Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB-NqrD subunit interface.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the major Na+ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na+-NQR. While the mechanisms underlying Na+-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na+-NQR. An in silico model of the NqrB-D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na+-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB-D interface underwent a non-physiological transition after 350 ns.

Computational modelling of protein interactions: energy minimization for the refinement and scoring of association decoys.

The prediction of protein-protein interactions based on independently obtained structural information for each interacting partner remains an important challenge in computational chemistry. Procedures where hypothetical interaction models (or decoys) are generated, then ranked using a biochemically relevant scoring function have been garnering interest as an avenue for addressing such challenges. The program PatchDock has been shown to produce reasonable decoys for modeling the association between pig alpha-amylase and the VH-domains of camelide antibody raised against it. We designed a biochemically relevant method by which PatchDock decoys could be ranked in order to separate near-native structures from false positives. Several thousand steps of energy minimization were used to simulate induced fit within the otherwise rigid decoys and to rank them. We applied a partial free energy function to rank each of the binding modes, improving discrimination between near-native structures and false positives. Sorting decoys according to strain energy increased the proportion of near-native decoys near the bottom of the ranked list. Additionally, we propose a novel method which utilizes regression analysis for the selection of minimization convergence criteria and provides approximation of the partial free energy function as the number of algorithmic steps approaches infinity.

Lovastatin protects human neurons against Abeta-induced toxicity and causes activation of beta-catenin-TCF/LEF signaling.

Alzheimer's disease (AD) is characterized by cognitive decline due to excess amyloid beta peptide (Abeta), neurofibrillary tangles, and neuronal loss. Abeta promotes neuronal apoptosis in AD by activating glycogen synthase kinase-3beta (GSK-3beta), leading to degradation of beta-catenin and inactivation of Wnt signaling. beta-Catenin interacts with the T-cell factor (TCF)/Lymphoid enhancer factor (LEF)-nuclear complex to mediate Wnt signaling and cell survival. Statins are associated with decreased prevalence of AD. Lovastatin has been shown to decrease the production of Abeta and to promote neuronal survival. The mechanisms of how statins promote neuronal survival are unclear. We propose that the neuroprotective effect of lovastatin may be due to inactivation of GSK-3beta activity, resulting in induction of Wnt signaling. Here, we report that lovastatin prevented Abeta-induced apoptosis in human SK-NSH cells. This was accompanied by reduction in active GSK-3beta, and increased nuclear translocation of beta-catenin, TCF-3, and LEF-1. Lovastatin treatment induced an increase in TCF/LEF-chloramphenicol acetyl transferase (CAT) gene reporter activity. More importantly, beta-catenin and TCF were required for the neuroprotective function of lovastatin. Our results suggest that lovastatin protects neuronal cells from Abeta-induced apoptosis and causes reduction in GSK-3beta activity, resulting in activation of Wnt signaling.

Alternative splicing of the first intron of the steroid receptor RNA activator (SRA) participates in the generation of coding and noncoding RNA isoforms in breast cancer cell lines.

The Steroid Receptor RNA Activator 1 (SRA1) has originally been described as a noncoding RNA specifically activating steroid receptor transcriptional activity. We have, however, identified, in human breast tissue, exon- 1 extended SRA1 isoforms containing two initiating AUG codons and encoding a protein we called SRAP. We recently reported a decreased estrogen receptor activity in breast cancer cells overexpressing SRAP, suggesting antagonist roles played by SRA1 RNA and SRAP. SRA1 appears to be the first example of a molecule active both at the RNA and at the protein level. No data are currently available regarding the mechanisms possibly involved in the generation of coding and noncoding functional SRA1 RNAs. Using 5'-Rapid Amplification of cDNA Extremities (5'-RACE), we have herein identified several putative transcription initiation sites surrounding the second methionine codon and used to generate coding SRA1 transcripts. In the process, we also identified an alternatively spliced noncoding SRA1 transcript still containing an intron-1 sequence. Using targeted RT-PCR approaches, we confirmed the presence in breast cancer cell lines of SRA1 RNAs containing a full as well as a partial intron-1 sequence and established that the relative proportion of these RNAs varied within breast cancer cell lines. Using a "minigene" strategy, we also showed that artificial RNAs containing the SRA1 intron-1 sequence are alternatively spliced in breast cancer cell lines. Interestingly, the splicing pattern of the minigene products parallels the one of the endogenous SRA1 transcripts. Altogether, our data suggest that the primary genomic sequence in and around intron-1 is sufficient to lead to a differential splicing of this intron. We propose that alternative splicing of intron-1 is one mechanism used by breast cancer cells to regulate the balance between coding and functional noncoding SRA1 RNAs.

