Molecular mechanism of environmental stress sensing by bacterial Zinc-containing Anti-Sigma factors

所有细胞的一个关键进化特性是它们感知和响应环境变化的能力。对于那些经常生活在恶劣和波动环境中的细菌来说，这一点尤其如此。一个活跃但仍然知之甚少的研究领域是细菌如何感知环境变化以及它们对这些变化做出反应的机制。这项建议的重点是这些问题的土壤生物链霉菌coelicolor，这是一个理想的模式，有两个原因。首先，它的基因组序列显示，这种生物体拥有很好的基因，这些基因编码的蛋白质可能参与对环境压力的反应，但这些蛋白质的作用方式尚未被描述。其次，链霉菌属是许多商业上重要的抗生素，抗癌剂和免疫抑制剂的来源，其生产可能与生物体对环境变化的反应能力有关，因此通过了解潜在的机制，我们可能能够影响这种治疗分子的生产。我们的建议集中在一组特定的压力传感器上，它们实际上是两种蛋白质的复合物：一种是sigma因子，它指导负责产生特定RNA分子（RNA聚合酶）的主要细胞酶产生蛋白质，使细胞能够对环境变化做出反应;另一种是anti-sigma因子，它与sigma因子结合并阻止其结合RNA聚合酶的能力。反西格马因子的工作是感知环境压力。已知这种传感机制涉及反西格玛因子的禁用，其释放西格玛因子以协调细胞对应激的反应。虽然它已经超过15年，因为我们第一次描述了现在被认为是一个广泛的组细胞质外功能（ECF）σ因子的存在（链霉菌单独有超过50个编码在其基因组中），我们仍然知道令人惊讶的是，反σ因子如何结合ECF σ因子或环境压力如何禁用它们。我们最近发现了链霉菌对一种特殊形式的氧化应激（衰老的主要原因）的反应机制，称为二硫化物应激。二硫化物是两个半胱氨酸氨基酸的硫原子之间形成的共价键，通常存在于从细胞分泌的蛋白质中（例如激素，如胰岛素），以帮助在恶劣的细胞外环境中稳定它们。然而，这种键对于细胞质的拥挤环境内的蛋白质是有毒的，在那里它们可以导致酶的失活和蛋白质的聚集。链霉菌通过使特定的抗σ因子（RsrA）失活来响应细胞内二硫键的出现，所述抗σ因子（RsrA）释放其σ因子σ R以产生抗氧化反应。我们已经确定了RsrA在其静息状态下的三维结构，即在其结合σ因子之前。与相关蛋白质复合物的比较表明，RsrA参与了一种新形式的分子识别，其中蛋白质基本上将其自身翻转以结合sigma R。我们还确定了RsrA失活形式的结构，其中内部二硫键阻断了蛋白质由内而外翻转的能力。这项提案旨在利用这些新的观察结果。我们将研究RsrA如何由内而外地结合其sigma因子。我们合理化，这种机制可能是其他形式的环境压力传感的基础上，这一大群细胞调节。因此，我们将揭示激活信号的选择几个反西格玛因子/西格玛因子配对，这还有待研究，并将它们与RsrA/西格玛R复杂。我们的目标是确定我们发现的机制是否代表了微生物环境压力传感的新范式。

A critical evolved property of all cells is their ability to sense and respond to environmental change. This is especially true of bacteria that often have to live in inhospitable and fluctuating environments. An active yet still poorly understood area of research is how bacteria sense environmental change and the mechanisms they deploy to respond to such changes. This proposal focuses on these questions in the soil-living organism Streptomyces coelicolor, which is an ideal model for two reasons. First, its genome sequence shows the organism is well armed with genes that encode proteins likely to be involved in responding to environmental stresses but the mode of action of these have yet to be described. Second, the genus Streptomyces is the source of a multitude of commercially important antibiotics, anticancer agents and immunosuppressants, the production of which are likely linked to the organism's ability to respond to environmental changes and so by understanding the underlying mechanisms we may be able to affect the production of such therapeutic molecules. Our proposal focuses on one specific group of stress sensors which are in fact complexes of two proteins: One is a sigma factor that directs the main cellular enzyme responsible for the production of specific RNA molecules (RNA polymerase) to produce proteins which allow the cell to respond to environmental change; the other is an anti-sigma factor that binds to the sigma factor and blocks its ability to bind RNA polymerase. It is the anti-sigma factor's job to sense the environmental stress. It is known that this sensing mechanism involves the disabling of the anti-sigma factor, which releases the sigma factor to coordinate the cellular response to the stress. Although it has been over 15 years since we first described the presence of what is now recognised as a widespread group of ExtraCytoplasmic Function (ECF) sigma factors (Streptomyces alone has over 50 of them encoded in its genome), we still know surprisingly little about how anti-sigma factors bind ECF sigma factors or how environmental stresses disable them. We have recently uncovered the mechanism by which Streptomyces responds to a particular form of oxidative stress (the main causative agent of ageing) known as disulfide stress. Disulfides are covalent bonds formed between the sulfur atoms of two cysteine amino acids, which are ordinarily found in proteins that get secreted from cells (e.g. hormones such as insulin) to help stabilise them in the harsh extracellular environment. Such bonds however are toxic for proteins inside the crowded environment of the cell cytoplasm where they can cause the inactivation of enzymes and the aggregation of proteins. Streptomyces responds to the appearance of intracellular disulfide bonds by inactivating a specific anti-sigma factor (RsrA), which releases its sigma factor, sigma R, to mount an anti-oxidative response. We have determined the three dimensional structure of RsrA in its resting state, i.e. before it binds sigma factor. Comparison to related protein complexes reveals that RsrA engages in a new form of molecular recognition in which the protein essentially turns itself inside-out to bind sigma R. We have also determined the structure of the deactivated form of RsrA in which an internal disulfide blocks the ability of the protein to turn inside-out. This proposal aims to capitalise on these novel observations. We will investigate how RsrA turns inside-out to bind its sigma factor. We rationalise that this mechanism could be the basis for other forms of environmental stress sensing by this large group of cellular regulators. We will therefore uncover the activation signals for a select few anti-sigma factor/sigma factor pairings, which have yet to be studied, and compare them to the RsrA/sigma R complex. Our goal is to determine if the mechanism we have discovered represents a new paradigm in environmental stress sensing in microbes.