AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects

Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases.     

Moulting hormone level during the last instar of Galleria mellonella larva

Using radioimmunoassay the moulting hormone titres of the greater wax moth were determined during the last larval instar. Two peaks were observed, one when the larvae start to spin and another just before the pupation. The second peak exhibits the higher MH level, equivalent to 3600 ng/g ecdysterone. By TLC-RIA analysis three compounds were detected: ecdysone, ecdysterone and a very polar metabolite (VPM). The pattern of MHs during the last larval instar is described and the possible changes in the activity of enzymes of MH metabolism and ecdysone-ecdysterone conversion is discussed.     

Phosphorylation of Y14 modulates its interaction with proteins involved in mRNA metabolism and influences its methylation

The multicomponent exon junction complex (EJC) is deposited on the spliced mRNA during pre-mRNA splicing and is implicated in several post-splicing events, including mRNA export, nonsense-mediated mRNA decay (NMD), and translation control. This report is the first to identify potential post-translational modifications of the EJC core component Y14. We demonstrate that Y14 is phosphorylated at its repeated arginine/serine (RS) dipeptides, likely by SR protein-specific kinases. Phosphorylation of Y14 abolished its interaction with EJC components as well as factors that function downstream of the EJC. A non-phosphorylatable Y14 mutant was equivalent to the wild-type protein with respect to its association with spliced mRNA and its ability in NMD activation, but the mutant sequestered EJC and NMD factors on ribosome-containing mRNA ribonucleoproteins (mRNPs). We therefore hypothesize that phosphorylation of Y14 occurs upon completion of mRNA surveillance, leading to dissociation of Y14 from ribosome-containing mRNPs. Moreover, we found that Y14 is possibly methylated at multiple arginine residues in the carboxyl-terminal domain and that methylation of Y14 was antagonized by phosphorylation of RS dipeptides. This study reveals antagonistic post-translational modifications of Y14 that may be involved in the remodeling of Y14-containing mRNPs.     

Exon selection in alpha-tropomyosin mRNA is regulated by the antagonistic action of RBM4 and PTB

RNA-binding motif protein 4 (RBM4) has been implicated in the regulation of precursor mRNA splicing. Using differential display analysis, we identified mRNAs that associate with RBM4-containing messenger RNPs in vivo. Among these mRNAs, alpha-tropomyosin (alpha-TM) is known to exhibit a muscle cell type-specific splicing pattern. The level of the skeletal muscle-specific alpha-TM mRNA isoform partially correlated with that of RBM4 in human tissues examined and could be modulated by ectopic overexpression or suppression of RBM4. These results indicated that RBM4 directly influences the expression of the skeletal muscle-specific alpha-TM isoform. Using minigenes, we demonstrated that RBM4 can activate the selection of skeletal muscle-specific exons, possibly via binding to intronic pyrimidine-rich elements. By contrast, the splicing regulator polypyrimidine tract binding protein (PTB) excluded these exons; moreover, RBM4 antagonized this PTB-mediated exon exclusion likely by competing with PTB for binding to a CU-rich element. This study suggests a possible mechanism underlying the regulated alternative splicing of alpha-TM by the antagonistic splicing regulators RBM4 and PTB.     

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.     

RNA-binding Motif Protein 4 Translocates to Cytoplasmic Granules and Suppresses Translation via Argonaute2 during Muscle Cell Differentiation

The RNA-binding motif protein 4 (RBM4) plays multiple roles in mRNA metabolism, including translation control. RBM4 suppresses cap-dependent translation but activates internal ribosome entry site-mediated translation. Because of its high expression level in muscle and heart, we investigated the function of RBM4 in myoblast cells. Here, we demonstrate that RBM4 is phosphorylated and translocates to the cytoplasm in mouse C2C12 cells upon cell differentiation. Notably, RBM4 is transiently deposited into cytoplasmic granules containing microtubule assembly factors as well as poly(A)⁺ RNAs. Moreover, RBM4 colocalizes with the components of micro-ribonucleoproteins, including the Argonaute2 (Ago2) protein, during muscle cell differentiation. RBM4 interacts directly with Ago2 and may recruit Ago2 to suppress translation of target mRNAs. Furthermore, RBM4 selectively associates with muscle cell-specific microRNAs and potentiates their translation repression activity by promoting micro-ribonucleoprotein association with target mRNAs. Altogether, our results suggest that RBM4 translocates to the cytoplasm and participates in translation suppression during muscle cell differentiation.     

Hepatitis B virus X protein differentially regulates cell cycle progression in X-transforming versus nontransforming hepatocyte (AML12) cell lines.

Hepatitis B virus (HBV) X protein (pX) is implicated in hepatocarcinogenesis of chronically infected HBV patients. To understand mechanism(s) of pX-mediated cellular transformation, we employed two tetracycline-regulated, pX-expressing cell lines, constructed in AML12 immortalized hepatocytes: one a differentiated (3pX-1) and the other a de-differentiated (4pX-1) hepatocyte cell line. Only 3pX-1 cells undergo pX-mediated transformation, via sustained Ras-Raf-mitogen-activated protein kinase pathway activation. pX-nontransforming 4pX-1 cells display sustained, pX-dependent JNK pathway activation. To understand how pX mediates different growth characteristics in 3pX-1 and 4pX-1 cells, we report, herein, comparative cell cycle analyses. pX-transforming 3pX-1 cells display pX-dependent G(1), S, and G(2)/M progression evidenced by cyclin D(1), A, and B(1) induction, and Cdc2 kinase activation. pX-nontransforming 4pX-1 cells display pX-dependent G(1) and S phase entry, followed by S phase pause and absence of Cdc2 kinase activation. Interestingly, 4pX-1 cells exhibit selective pX-induced expression of cyclin-dependent kinase inhibitor p21(Cip1), tumor suppressor p19(ARF), and proapoptotic genes bax and IGFBP-3. Despite the pX-mediated induction of growth arrest and apoptotic genes and the absence of pX-dependent Cdc2 activation, 4pX-1 cells do not undergo pX-dependent G(2)/M arrest or apoptosis. Nocodazole-treated, G(2)/M-arrested 4pX-1 cells exhibit pX-dependent formation of multinucleated cells, similar to human T-cell lymphotropic virus type I Tax-expressing cells. We propose that in 4pX-1 cells, pX deregulates the G(2)/M checkpoint, thus rescuing cells from pX-mediated apoptosis.     

Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10³ and 10⁴ times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D⁵) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K⁵). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine.