous fungus N. crassa. In addition, very little is known about ORPs in fungi: for example, whether they can bind a chromophore, absorb light, etc. Thus, the principal investigators are in the position of making pivotal discoveries in this field using N. crassa as an experimental organism.

视黄素是一种具有七个跨膜螺旋的蛋白质，可以与视网膜的全反式或11顺式异构体结合，形成被称为视紫红质的光吸收色素。此前，视蛋白编码基因只从动物和古菌中克隆出来。首席研究人员最近从真核微生物中首次发现了视蛋白基因，粗糙脉孢菌NOP-1。NOP-1蛋白序列与古生菌视蛋白最相似，保守了所有22个视网膜结合口袋残基，包括与视网膜形成Schiff碱基的赖氨酸残基。NOP-1还与几个来自不同真菌物种的预测蛋白相似，包括酿酒酵母Hsp30p。除了两个预测的丝状真菌蛋白外，所有其他相关真菌蛋白都缺乏Schiff碱基赖氨酸残基，我们将其称为视蛋白相关蛋白(ORPs)。NOP-1已在巴斯德毕赤酵母中过表达，并测定了其光谱性质。与古生菌视蛋白类似，NOP-1通过Schiff碱基连接全反式视黄醇。所得色素的最大吸收波长为534 nm(绿色)。NOP-1色素的光周期相对较长，与古细菌感官视紫红质相似，表明NOP-1在粗毛拟青霉中作为感官受体发挥作用。NOP-1信息最丰富的条件下，有利于无性产孢子(分生孢子)在新月。虽然NOP-1突变体在30oC时没有明显的表型，但这些菌株在高温(37-42oC)的生长温度下，在无性孢子形成结构和细胞活力方面表现出绿光依赖性缺陷。此外，首席调查者还发现了一种编码粗毛拟青霉ORP(ORP-1)的EST。预测的ORP-1蛋白序列与杂色云芝HSP30最相似。与NOP-1相比，ORP-1只显示了22个视网膜结合口袋残基中的50%与古生菌视紫红质结合，包括用异亮氨酸取代希夫碱基赖氨酸残基。NOP-1与古生菌视紫红质的序列相似性；NOP-1与酿酒酵母Hsp30p的进化关系；NOP-1的光依赖性表达；NOP-1的感觉性视紫红质样光周期；NOP-1突变体在寡霉素存在下的光依赖性分生缺陷，以及高温下细胞生长的光依赖性缺陷，导致了以下假设：在粗毛拟青霉的细胞胁迫反应中，Opsins和ORPs可能分别通过光依赖和独立的途径调节细胞的生长、存活和发育。本项目的研究目标是：1)确定NOP-1在粗毛藻中的定位和天然发色团，进一步研究NOP-1在胁迫和发育调控中的作用；2)鉴定调控NOP-1基因表达的基因和其表达受NOP-1调控的基因；3)建立和分析ORP-1突变体，并过表达和鉴定编码的ORP-1蛋白；4)鉴定其他编码Opsins和ORPs的基因，以及其他NOP-1途径成分。在这一合作研究项目期间，一个实验室将侧重于目标1和目标3，而另一个实验室将侧重于目标2和目标4。这一合作研究项目将加强和扩大现有的合作，并利用两个主要研究人员可获得的互补技能和资源。一位首席研究员在基因组分析、分子进化、应激反应的遗传学和生化以及丝状真菌的遗传学方面拥有广泛的专业知识。另一位在蛋白质生物化学、真菌基因突变分析、胁迫反应和通过G蛋白和组氨酸激酶的信号转导方面拥有丰富的经验。前者可以利用脉孢子虫基因组项目的资源，包括自动测序和微阵列设施。后者与古生菌视紫红质生物物理分析领域的一位领导者关系密切，他已经成为过去，现在是合作者。真核微生物中存在七螺旋视蛋白受体已有几十年的历史。这些生物中的许多利用光信号来调节细胞的生长、繁殖、趋光性或生物钟。因此，粗毛拟青霉中视蛋白的鉴定对丝状真菌和其他真核生物具有巨大的影响。此外，在酿酒酵母中缺乏视蛋白表明了在模式丝状真菌N.crassa中研究这类蛋白质的重要性。此外，人们对真菌中的ORP知之甚少：例如，它们是否能结合生色团、吸收光线等。因此，主要研究人员能够利用粗毛拟青霉作为实验生物体在这一领域取得关键发现。