Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals.

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.     

Straight-chain non-polar amino acids are good helix-formers in water

For comparison with earlier data on naturally occurring non-polar amino acids (Ala, Leu, Phe, Val, Ile), the comparative helix-forming tendencies have been measured for non-polar amino acid residues that have unbranched side-chains, with an ethyl, propyl or butyl group, and also for methionine. The substitutions are made in a 17-residue alanine-based peptide. The results show that straight-chain non-polar amino acids have high helix-forming tendencies compared to beta-branched non-polar amino acids. Restriction of side-chain conformations in the helix, with a corresponding reduction in conformational entropy, is the likely explanation. There is a small increase in helix-forming tendency as the side-chain increases in length from ethyl to butyl, which suggests that a helix-stabilizing hydrophobic interaction is being detected.     

Neto2 Interacts with the Scaffolding Protein GRIP and Regulates Synaptic Abundance of Kainate Receptors

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ∼40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses.     

MDBK nuclear factor-binding site of various serotypes of adenovirus DNA

A fraction with the ability to bind the terminal fragment of equine adenovirus (EAd) DNA was prepared from MDBK cell nuclei. The fraction (MDBK nuclear factor) bound to the terminal fragment of all human and animal adenovirus DNAs examined except avian adenovirus EDS-76. However, the terminal fragments of two animal adenoviruses, EAd and bovine adenovirus type 3 (BAd3), showed higher affinity for the nuclear factor than the others. The MDBK nuclear factor-binding site determined by footprinting analysis was the sequence located between nucleotides 22 and 46 in EAd, between 36 and 53 in canine adenovirus type 2, and between 20 and 46 in BAd3, counting from the terminus. The respective binding site contained a sequence resembling the consensus sequence. The binding site of Ad4 DNA was not within the inverted terminal repetition, but was located at least 550 base pairs apart from the terminus.     

Solution-Phase, parallel synthesis and pharmacological evaluation of acylguanidine derivatives as potential sodium channel blockers

Solution-phase synthesis of various acylguanidine derivatives and the evaluation of a small library of compounds as potential sodium channel blockers are described.