No abstract

Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intraamygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E-eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory. T he synthesis of new proteins within relevant neuronal circuits is widely agreed to be a basic requirement for long-term memory (LTM) storage. Translation is important for stabilizing active memories because it triggers the production of new proteins that are required for persistent molecular and synaptic changes during both consolidation (after learning) and reconsolidation (after memory reactivation). However, the role of translation in memory formation has been explored only in the context of overall cellular protein translation. There are at least two forms of protein synthesis that could in principle be exploited for either memory consolidation or reconsolidation. The primary mode of translation initiation requires formation of a multiprotein complex of eukaryotic initiation factors (eIFs) bound to the 5′ methylated-GTP cap of target mRNAs (1, 2). Specifically, the interaction between eIF4E and eIF4G facilitates eIF4A RNA helicase activity, recruitment of the 40S ribosomal subunit, scanning, and peptide elongation (3, 4). Molecules that block the formation of eIF4F (eIF4E + eIF4G + eIF4A), such as the endogenous regulator 4E-binding protein, which binds to and sequesters eIF4E, therefore effectively inhibits cap-dependent translation. Likewise, the small molecule, 4EGI-1, which selectively disrupts eIF4E-eIF4G interactions (eIF4F formation) in vitro (5), also inhibits cap-dependent translation. The second route that mRNAs can be translated occurs via internal ribosomal entry sites (IRES), which are unaffected by disruptions to the 5′ cap translation machinery, such as blockade of eIF4E-eIF4G interactions (5). A role for eIF4E-eIF4G interactions during hippocampal synaptic plasticity has been shown (6-8), but they have not yet been demonstrated for memory formation. The ability to dissociate mechanisms of translation control is relevant to the study of associative learning because little is known about the relative roles of cap-dependent and IRES-mediated translation in mammalian brain function. For example, there is evidence that an IRES mediates translation of fragile X mental retardation protein, a protein that is absent in ...

Symmetrical N,N’-diarylureas: 1,3-bis(3,4-dichlorophenyl)-, 1,3-bis[4-chloro-3(trifluoromethyl) phenyl]- and 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea, were identified as potent activators of the eIF2α kinase heme regulated inhibitor. They reduce the abundance of the eIF2·GTP·tRNAiMet ternary complex and inhibit cancer cell proliferation. An optimization process was undertaken to improve their solubility while preserving their biological activity. Non-symmetrical hybrid ureas were generated by combining one of the hydrophobic phenyl moieties present in the symmetrical ureas with the polar 3-hydroxy-tolyl moiety. O-alkylation of the later added potentially solubilizing charge bearing groups. The new non-symmetrical N,N’-diarylureas were characterized by ternary complex reporter gene and cell proliferation assays, demonstrating good bioactivities. A representative sample of these compounds potently induced phosphorylation of eIF2α and expression of CHOP at the protein and mRNA levels. These inhibitors of translation initiation may become leads for the development of potent, non-toxic, and target specific anti-cancer agents.

For almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S ribosomal RNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (E. coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (K d s) of the amidated peptide HPVHHYQ-NH 2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH 2 exhibits moderate affinity for the A-site RNA, with an average K d value of 16 μM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493.It has been half a century since the first antibiotic penicillin was discovered and successfully applied to the treatment of infectious diseases; however, infectious diseases still remain the third-leading cause of death in the United States and the second-leading cause of death worldwide (1). Widespread and intensive use of antibiotics in hospitals and agriculture has led to significant levels of resistance (2,3). The emergence of antibiotic resistance, especially multidrug resistance, is a growing threat to human health (4). Therefore, a significant driving force exists for the development of new antimicrobial agents, especially those to combat multidrug-resistant pathogens.The majority of antibiotics used in the clinic target protein synthesis, which occurs at the core of the ribosome (5). High-resolution X-ray crystal structures of rRNA fragments, 30S and 50S subunits, and whole ribosomes, either free or complexed with antibiotics, have provided important information that will help in the design of new anti-infective compounds (6-10). † This work was supported by NIH grant AI061192. SUPPORTING INFORMATION AVAILABLEFigures S1-S4 show MALDI-TOF characterization of HPVHHYQ-NH 2 , ESI-MS data (salt dependence), and binding curve for RNase footprinting data (A1493). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 September 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH...

[structure: see text] Isothermal titration calorimetry (ITC) is used to study the thermodynamic consequences of systematically modifying the hydrophobic character of a single residue in a series of protein-binding ligands. By substituting standard and nonproteinogenic aliphatic amino acids for the C-terminal valine of the hexapeptide KKETEV, binding to the third PDZ domain (PDZ3) of the PSD-95 protein is characterized by distinct changes in the Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) parameters. One notable observation is that peptide binding affinity can be improved with a nonstandard residue.

Helix 69 of E. coli 23S rRNA has important roles in specific steps of translation, such as subunit association, translocation, and ribosome recycling. An M13 phage library was used to identify peptide ligands with affinity for helix 69. One selected sequence, NQVANHQ, was shown through a bead assay to interact with helix 69. Electrospray ionization mass spectroscopy revealed an apparent dissociation constant for the amidated peptide and helix 69 in the low micromolar range. This value is comparable to that of aminoglycoside antibiotics binding to the A site of 16S rRNA or helix 69. Helix 69 variants (human) and unrelated RNAs (helix 31 or A site of 16S rRNA) showed two- to four-fold lower affinity for NQVANHQ-NH2. These results suggest that the peptide has desirable features for development as a lead compound for novel antimicrobials.

A series of multivalent peptides, with the ability to simultaneously bind two separate PDZ domain proteins, has been designed, synthesized, and tested by isothermal titration calorimetry (ITC). The monomer sequences, linked with succinate, varied in length from five to nine residues. The thermodynamic binding parameters, in conjunction with results from mass spectrometry, indicate that a ternary complex is formed in which each peptide arm binds two equivalents of the third PDZ domain (PDZ3) of the neuronal protein PSD-95.