UV exposure in tidal marshes strongly depends on the sea level.

<p>The schematic qualitatively depicts the fluctuations in UV exposure for <i>N. vectensis</i> as a function of the sea level. Most of the DNA-damaging UV light drops within the first 1–2 meters to about 10% of UV irradiation present at the water surface. DNA damage caused by UV-irradiation decreases by several magnitudes within the first meters of coastal sea water.</p

UV-induced germ cell death in adult starlet sea anemones.

<p>A The immunofluorescence image depicts the germ cell compartment of the mesenteria in a transverse section of an adult polyp. The epithelium of the body column consisting of ectoderm and endoderm is visible in the lower left corner. TUNEL staining (green) indicates the number of apoptotic cells in the mesenteria of adult polyps after 1200 J/m² UV irradiation. Massive fragmentation of genomic DNA is detected in the germ cell compartment, whereas only few TUNEL positive cells were visible in the epithelium. Genomic DNA is stained with propidium iodide (red). Scale bar 150 µm. B Cell death in the germ cell compartment was quantified following different doses of UV irradiation by counting all TUNEL-positive gametes versus the total number of gametes. Error bars: standard deviation; * equals P<0.05, ** equals P<0.01, *** equals P<0.001. C Electromobility shift assay (EMSA) with whole protein lysates of irradiated (UV- or γ-IR) or non-irradiated adult <i>N. vectensis</i> polyps revealed DNA-binding activity for the canonical p53 binding sequence. Lysate was either incubated with a radioactively labeled control oligonucleotide with random DNA sequence (left section of the EMSA) or with a radioactively labeled oligonucleotide containing the consensus p53 DNA binding sequence (right section of the EMSA). Two different protein complexes (arrow and arrowhead) were detected using whole animal lysates from adult polyps when incubated with the p53 DNA binding sequence encoding oligonucleotide. SAOS-2 cells transfected with human TAp63α or vector only were analyzed in parallel as controls.</p

Protein sequence comparisons of the DNA binding domains of the human and <i>N. vectensis</i> p53 protein family.

<p>A Protein sequence comparison of the DNA binding domain sequences between the human (hu) and <i>N. vectensis</i> p53 protein family (nv). Black circles indicate all amino acids with direct contact to DNA identified in human p53 and black rectangles mark amino acids that complex a zinc atom. All of these amino acids are conserved in all <i>N. vectensis</i> p53 paralogs. B Protein sequence identities of the DNA binding domain within the p53 protein family of the species <i>H. sapiens</i> and <i>N. vectensis</i> are depicted. The thick double arrow indicates the range of protein sequence identities of all possible cross species sequence comparisons.</p

Phylogeny of p53-like proteins.

<p>A Schematic depicting a rooted phylogenetic tree for the p53 protein family of different species (nvpEC53, nvp63, nvpVS53: <i>Nematostella vectensis</i>, Cnidaria. Hup53, hup63, hup73: <i>Homo sapiens</i>, Deuterostomia. Lfp53: <i>Loligo forbesi</i>; map63/p73: <i>Mya arenaria</i>, Ecdysozoa. Dmp53: <i>Drosophila melanogaster</i> and CEP-1: <i>Caenorhabditis elegans</i>, Lophotrochozoa). B An unrooted phylogenetic tree with bootstrap values from 1000 iterations is depicted.</p

UV irradiation induced apoptosis is nvp63 dependent.

<p>A Schematic representation of the experimental protocol for the siRNA treatment of adult polyps. 24 h starved adult polyps were incubated in artificial sea water containing 10 µM double stranded siRNA for 48 h at room temperature. Subsequently, protein expression was analyzed by Western blotting. B Western blots depict the expression of nvvasa, nvGAPDH, and nvβ-actin after treatment of adult polyps with the indicated siRNA. Bar graphs indicate the amount of expression relative to cytoplasmic β-actin and after normalization to the scrambeled siRNA value. C Nvvasa1 is efficiently suppressed by RNA interference in the experimental setup. Adult polyps were incubated with a non-vasa siRNAs, a mixture of nvvasa1 and nvvasa2 siRNAs in equal amounts, or with nvvasa1 siRNAs or nvvasa2 siRNAs alone. The relative signal intensities normalized to nvGAPDH are visualized in the bar graphs below the Western blot signals. D Quantitative RT-PCR determined the knock down of nvp63 mRNA in adult polyps by the siRNA nvp63si1. The amount of nvp63 mRNA was normalized to nv18S-rRNA expression. E UV-induced apoptosis was determined at 12 J/m² after incubation of adult polyps with the siRNAs indicated. Nvp63 specific siRNAs suppressed UV-irradiation induced death of gametes. * equals P<0.05, ** equals P<0.01. F Cell death was detected by TUNEL staining (green) on mesenterial tissue sections from adult polyps incubated with either scrambled siRNA or nvp63 specific siRNAs. Genomic DNA was visualized with Hoechst 3342 (colored in red).</p

Nvp63 drives gene transcription in a heterologous expression system.

