Amyloid-ß regulates gap junction protein connexin 43 trafficking in cultured primary astrocytes.

Altered expression and function of astroglial gap junction protein connexin 43 (Cx43) has increasingly been associated to neurotoxicity in Alzheimer disease (AD). Although earlier studies have examined the effect of increased ß-amyloid (Aß) on Cx43 expression and function leading to neuronal damage, underlying mechanisms by which Aß modulates Cx43 in astrocytes remain elusive. Here, using mouse primary astrocyte cultures, we have examined the cellular processes by which Aß can alter Cx43 gap junctions. We show that Aß25-35 impairs functional gap junction coupling yet increases hemichannel activity. Interestingly, Aß25-35 increased the intracellular pool of Cx43 with a parallel decrease in gap junction assembly at the surface. Intracellular Cx43 was found to be partly retained in the endoplasmic reticulum-associated cell compartments. However, forward trafficking of the newly synthesized Cx43 that already reached the Golgi was not affected in Aß25-35-exposed astrocytes. Supporting this, treatment with 4-phenylbutyrate, a well-known chemical chaperone that improves trafficking of several transmembrane proteins, restored Aß-induced impaired gap junction coupling between astrocytes. We further show that interruption of Cx43 endocytosis in Aß25-35-exposed astrocytes resulted in their retention at the cell surface in the form of functional gap junctions indicating that Aß25-35 causes rapid internalization of Cx43 gap junctions. Additionally, in silico molecular docking suggests that Aß can bind favorably to Cx43. Our study thus provides novel insights into the cellular mechanisms by which Aß modulates Cx43 function in astrocytes, the basic understanding of which is vital for the development of alternative therapeutic strategy targeting connexin channels in AD.

Probing the functional conformations of an atypical proline-rich fusion peptide.

The left-handed polyproline II (PPII) type helical structures are thought to play a very important role in many essential biological processes, particularly in recognition mechanisms. However, reliable characterisation of PPII conformation in solution can be experimentally challenging. Computational simulation of these structures offers an attractive alternative, but the accuracy of the results is dependent on the accuracy of the force field employed. In this report, we present the results of simulation of the structural and dynamical properties of a proline-rich viral fusion peptide for which a solution NMR study reported a substantial stretch of PPII conformation in the central region. The suggested mode of action of the p15 fusion peptide depended on the exposure of the flanking N-terminal hydrophobic residues to solvent thereby facilitating their interaction with the target membrane. Our simulations with a set of four force field and water model combinations consisting of (AMBER ff99SB*-ILDNP + TIP3P), (OPLS-AA + SPC/E), (AMBER ff03ws + TIP4P/2005 water with scaled protein-water interactions) and (CHARMM36m + TIP3P) showed a general agreement with the NMR results for all the four force field and water model combinations. The central region encompassing positions 9-15 showed a large PPII propensity, reduced flexibility and lower conformational entropy. The PPII conformations were stable and satisfied the Burgi-Dunitz criteria without the participation of any significant water bridging interaction. However, comparison of the experimentally observed chemical shifts with the distribution of shifts predicted from the simulated ensembles showed a much better agreement for the CHARMM36m + TIP3P and AMBER ff03ws + TIP4P/2005 combinations. The models based on these two force fields also generated conformations which were in much better agreement with the NMR model than the much more compact structures predicted by the AMBER ff99SB*-ILDNP and OPLS-AA force fields and predicted a substantially larger solvent accessible surface area in accordance with the suggested mechanism of action of the peptide.

Plant Polypeptide Hormone Systemin Prefers Polyproline II Conformation in Solution.

Systemin, an 18 amino-acid-signaling peptide, was the first plant polypeptide hormone to be discovered. Earlier structural studies involving NMR spectroscopy indicated a lack of definite structure in solution while circular dichroism spectroscopy suggested the presence of left-handed polyproline II (PPII) conformation. Here, we report the results of molecular dynamics simulations of the peptide in explicit solvent with two different force fields, namely, ff99SBildn and ff99IDPs, both of which showed a large propensity for PPII-like conformations in spite of showing differing features for other conformational characteristics. More remarkably, the conformations with predicted chemical shifts that agreed better with the NMR observations had a larger than average PPII content, especially for the ff99IDPs force field. An independent docking calculation of the molecule with the putative receptor SR160 also retained this conformational preference for PPII structure. The results suggest PPII to be an important class of conformation for systemin which may have a role in its bioactivity.