The role of transforming growth factor beta signaling in messenger RNA stability.

Transforming growth factor beta (TGF-beta) is a biologically multipotent regulatory protein implicated in functions that include the regulation of cellular growth, differentiation, extracellular matrix formation, and wound healing. It also plays a role in the pathologies of Alzheimer's disease, cancer and autoimmune disorders. TGF-beta modulates gene expression by affecting transcriptional activation and mRNA turnover rate. Steady-state mRNA levels depend on both the transcriptional activity and mRNA half-life. The stability of mRNA can be modified by the binding of trans-acting factors to cis-elements on the message. These can protect the mRNA from cleavage by RNAses, or they may promote mRNA cleavage. Changes in mRNA stability can lead to changes in the proteome and subsequently in cellular metabolism. The SMAD family of proteins has been implicated in the transduction of the TGF-beta signal, where they regulate transcriptional activity. This review attempts to provide new insights into the role played by TGF-beta in the regulation of mRNA turnover.

Myogenic signaling by lithium in cardiomyoblasts is Akt independent but requires activation of the beta-catenin-Tcf/Lef pathway.

Rat H9c2 cardiomyoblasts can proliferate and maintain an undifferentiated state in the presence of serum. These cardiomyoblasts have been used as a cellular model to study myogenic differentiation after serum withdrawal. Here, we examined the effects of lithium, a known inhibitor of glycogen synthase kinase-3beta and activator of Wnt pathway in myogenic differentiation. We show that in the presence of serum, lithium induced the differentiation of H9c2 cells as measured by multinucleated myotube formation and expression of the muscle-specific proteins, myogenin and skeletal alpha-actin. This differentiation was preceded by nuclear accumulation of beta-catenin, which was associated with increased Tcf/Lef-dependent transcription. We also observed that lithium mediated the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target Akt. Inhibition of PI3-kinase by LY294002 and over-expression of dominant-negative PI3-kinase caused a marked reduction in beta-catenin levels. This inhibition was associated with decreased beta-catenin-Tcf/Lef-dependent transcription, lack of multinucleated myotube formation, and expression of the muscle-specific proteins. In contrast, expression of dominant-negative Akt failed to inhibit the effects of lithium. We conclude that the capacity of lithium to overcome the inhibitory effects of serum and to induce the differentiation of H9c2 cardiomyoblasts is mediated, in part, by the stabilization and nuclear translocation of beta-catenin in a PI3-kinase-dependent but Akt-independent manner. Once activated, beta-catenin then interacts with the Lef/Tcf complex to regulate expression of myogenic-inducing genes.

Late Simian virus 40 transcription factor is a target of the phosphoinositide 3-kinase/Akt pathway in anti-apoptotic Alzheimer's amyloid precursor protein signalling.

The association of familial Alzheimer's disease (FAD) with mutations in Alzheimer's amyloid precursor protein (APP) suggests important functions for APP in the central nervous system. Mutations in APP impair its function to confer resistance to apoptosis in cells under stress, and this may contribute to neurodegeneration in Alzheimer's disease (AD) brain, but the mechanisms involved are unknown. We examined the role of the late Simian virus 40 transcription factor (LSF), in anti-apoptotic APP pathways. We show that in APP-deficient B103 cells, expression of wild-type human APP (hAPPwt), but not of FAD-mutant APP, inhibited staurosporine (STS)-induced apoptosis. This inhibition was further enhanced by expression of LSFwt, although LSFwt alone was not sufficient to inhibit STS-induced apoptosis. In contrast, expression of dominant-negative LSF led to a marked increase in STS-induced cell death that was significantly blocked by hAPPwt. These effects of APP were accompanied by LSF nuclear translocation and dependent gene transcription. The activation of LSF is dependent on the expression of hAPPwt and is inhibited by the expression of dominant-negative forms of either phosphoinositide 3-kinase or Akt. These results demonstrate that LSF activation is required for the neuroprotective effects of APP via phosphoinositide 3-kinase/Akt signalling. Alterations in this pathway by aberrations in APP and/or LSF could promote neuronal loss in AD brain, due to secondary insults. Thus a link is established between APP and LSF and AD.