S-Acylation of transmembrane proteins in the early secretory pathway

真核细胞由许多不同的隔室和途径组成，它们专门执行特定的功能。分泌途径对细胞中产生的数千种不同的膜蛋白进行修饰和分类。随着它们的合成，膜蛋白进入分泌途径，通过内质网和高尔基体进入质膜。在这些运输步骤中，蛋白质经历了许多化学修饰，如糖基或脂基的附着--这些修饰通常在将蛋白质引导到特定细胞位置或增强其折叠和稳定性方面发挥重要作用。虽然糖基与蛋白质结合的分泌途径中的糖基化途径已经得到了广泛的研究，但对蛋白质的S-酰化(脂基的结合)却知之甚少。特别是，成千上万的膜蛋白是如何选择通过分泌途径进行S酰化的，内质网和高尔基体是如何协调这一过程的？S酰化是由“zdhhc”酶介导的，在人类中有23种这样的酶。绝大多数zDHHC酶存在于内质网和高尔基体中，我们之前的工作表明，这些酶要么专门介导特定和有限的蛋白质池的S酰化，要么具有广泛的特异性，允许它们修改一组不同的蛋白质-然而，单个zDHHC酶修饰的蛋白质池定义不明确。这个项目将以隔室为中心来看待S-酰化反应，并使用允许在内质网可逆捕获膜蛋白的方法，使我们能够对它们在这个隔室和高尔基体之间的移动进行精细控制。这将与高灵敏度的S酰化分析相结合，绘制出不同膜蛋白的特定隔室的S酰化模式(即它们是在内质网还是在该隔室释放后被S酰化)。为了了解不同区段如何选择膜蛋白进行S酰化反应，我们将详细分析支撑特定区段特定S酰化模式的氨基酸序列要求。最重要的是，我们将研究内质网中存在的八种不同的zDHHC酶，以破译它们如何对该细胞器的S酰化能力和特异性做出贡献。这里将特别关注zDHHC6酶，我们最近的工作表明它可能是一种广泛的特异性酶，介导了内质网一系列膜蛋白的S酰化。这些互补分析将为我们理解大量多样的膜蛋白的S酰化是如何由不同的细胞室协调的提供重要的突破，并将对通过分泌途径运输过程中的蛋白质修饰产生新的基本见解。除了这一基本的新知识，这项工作可能会产生更长期的影响，因为人们越来越有兴趣将S-酰化途径作为治疗不同人类疾病的靶点，包括癌症、神经疾病和传染病。了解不同区段和酶异构体在不同类别膜蛋白的S-酰化反应中的作用，将有助于确保这一过程能够充分和适当地作为治疗靶点。

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.