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Understanding The Interaction Mechanism Of Nogo-66 With (i) NgR1 And (ii) Membrane Phosphocholine And The Structure Determination JSRV Envelope Protein Cytoplasmic Tail And Transmembrane Domain

  • Author(s): Alhoshani, Ali Rashed
  • Advisor(s): Cocco, Melanie
  • et al.
Abstract

Several myelin-associated proteins, the neurite outgrowth inhibitor (Nogo), myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp) contribute to inhibiting central nerve system (CNS) regeneration after injuries, mainly by blocking axonal regrowth. The neurite outgrowth inhibitor (Nogo A) is a multi-domain transmembrane (TM) protein. The loop domain, connecting the two hydrophobic C-terminal TM helices, is referred to as Nogo66. The inhibitory effect of Nogo is established when Nogo66 binds to its receptor (NgR) on the axon. Based on mutagenesis studies performed in our lab, residues Ser 38, Asn 39, Ser 40, Leu 42, Arg 53 and Arg 54 affected the binding of Nogo66 to NgR. Arg 53 and Arg 54 are among the most affected residues. On the other hand, combinatorial studies on NgR, Asp 111, Asp 114, Asp 163 are the most residues contributing to the interaction of NgR with Nogo66 (1). To confirm this observation, High Ambiguity Driven Docking (HADDOCK) expert interface server was used (2). As starting structures for docking studies, I used the crystal structure of NgR (PDB id 1OZN) and the NMR solution structure of Nogo66 (PDB id 2KO2). The complex structures that resulted from the HADDOCK studies were in agreement with our mutagenesis studies and provided insight into the mechanism of inhibition. Following the docking analysis, I used an aptamer to inhibit its function and help promote the axonal regeneration after injuries. One of the identified sequences (Ali 3) showed some specificity and affinity.

Human reticulon 4 (RTN-4) has been identified as the neurite outgrowth inhibitor (Nogo). This protein contains a span of 66 amino acids (Nogo-66) flanked by two membrane helices at the C-terminus. We previously determined the NMR structure of Nogo-66 in a native-like environment and defined the regions of Nogo-66 expected to be membrane embedded. We hypothesize that aromatic groups and a negative charge hyperconserved among RTNs (Glu26) drive the remarkably strong association of Nogo-66 with a phosphocholine surface. Glu26 is an isolated charge with no counter ion provided by nearby protein groups. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point. To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, association with lipid and affinity for the Nogo receptor. In an aqueous environment, Nogo-66 Glu26Ala is more helical than WT and binds the Nogo receptor with higher affinity. Thus, we can conclude that in the absence of a neutralizing positive charge provided by lipid, the glutamate anion is destabilizing to the Nogo-66 fold. Although the Nogo-66 Glu26Ala free energy of transfer from water into lipid is similar to that of WT, NMR data reveal a dramatic loss of tertiary structure for the mutant in DPC micelles. These data show that Glu26 has a key role in defining the structure of Nogo-66 on a phosphocholine surface.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a transmissible lung cancer in sheep, ovine pulmonary adenocarcinoma (OPA). OPA resembles bronchiole-alveolar carcinoma in humans, and it is an excellent animal model for this disease. A unique feature of JSRV is that the viral envelope (Env) protein also functions as an oncogene, in that the expression of the JSRV Env protein causes morphological transformation of fibroblast and epithelial cell lines, and vectored Env expression induces epithelial tumors in several animals. Previous studies showed that the region containing the short 46 amino acid C-terminal cytoplasmic tail (CT) of JSRV Env is essential for the ability of JSRV to transform cells. Residues in the cytoplasmic tail include a tyrosine (Y590), which is present in a consensus motif YXXM, which could potentially bind the regulatory subunit of phosphatidyl inositol 3-kinase (PI3K) if the Y590 is phosphorylated. Alanine scanning mutagenesis on the JSRV transmembrane (TM) and CT has been conducted. Mutation of some residues abolished Env transformation potential, while mutation of other residues had no effect or partial effects. To further understand the mechanism of JSRV transformation, structure-function analysis of the TM cytoplasm tail (CT) is important. We have determined the structure of the JSRV CT using NMR spectroscopy. This data allows us to interpret the alanine scanning mutagenesis, and allows better understanding of previous studies. Interestingly, using both CD and NMR, we find that the CT is only structured in the presence of a phosphocholine surface. The results validated some aspects of the predicted structure, and they also provided a basis for evaluating models of transformation.

p53 is a transcription factor with tumor suppressor activity that is triggered by various cellular stresses and functions to control genomic instability. Since p53 functions as a tetramer, any defect in either p53 allele would result in cancer. The importance of function of p53 and rescuing its mutations for cancer prevention has motivated researchers to find second-site suppressors, short peptides, and small molecules to restore wild type conformation. Bachmann and co-workers have identified the global suppressor motif involving amino acid N235, Y239, R240 restore the function by rescuing more than 50 % of the most p53 cancer mutations (Baroni, Wang et al. 2004). In order to understand the rescue mechanism I use NMR and monitor Hydrogen exchange (HX) to determine the most stable residues. I then calculated a protection factor (P) for each residue. p53 rescue mutants showed more protection than wild type, consistent with their mechanism of increasing stability. The average of protection factors for stable residues in N239Y compare to those in N235K and wild type i.e. N239Y (4.39 x106)> N235K (1.68 x106)>Wild type (6.94 x105). The extreme protection in Asn-268 and Asp-259 in N239 and N235K is due to the involvement in a hydrogen bond network, which furthers their stability.

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