Expanding the genetic code of Drosophila melanogaster.

Genetic code expansion for unnatural amino acid mutagenesis has, until recently, been limited to cell culture. We demonstrate the site-specific incorporation of unnatural amino acids into proteins in Drosophila melanogaster at different developmental stages, in specific tissues and in a subset of cells within a tissue. This approach provides a foundation for probing and controlling processes in this established metazoan model organism with a new level of molecular precision.

Mechanistic differences in the transcriptional activation of p53 by 14-3-3 isoforms.

p53 maintains genome integrity by initiating the transcription of genes involved in cell-cycle arrest, senescence, apoptosis and DNA repair. The activity of p53 is regulated by both post-translational modifications and protein-protein interactions. p53 that has been phosphorylated at S366, S378 and T387 binds 14-3-3 proteins in vitro. Here, we show that these sites are potential 14-3-3 binding sites in vivo. Epsilon (epsilon) and gamma (gamma) isoforms required phosphorylation at either of these sites for efficient interaction with p53, while for sigma (sigma) and tau (tau) these sites are dispensable. Further, sigma and tau bound more weakly to p53 C-terminal phosphopeptides than did epsilon and gamma. However, the four isoforms bound tightly to di-phosphorylated p53 C-terminal peptides than did the mono-phosphorylated counterparts. Interestingly, all the isoforms studied transcriptionally activated wild-type p53. sigma and tau stabilized p53 levels in cells, while epsilon and gamma stimulated p53-DNA binding activity in vitro. Overall, the results suggest that structurally and functionally similar 14-3-3 isoforms may exert their regulatory potential on p53 through different mechanisms. We discuss the isoform-specific roles of 14-3-3 in p53 stabilization and activation of specific-DNA binding.

Molecular basis of the interactions between the p73 N terminus and p300: effects on transactivation and modulation by phosphorylation.

The transcription factor p73 belongs to the p53 family of proteins and can transactivate a number of target genes in common with p53. Here, we characterized the interaction of the p73 N terminus with four domains of the transcriptional coactivator p300 and with the negative regulator Mdm2 by using biophysical and cellular measurements. We found that, like p53, the N terminus of p73 contained two distinct transactivation subdomains, comprising residues 10-30 and residues 46-67. The p73 N terminus bound weakly to the Taz1, Kix, and IBiD domains of p300 but with submicromolar affinity for Taz2, in contrast to previous reports. We found weaker binding of the p73 N terminus to the p300 domains in vitro correlated with a significant decrease in transactivation activity in a cell line for the QS and T14A mutants, and tighter binding of the phosphomimetic T14D in vitro correlated with an increase in vivo. Further, we found that phosphorylation of T14 increased the affinity of the p73 N terminus for Taz2 10-fold. The phosphomimetic p73alpha T14D caused increased levels of transactivation.

Pygopus and Legless target Armadillo/beta-catenin to the nucleus to enable its transcriptional co-activator function.

Wnt signalling controls the transcription of genes that function during normal and malignant development. Stimulation by canonical Wnt ligands activates beta-catenin (or Drosophila melanogaster Armadillo) by blocking its phosphorylation, resulting in its stabilization and translocation to the nucleus. Here, Armadillo/beta-catenin binds to TCF/LEF transcription factors and recruits chromatin-modifying and -remodelling complexes to transcribe Wnt target genes. The transcriptional activity of Armadillo/beta-catenin depends on two conserved nuclear proteins recently discovered in Drosophila, Pygopus (Pygo) and Legless/BCL-9 (Lgs). Lgs functions as an adaptor between Pygo and Armadillo/beta-catenin, but how Armadillo/beta-catenin is controlled by Pygo and Lgs is not known. Here, we show that the nuclear localization of Lgs entirely depends on Pygo, which itself is constitutively localized to the nucleus; thus, Pygo functions as a nuclear anchor. Pygo is also required for high nuclear Armadillo levels during Wingless signalling, and together with Lgs increases the transcriptional activity of beta-catenin in APC mutant cancer cells. Notably, linking Armadillo to a nuclear localization sequence rescues pygo and lgs mutant fly embryos. This indicates that Pygo and Lgs function in targeting Armadillo/beta-catenin to the nucleus, thus ensuring its availability to TCF during Wnt signalling.

Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53.

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1-61) of p53 via an extended binding interface, with dissociation constant of 12.7 (+/- 0.7) microM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1-40) and TAD2 (residues 41-61) subdomains of p53. HmtSSB enhances intrinsic 3'-5' exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) present at 3'-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.

Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53.

Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K(d) of 1.95 +/- 0.19 microM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM-p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices alpha1 and alpha2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM-p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM-p53 interaction are discussed.

Tagging and Enriching Proteins Enables Cell-Specific Proteomics.

