Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility, and premature aging. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with aging. Here we show that the FoxO transcription factor DAF-16 is activated in response to DNA damage during development while the DNA damage responsiveness of DAF-16 declines with aging. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16 mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists.

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.

SummaryDNA damage causally contributes to aging and age-related diseases. Mutations in nucleotide excision repair (NER) genes cause highly complex congenital syndromes characterized by growth retardation, cancer susceptibility, and accelerated aging in humans. Orthologous mutations in Caenorhabditis elegans lead to growth delay, genome instability, and accelerated functional decline, thus allowing investigation of the consequences of persistent DNA damage during development and aging in a simple metazoan model. Here, we conducted proteome, lipidome, and phosphoproteome analysis of NER-deficient animals in response to UV treatment to gain comprehensive insights into the full range of physiological adaptations to unrepaired DNA damage. We derive metabolic changes indicative of a tissue maintenance program and implicate an autophagy-mediated proteostatic response. We assign central roles for the insulin-, EGF-, and AMPK-like signaling pathways in orchestrating the adaptive response to DNA damage. Our results provide insights into the DNA damage responses in the organismal context.

Herein, we demonstrate that with the widespread theme of residue patterning and stereochemical restraints of self-complimenting proteinogenic amino acids, a new and rich class of homomeric dipeptides exhibiting two-dimensional fluid aggregates with hierarchical ordering can be obtained. In particular, a simple way of achieving a class of functional dipeptides, wherein the first and the second residues chosen are L-/D-alanines and L-/D-leucines, has been accomplished. The supramolecules synthesized can be regarded as intermediates between polycatenars and taper-shaped amphiphiles because they possess two lipophilic segments interlinked by a peptide unit (spacer). Two pairs of enantiomers and their respective diastereomers derived from these amino acids are evidenced to self-organize into a helical columnar phase through hydrogen bonding by means of FTIR, UV/Vis, and chiroptical circular dichroism (CD) spectral analyses as well as by optical, calorimetric, electrical switching, and X-ray studies. The CD and X-ray studies have revealed that the form chirality (handedness) and the magnitude of out-of-plane fluctuations of the lattice planes of the fluid supramolecular columnar structures are solely directed by the stereochemistry encoded in the spacer. Of special significance, the less frequently found oblique helical columnar phase formed by a pair of enantiomers derived from L-/D-alanines, unlike those derived from other amino acids, exhibit ferroelectric behavior; the measured spontaneous polarization is as high as 440 nC cm(-2). Besides, all these supramolecules form stable organogels in ethanol and the CD and SEM studies on a representative gel suggest the presence of helical structures.

The Curtius rearrangement of Fmoc-amino acid azides 1 was carried out in toluene by refluxing the solution for 30 min. The resulting isocyanates 2 have been isolated as crystalline solids and are fully characterized by IR, (1)H NMR, (13)C NMR, and mass spectra. They are found to be stable for several months when stored at 4 degrees C. The acyl azides of Asp, Glu, Ser, Tyr, and Lys with side-chain protection having tert-butyl, benzyl, and Boc groups were also converted to the corresponding isocyanates 2h-m. The rearrangement of Fmoc-amino acid azides in toluene to isocyanates 2 under microwave irradiation was also accomplished. The direct exposure of solid azides to microwaves for 60 s led to the completion of the rearrangement. The resulting isocyanates, after recrystallization, were found to be analytically pure. The scale-up of the rearrangement, under microwave irradiation as tested up to 0.75 mol, posed no problems and led to the isolation of the isocyanates in 91-96% yield. The utility of isocyanates as building blocks in the synthesis of urea peptides 4 is demonstrated. Further, the coupling of isocyantes 2 directly with N,O-bis(trimethylsilyl) derivatives of amino acids 6 resulted in urea peptide acids 7 with good yield in high purity. Thus, the synthesis of urea peptide acids 7d-g containing Asp, Glu, Ser, and Tyr with a free side-chain functional group have been carried out.

Synthesis and biological evaluations of a potential dual chemotherapeutic photosensitizer for photodynamic therapy based on the first exocyclically platinated tetrapyridinoporphyrazine.

The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis.

Novel tetraplatinated metalloporphyrin-based photosensitizers (PSs) show excellent phototoxic indexes up to 5800 against HeLa cells, which is, to the best of our knowledge, the highest value reported for any porphyrin...