<p>A The direct binding for nvp63, human TAp63α, and human TAp63γ to the consensus p53 DNA binding sequence was determined <i>in vitro</i>. Lysates of SAOS-2 cells transfected with myc-tagged nvp63, TAp63α, or TAp63γ were incubated with biotinylated DNA encoding the canonical p53 binding sequence or random sequence. DNA-protein complexes bound to streptavidin were detected with an anti-myc-tag antibody. The value determined for the binding to the random DNA sequence was subtracted from the value obtained for the binding to the p53 binding sequence for each protein lysate. All obtained values are normalized to control. The dashed line indicates the signal obtained for the lysate with the random DNA sequence and the signal obtained with the p53 consensus DNA binding sequence. B Co-transfection of a luciferase reporter plasmid containing several repeats of the canonical p53 binding sequence allowed the determination of the level of nvp63-mediated transactivation. The mutation R190A in nvp63 abolished while the deletion of the C-terminal 34 amino acids (ΔC) induced reporter gene transcription. A schematic depicts the nvp63 protein and mutated variants. The transactivation domain (TAD), the DNA binding domain (DBD), the oligomerization domain (OD), and the transactivation inhibitory domain (TID) are boxed in yellow, red, green and light grey respectively. The black triangle and bar in the DNA binding domain indicates the position of the point mutation (R190A). C UV irradiation (1200 J/m²) of nvp63 expressing SAOS-2 cells increased the transactivation ability of nvp63. The fold induction in nvp63-transfected SAOS-2 cells is normalized to controls.</p

Interference-Free Proteome Quantification with MS/MS-based Isobaric Isotopologue Detection

Chemical labeling of peptides prior to shotgun proteomics allows relative quantification of proteins in biological samples independent of sample origin. Current strategies utilize isobaric labels that fragment into reporter ions. However, quantification of reporter ions results in distorted ratio measurements due to contaminating peptides that are co-selected in the same precursor isolation window. Here, we show that quantitation of isobaric peptide fragment isotopologues in tandem mass spectra reduces precursor interference. The method is based on the relative quantitation of isobaric isotopologues of dimethylated peptide fragments in tandem mass spectra following higher energy collisional dissociation (HCD). The approach enables precise quantification of a proteome down to single spectra per protein and quantifies >90% of proteins in a MudPIT experiment and accurately measures proteins in a model cell line for cystic fibrosis

Quantification of CFTR, Hsc70 and Hsp90 in CFTR-containing complexes.

<p><b>A.</b> Absolute abundance (ng/µl) of Hsp90 calculated by ¹⁵N protein labeling, AQUA labeling and Western blotting (WB). <b>B.</b> Absolute abundance (ng/µl) of Hsc70 calculated by ¹⁵N protein labeling, AQUA labeling and Western blotting (WB). In all panels, data is shown as mean ± SD, n≥3.</p

Quantification of ΔF508-CFTR interaction with core chaperones following temperature shift

<p>. <b>A.</b> Western blot analysis of HEK293 cells stably expressing ΔF508-CFTR cultured at 37°C or 30°C in the presence of 50 μM cyclohexamide (CHX) or vehicle control for the indicated time. <b>B.</b> Absolute quantification of ΔF508 CFTR and interacting chaperones at 37°C (black) or 30°C for 16 h (white). Absolute protein abundance of CFTR, Hsp90, Hsc70, and Hsp40 in CFTR-containing complexes is shown and expressed in pmols. <b>C.</b> Immunoblot and densitometric analysis for CFTR, Hsp90, Hsc/p70 and Hsp40 in CFTR-containing immunoprecipitates. In the densitometric analysis, the relative protein amount is shown in arbitrary units (a.u.). In all panels, data is shown as mean ± SD, n = 3 and asterisks represent p value <0.05 as determined by two-tailed t-test using the ΔF508-CFTR at 37°C sample as the reference.</p

Minimal sequential ordering of intra- and inter-domain folding events responsible for CFTR folding and trafficking.

<p>Intra-domain folding of NBD1 is dictated by the Hsp90 system (step 1). A structural rearrangement occurs in response to the binding of cytoplasmic loop 4 (CL4) to the F508 containing hydrophobic pocket present WT NBD1 (step 2). The binding of CL4 provides a stabilizing effect on NBD1, releasing Hsp90 and promoting H8–H9 helix-coil transition. This H8–H9 transition would expose the NBD2-binding interface of NBD1 and allow NBD1 to ‘chaperone’ <i>in trans</i> the folding of NBD2 (step 3).</p