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

所有细胞的一个重要进化特性是它们感知和响应环境变化的能力。对于那些经常不得不生活在恶劣和波动的环境中的细菌来说尤其如此。细菌如何感知环境变化以及它们如何应对这种变化的机制是一个活跃但仍然知之甚少的研究领域。本研究的重点是土壤生物链霉菌（Streptomyces colicolor）的这些问题，这是一个理想的模型，有两个原因。首先，它的基因组序列表明，这种生物体拥有编码蛋白质的基因，这些蛋白质可能参与对环境压力的反应，但这些蛋白质的作用模式尚未被描述。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，它们的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些治疗分子的生产。我们的建议侧重于一组特定的压力传感器，它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；另一种是与sigma因子结合并阻断其结合RNA聚合酶的能力的抗sigma因子。反西格玛因子的工作是感知环境压力。众所周知，这种感知机制涉及到抗sigma因子的失能，从而释放sigma因子来协调细胞对压力的反应。尽管自我们首次描述现在被认为是广泛存在的细胞质外功能（ECF） sigma因子（链霉菌在其基因组中编码超过50个）的存在以来已经超过15年了，但我们对抗sigma因子如何结合ECF sigma因子或环境压力如何使其失能仍然知之甚少。我们最近发现了链霉菌对一种特殊形式的氧化应激（衰老的主要诱因）作出反应的机制，这种氧化应激被称为二硫化物应激。二硫化物是在两个半胱氨酸氨基酸的硫原子之间形成的共价键，通常存在于细胞分泌的蛋白质（如胰岛素等激素）中，以帮助它们在恶劣的细胞外环境中保持稳定。然而，这种键对细胞质拥挤环境中的蛋白质是有毒的，在那里它们会导致酶的失活和蛋白质的聚集。链霉菌通过灭活一种特定的抗西格玛因子（RsrA）来响应细胞内二硫键的出现，该因子释放其西格玛因子，西格玛R，以引起抗氧化反应。我们已经确定了RsrA在静息状态下的三维结构，即在它结合sigma因子之前。与相关蛋白复合物的比较表明，RsrA参与了一种新的分子识别形式，在这种形式中，蛋白质本质上是将自身翻过来结合sigma r。我们还确定了RsrA失活形式的结构，其中内部二硫化物阻止了蛋白质翻过来的能力。这项提议旨在利用这些新的观察结果。我们将研究RsrA如何从内到外结合其sigma因子。我们认为，这种机制可能是这一大群细胞调节因子对其他形式的环境压力感知的基础。因此，我们将揭示一些尚未研究的抗sigma因子/sigma因子配对的激活信号，并将其与RsrA/sigma R复合物进行比较。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感知的新范式。

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.