Cell-specific proteomics in multicellular systems and whole animals is a promising approach to understand the differentiated functions of cells and tissues. Here, we extend our stochastic orthogonal recoding of translation (SORT) approach for the co-translational tagging of proteomes with a cyclopropene-containing amino acid in response to diverse codons in genetically targeted cells, and create a tetrazine-biotin probe containing a cleavable linker that offers a way to enrich and identify tagged proteins. We demonstrate that SORT with enrichment, SORT-E, efficiently recovers and enriches SORT tagged proteins and enables specific identification of enriched proteins via mass spectrometry, including low-abundance proteins. We show that tagging at distinct codons enriches overlapping, but distinct sets of proteins, suggesting that tagging at more than one codon enhances proteome coverage. Using SORT-E, we accomplish cell-specific proteomics in the fly. These results suggest that SORT-E will enable the definition of cell-specific proteomes in animals during development, disease progression, and learning and memory.

Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal.

Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.

Molecular basis for modulation of the p53 target selectivity by KLF4.

The tumour suppressor p53 controls transcription of various genes involved in apoptosis, cell-cycle arrest, DNA repair and metabolism. However, its DNA-recognition specificity is not nearly sufficient to explain binding to specific locations in vivo. Here, we present evidence that KLF4 increases the DNA-binding affinity of p53 through the formation of a loosely arranged ternary complex on DNA. This effect depends on the distance between the response elements of KLF4 and p53. Using nuclear magnetic resonance and fluorescence techniques, we found that the amino-terminal domain of p53 interacts with the KLF4 zinc fingers and mapped the interaction site. The strength of this interaction was increased by phosphorylation of the p53 N-terminus, particularly on residues associated with regulation of cell-cycle arrest genes. Taken together, the cooperative binding of KLF4 and p53 to DNA exemplifies a regulatory mechanism that contributes to p53 target selectivity.

Structural consequences of nucleophosmin mutations in acute myeloid leukemia.

Mutations affecting NPM1 (nucleophosmin) are the most common genetic lesions found in acute myeloid leukemia (AML). NPM1 is one of the most abundant proteins found in the nucleolus and has links to the MDM2/p53 tumor suppressor pathway. A distinctive feature of NPM1 mutants in AML is their aberrant localization to the cytoplasm of leukemic cells. This mutant phenotype is the result of the substitution of several C-terminal residues, including one or two conserved tryptophan residues, with a leucine-rich nuclear export signal. The exact molecular mechanism underlying the loss of nucleolar retention, and the role of the tryptophans, remains unknown. In this study we have determined the structure of an independently folded globular domain in the C terminus of NPM1 using NMR spectroscopy, and we report that the conserved tryptophans are critical for structure. This domain is necessary for the nucleolar targeting of NPM1 and is disrupted by mutations in AML with cytoplasmic NPM1. Furthermore, we identify conserved surface-exposed lysine residues that are functionally rather than structurally important for nucleolar localization. This study provides new focus for efforts to understand the pathogenesis of AML with cytoplasmic NPM1 and may be used to aid the design of small molecules that target the C-terminal domain of NPM1 to act as novel anti-proliferative and anti-leukemia therapeutics.

The novel p53 isoform "delta p53" is a misfolded protein and does not bind the p21 promoter site.

The tumor suppressor p53 can be expressed as different isoforms because of promoter selection and mRNA editing. One isoform, "delta p53" (Delta p53), results from what would be an unusual alternative splicing of exons 7/8 of the p53 gene, conserving the reading frame and generating a novel protein with proposed transcriptional activity essential for the intra S-phase checkpoint. Here, we show that the deletion of the 66 residues that correspond to strand beta10 and the C-terminal helix of the core domain and the interconnecting linker to the tetramerization domain occurring in the Delta p53 isoform leads to a misfolded and unstable protein, prone to form soluble aggregates, which does not bind the p21 promoter site. The complex of coexpressed Delta p53 and flp53 is soluble in vitro and binds poorly to DNA. Our results provide a structural explanation for the dominant-negative effect of Delta p53 and its lack of transcriptional activity.

Pygopus residues required for its binding to Legless are critical for transcription and development.

Pygopus and Legless/Bcl-9 are recently discovered core components of the Wnt signaling pathway that are required for the transcriptional activity of Armadillo/beta-catenin and T cell factors. It has been proposed that they are part of a tri-partite adaptor chain (Armadillo>Legless>Pygopus) that recruits transcriptional co-activator complexes to DNA-bound T cell factor. Here, we identify four conserved residues at the putative PHD domain surface of Drosophila and mouse Pygopus that are required for their binding to Legless in vitro and in vivo. The same residues are also critical for the transactivation potential of DNA-tethered Pygopus in transfected mammalian cells and for rescue activity of pygopus mutant embryos. These residues at the Legless>Pygopus interface thus define a specific molecular target for blocking Wnt signaling during development and cancer.