Nedd4‐1 binds and ubiquitylates activated FGFR1 to control its endocytosis and function

Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4‐1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4‐1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non‐canonical sequence (non‐PY motif) on FGFR1. Deletion of this recognition motif (FGFR1‐Δ6) abolishes Nedd4‐1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4‐1 knockdown. Accordingly, FGFR1‐Δ6, or Nedd4‐1 knockdown, exhibits sustained FGF‐dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1‐Δ6 in human embryonic neural stem cells strongly promotes FGF2‐dependent neuronal differentiation. Furthermore, expression of this FGFR1‐Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4‐1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development.     

Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance

Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na⁺ channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 μM range.     

Versatile communication strategies among tandem WW domain repeats

Interactions mediated by short linear motifs in proteins play major roles in regulation of cellular homeostasis since their transient nature allows for easy modulation. We are still far from a full understanding and appreciation of the complex regulation patterns that can be, and are, achieved by this type of interaction. The fact that many linear-motif-binding domains occur in tandem repeats in proteins indicates that their mutual communication is used extensively to obtain complex integration of information toward regulatory decisions. This review is an attempt to overview, and classify, different ways by which two and more tandem repeats cooperate in binding to their targets, in the well-characterized family of WW domains and their corresponding polyproline ligands.     

GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2

Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, which bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of inhibition leads to hypertension. ENaC channels are maintained in the active state by G-protein-coupled receptor kinase 2 (GRK2), an enzyme implicated in the development of essential hypertension. Here, we report that GRK2 interacts not only with ENaC, but also with both Nedd4 and Nedd4-2. Additionally, GRK2 is capable of phosphorylating both Nedd4 and Nedd4-2 at multiple sites. Of possible significance is the phosphorylation of the threonine at position 466 in Nedd4, which is located in the area of the ww3 domain that binds ENaC. These results support and extend the role of GRK2 in sodium transport regulation.     

Understanding the folding and stability of a designed WW domain protein with replica exchange molecular dynamics simulations

WW domain proteins are usually regarded as simple models for understanding the folding mechanism of β-sheet. CC45 is an artificial protein that is capable of folding into the same structure as WW domain. In this article, the replica exchange molecular dynamics simulations are performed to investigate the folding mechanism of CC45. The analysis of thermal stability shows that β-hairpin 1 is more stable than β-hairpin 2 during the unfolding process. Free energy analysis shows that the unfolding of this protein substantially proceeds through solvating the smaller β-hairpin 2, followed by the unfolding of β-hairpin 1. We further propose the unfolding process of CC45 and the folding mechanism of two β-hairpins. These results are similar to the previous folding studies of formin binding protein 28 (FBP28). Compared with FBP28, it is found that CC45 has more aromatic residues in N-terminal loop, and these residues contact with C-terminal loop to form the outer hydrophobic core, which increases the stability of CC45. Knowledge about the stability and folding behaviour of CC45 may help in understanding the folding mechanisms of the β-sheet and in designing new WW domains.     

Structure of the dodecahedral penton particle from human adenovirus type 3

The sub-viral dodecahedral particle of human adenovirus type 3, composed of the viral penton base and fiber proteins, shares an important characteristic of the entire virus: it can attach to cells and penetrate them. Structure determination of the fiberless dodecahedron by cryo-electron microscopy to 9 Angstroms resolution reveals tightly bound pentamer subunits, with only minimal interfaces between penton bases stabilizing the fragile dodecahedron. The internal cavity of the dodecahedron is approximately 80 Angstroms in diameter, and the interior surface is accessible to solvent through perforations of approximately 20 Angstroms diameter between the pentamer towers. We observe weak density beneath pentamers that we attribute to a penton base peptide including residues 38-48. The intact amino-terminal domain appears to interfere with pentamer-pentamer interactions and its absence by mutation or proteolysis is essential for dodecamer assembly. Differences between the 9 Angstroms dodecahedron structure and the adenovirus serotype 2 (Ad2) crystallographic model correlate closely with differences in sequence. The 3D structure of the dodecahedron including fibers at 16 Angstroms resolution reveals extra density on the top of the penton base that can be attributed to the fiber N terminus. The fiber itself exhibits striations that correlate with features of the atomic structure of the partial Ad2 fiber and that represent a repeat motif present in the amino acid sequence. These new observations offer important insights into particle assembly and stability, as well as the practicality of using the dodecahedron in targeted drug delivery. The structural work provides a sound basis for manipulating the properties of this particle and thereby enhancing its value for such therapeutic use.     

蛋白转导域内在化机制的研究进展

蛋白转导域（protein transduction domain,PTD）可以携带外源生物大分子进入细胞,在分子生物学、细胞生物学的基础研究及生物技术应用中,都展示出良好的前景,应用广泛,但机制不甚明确。已知的PTD均有其关键的特定氨基酸存在和较强的正电荷分布,并具有独特的二级结构及空间构象,这些特殊的结构特征对其内在化机制起决定作用。目前认为巨胞饮作用是PTD入胞的主要机制,PTD在经过细胞表面糖胺聚糖紧密结合快速作用及电荷作用后,由脂筏蛋白介导的巨胞饮作用内在化,然后巨胞饮体脂质双层破裂,使蛋白转导域．大分子释放入胞浆及胞核。     

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4‐1) and Nedd4L (Nedd4‐2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4‐1 and Nedd4‐2, and rat‐Nedd4‐1, using protein microarrays spotted with ～8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4‐1 and Nedd4‐2, others were specific to only one, with several Tyr kinases preferred by Nedd4‐1 and some ion channels by Nedd4‐2; this was subsequently validated in vivo. Accordingly, Nedd4‐1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4‐1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.     

TAT蛋白转导域：蛋白质治疗的新曙光

TAT蛋白转导域是源自人类免疫缺陷病毒Tat蛋白的一段碱性氨基酸多肽,能够将与之共价连接的多肽、蛋白、核酸等生物大分子快速而高效地转导入细胞内部,在药物转运和疾病治疗等领域有着巨大的应用潜力.TAT蛋白转导域首先通过电荷相互作用吸附于细胞膜,然后通过脂筏介导的巨胞饮作用进入细胞.随着体外研究的不断成熟,应用TAT蛋白转导域治疗人类肿瘤、卒中、炎症等疾病的动物模型也获得了成功,TAT蛋白转导域进入临床指日可待.     

WW结构域——蛋白质-蛋白质相互作用的一种模式

WW结构域是由38～40个氨基酸残基严密组织形成一个连贯、紧凑的结构域;它以包含两个色氨酸残基为主要特征,能专一地与含有XPPXY保守序列的蛋白质相互作用.这种相互作用涉及许多细胞内事件,如非受体信号传导、转录调节、蛋白质降解等等,并且这种相互作用的变化会直接或间接影响到人体的正常生理代谢功能而引起疾病.