S-Acylation of transmembrane proteins in the early secretory pathway

真核细胞由许多不同的区室和通路组成，这些区室和通路专门执行特定的功能。分泌途径修饰和分类细胞中产生的数千种不同的膜蛋白。合成后，膜蛋白进入分泌途径，通过内质网和高尔基体区室到达质膜。在这些运输步骤中，蛋白质经历了许多化学修饰，例如糖或脂质基团的连接-这些修饰通常在将蛋白质引导到特定的细胞位置或增强其折叠和稳定性方面发挥重要作用。尽管已经广泛研究了分泌途径中介导糖基与蛋白质连接的糖基化途径，但对蛋白质的S-酰化（脂质基团的连接）了解甚少。特别是，通过分泌途径的数千种膜蛋白是如何被选择进行S-酰化的，内质网和高尔基体是如何协调这一过程的？S-酰化由“zDHHC”酶介导，并且在人类中存在23种这些酶。绝大多数zDHHC酶存在于内质网和高尔基体中，我们以前的工作表明，这些酶要么专门介导特定和限制性蛋白质库的S-酰化，要么具有广泛的特异性，允许它们修饰不同的蛋白质-然而，由单个zDHHC酶修饰的蛋白质库定义不清。这个项目将采取一个隔间为中心的观点S-酰化和使用的方法，允许可逆的捕获膜蛋白在内质网，使我们能够施加精细的控制他们之间的运动，这个隔间和高尔基体。这将与S-酰化的高灵敏度测定相结合，以绘制出不同阵列的膜蛋白的隔室特异性S-酰化模式（即，它们是否在内质网或从该隔室释放后被S-酰化）。为了了解膜蛋白是如何被选择为S-酰化由不同的隔间，我们将进行详细的分析的氨基酸序列的要求，支持隔间特定的S-酰化模式。除此之外，我们将研究内质网中存在的八种不同的zDHHC酶，以破译它们如何有助于该细胞器的S-酰化能力和特异性。这里的一个具体重点将是酶zDHHC 6，我们最近的工作表明，它可能是一种广泛特异性的酶，介导内质网上一系列膜蛋白的S-酰化。这些互补分析将为我们理解S-酰化是如何发生的提供重要突破。大量不同的膜蛋白的酰化由不同的细胞区室协调，并将产生新的基本见解在通过分泌途径运输过程中转化为蛋白质修饰。除了这些基本的新知识，这项工作可能会产生长期的影响，因为人们越来越有兴趣靶向蛋白S-酰化途径作为治疗不同的人类疾病，包括癌症，神经系统疾病和传染病。了解不同类别膜蛋白的S-酰化中不同隔室和酶同工型的作用将有助于提供重要信息，确保该过程可以充分和适当地用作治疗靶点。

Eukaryotic cells are composed of many different compartments and pathways that are specialised to perform specific functions. The secretory pathway modifies and sorts the thousands of different membrane proteins that are produced in a cell. Following their synthesis, membrane proteins enter the secretory pathway and move through the endoplasmic reticulum and Golgi compartments en route to the plasma membrane. During these transport steps, proteins undergo a number of chemical modifications such as the attachment of sugar or lipid groups - and these modifications often play an important role in directing proteins to a specific cellular location or enhancing their folding and stability. Whereas glycosylation pathways in the secretory pathway that mediate the attachment of sugar groups to proteins have been extensively studied, the S-acylation of proteins (attachment of lipid groups) is poorly understood. In particular, how are the thousands of membrane proteins passing through the secretory pathway selected for S-acylation and how do the endoplasmic reticulum and Golgi coordinate this process?S-Acylation is mediated by "zDHHC" enzymes and there are twenty-three of these enzymes in humans. The vast majority of zDHHC enzymes are present at the endoplasmic reticulum and Golgi and our previous work suggested that these enzymes are either specialised to mediate the S-acylation of a specific and restricted pool of proteins or instead have a broad specificity that allows them to modify a diverse set of proteins- however the pools of proteins modified by individual zDHHC enzymes are poorly defined. This project will take a compartment-centric view of S-acylation and use methods that permit the reversible trapping of membrane proteins at the endoplasmic reticulum, allowing us to exert fine control over their movement between this compartment and the Golgi. This will be combined with high-sensitivity assays of S-acylation to map out the compartment-specific S-acylation patterns of a diverse array of membrane proteins (i.e. whether they are S-acylated at the endoplasmic reticulum or after release from this compartment). To understand how membrane proteins are selected for S-acylation by different compartments, we will undertake a detailed analysis of the amino acid sequence requirements that underpin compartment-specific S-acylation patterns. On top of this, we will study eight different zDHHC enzymes present at the endoplasmic reticulum to decipher how they contribute to the S-acylation capacity and specificity of this organelle. A specific focus here will be on the enzyme zDHHC6, which our recent work suggests may be a broad specificity enzyme that mediates S-acylation of a range of membrane proteins at the endoplasmic reticulum.These complementary analyses will provide an important breakthrough in our understanding of how the S-acylation of a large and diverse array of membrane proteins is coordinated by different cellular compartments and will generate new fundamental insight into protein modification during transport through the secretory pathway. In addition to this fundamental new knowledge, this work is likely to have longer-term impact as there is growing interest in targeting protein S-acylation pathways as a treatment for different human diseases including cancer, neurological disorders and infectious diseases. Understanding the roles of different compartments and enzyme isoforms in S-acylation of different classes of membrane protein will contribute important information that ensures that this process can be fully and appropriately exploited as a therapeutic target.