Linking functions: an additional role for an intrinsically disordered linker domain in the transcriptional coactivator CBP

The multi-domain transcriptional coactivators CBP/p300 integrate a multitude of signaling inputs, interacting with more than 400 proteins via one or more of their globular domains. While CBP/p300 function is typically considered in terms of these structured domains, about half of the protein consists of intrinsically disordered regions (IDRs) of varying length. However, these IDRs have only been thought of as linkers that allow flexible spatial arrangement of the structured domains, but recent studies have shown that similar IDRs mediate specific and critical interactions in other proteins. To examine the roles of IDRs in CBP, we performed yeast-two-hybrid screenings of placenta and lung cancer cDNA libraries, which demonstrated that the long IDR linking the KIX domain and bromodomain of CBP (termed ID3) can potentially bind to several proteins. The RNA-binding Zinc-finger protein 106 (ZFP106) detected in both libraries was identified as a novel substrate for CBP-mediated acetylation. Nuclear magnetic resonance (NMR) spectroscopy combined with cross-linking experiments and competition-binding assays showed that the fully disordered isolated ID3 transiently interacts with an IDR of ZFP106 in a fashion that disorder of both regions is maintained. These findings demonstrate that beside the linking function, ID3 can also interact with acetylation substrates of CBP

Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade

Quantification of Intrinsically Disordered Proteins: A Problem Not Fully Appreciated

Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial Qubit(TM) Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions

Expanding the DNA alphabet in the fruit fly: Uracil enrichment in genomic DNA

DNA integrity is under the control of multiple pathways of nucleotide metabolism and DNA damage recognition and repair. Unusual sets of protein factors involved in these control mechanisms may result in tolerance and accumulation of non-canonical bases within the DNA. We investigate the presence of uracil in genomic DNA of Drosophila melanogaster. Results indicate a developmental pattern and strong correlations between uracil-DNA levels, dUTPase expression and developmental fate of different tissues. The intriguing lack of the catalytically most efficient uracil-DNA glycosylase in Drosophila melanogaster may be a general attribute of Holometabola and is suggested to be involved in the specific characteristics of uracil-DNA metabolism in these insects. © 2013 Landes Bioscience

Structural disorder and local order of hNopp140

Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here we investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. Structural disorder of hNopp140 is apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatography, 1HNMR spectrum and differential scanning calorimetry scan. hNopp140 has a significant tendency to aggregate and the change of its circular dichroism spectrum in the presence of 0–80% TFE suggests a tendency to formlocal helical structures.Wide-line NMRmeasurements suggest the overall disordered character of the protein. In all, our data suggest that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB)

Tolerance and stability of uracil-containing DNA in <i>D. melanogaster</i>.

<p>(A) Dose-response curve upon FdUR treatment followed by Alamar Blue assay. (B) FdUR leads to uracil accumulation in DNA of <i>Drosophila</i> S2 cells. Data indicate that increased level of uracil is well-tolerated in <i>Drosophila</i>, but not in human cells. Data are presented as mean ± s.e.m. (C) <i>Drosophila</i> S2 (top panels) and human HeLa cells (bottom panels) were transfected with normal plasmid (left panels) or uracil-containing plasmid (right panels). Expression of YFP in <i>Drosophila</i> S2 cells or dsRedMonomer in HeLa cells indicates stability of the DNA. (D) Microinjection of uracil-plasmid into <i>Drosophila</i> embryo. Non-injected embryos served as control sample.</p

Silencing of dUTPase in <i>Drosophila</i> larvae and pupae.

<p>Western blots in (A) show that the protein level of dUTPase is under detection limit in silenced animals. Actin served as loading control. (B) Curves show the relative number of silenced and non-silenced animals that have undergone puparium formation at the given time point after egg deposition. Inflection points of the curves represent the mean time of puparium formation characteristic for the given population. dUTPase silencing did not perturb the time interval required for puparium formation. (C) Graph shows the number of counted dead animals relative to number of hatched curly winged control flies. Among these dead animals, three groups with distinct morphological traits characteristic for wandering larvae (w3L), prepupae (preP), and pupal stage P5 (P5) were identified and counted. (D) Genomic uracil content of dUTPase silenced and control tissues from 3^rd larvae.</p

Occurrence of genes encoding dUTPase and UNG in different insects.

<p>The gene for dUTPase is ubiquitous, but the gene of the major uracil–DNA glycosylase, ung is not encoded in the genome of Holometabola species.</p>*<p>In the genome of <i>Aedes aegypti</i> strain Liverpool, an unexpected ung sequence was found, showing very high (87%–94%) similarity to the ung gene of Comamonadaceae, a family of Proteobacteria, arguing for its bacterial origin.</p