We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery.

In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. We have developed a cell-free system capable of processing and joining noncompatible DNA ends. The system had key features of NHEJ in vivo, including dependence on Ku, DNA-PKcs, and XRCC4/Ligase4. The NHEJ reaction had striking properties. Processing of noncompatible ends involved polymerase and nuclease activities that often stabilized the alignment of opposing ends by base pairing. To achieve this, polymerase activity efficiently synthesized DNA across discontinuities in the template strand, and nuclease activity removed a limited number of nucleotides back to regions of microhomology. Processing was suppressed for DNA ends that could be ligated directly, biasing the reaction to preserve DNA sequence and maintain genomic integrity. DNA sequence internal to the ends influenced the spectrum of processing events for noncompatible ends. Furthermore, internal DNA sequence strongly influenced joining efficiency, even in the absence of processing. These results support a model in which DNA-PKcs plays a central role in regulating the processing of ends for NHEJ.

Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy but cause high incidence of angioedema. We examined whether dual inhibition of angiotensin AT1 receptor (ARB) and NEP (ARB-NEPI, valsartan-candoxatril) provides similar efficacy to omapatrilat without the risk of angioedema. Activity of test compounds at the targets was assayed using fluorescence-based enzyme assays (ACE, NEP, aminopeptidase P) or competition binding assays (AT1). Target engagement in vivo (ACE, AT1, and NEP) was quantified by measuring inhibition of angiotensin-pressor responses and potentiation of atrial natriuretic peptide-induced urinary cyclic guanosine monophosphate (cGMP) output in rats. Tracheal plasma extravasation (TPE) was used as a surrogate to assess propensity of compounds to promote upper airway angioedema. Antihypertensive efficacy in renin-dependent and -independent states was measured in spontaneously hypertensive rats and deoxycorticosterone acetate salt hypertensive rats, respectively. Administration of omapatrilat and coadministration of valsartan and candoxatril blocked angiotensin induced vasopressor responses and potentiated atrial natriuretic peptide-induced increase in urinary cGMP output. In spontaneously hypertensive rats, valsartan, omapatrilat, and valsartan-candoxatril combination all produced reduction in blood pressure to a similar extent, whereas candoxatril was ineffective. In deoxycorticosterone acetate rats, omapatrilat, candoxatril, and valsartan-candoxatril combination but not valsartan produced reduction in blood pressure. Antihypertensive doses of omapatrilat produced robust increases in TPE; by contrast, valsartan, candoxatril, or their combination did not increase TPE. Pretreatment with icatibant, a bradykinin B2 antagonist, abolished omapatrilat-induced TPE but not its antihypertensive effects. On the background of NEP inhibition, suppression of the renin-angiotensin system through ARB and ACE inhibition shows a similar antihypertensive efficacy but exerts differential effects on bradykinin metabolism and TPE indicative of reduced risk of angioedema. Thus, dual AT1 receptor blockade and NEP inhibition is potentially an attractive approach to retain the excellent antihypertensive effects of omapatrilat but with a superior safety profile.

Nonhomologous end-joining (NHEJ) repairs DNA doublestrand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence. Nonhomologous end-joining (NHEJ)2 preserves chromosomal integrity by repairing DNA double-strand breaks. Processing of DNA ends is a key step in NHEJ since double-strand breaks often create ends that are not directly ligatable. For example, ionizing radiation generates nucleotides with aberrant structures such as 3Ј-phosphate or 3Ј-phosphoglycolate groups, which must be removed by nuclease activity before ligation can occur.Processing of DNA ends in vivo has been studied in the context of V(D)J recombination. The RAG1/RAG2 dimer cleaves two sites in the immunoglobulin locus to create two hairpin coding ends and two blunt signal ends (1). Nuclease activity opens the hairpin ends to leave 3Ј overhangs, which are filled in to generate P-nucleotide addition (2). To fill in 3Ј overhangs, a polymerase must synthesize DNA from a primer on the opposing end. Nucleotide deletion also occurs, but nuclease activity is limited to less than 20 nucleotides, often back to regions of microhomology (3, 4). Strikingly, the blunt signal ends are joined without processing. Thus, the NHEJ reaction is strongly biased toward the preservation of DNA sequence, suppressing processing if it is not needed and limiting the extent of processing when it is required.The NHEJ reaction requires core proteins and processing enzymes (5). The core proteins include Ku, DNA-PKcs, XRCC4/Ligase IV (XL), and Cernunnos (also named XRCC4-like factor, XLF) (6, 7). Proposed processing enzymes include the nuclease Artemis (8) and DNA polymerases and (9 -12). The biochemical properties of these proteins suggest that NHEJ repairs double-strand breaks in an ordered series of steps. Ku binds to DNA ends and translocates inward, recruiting DNAPKcs (13, 14). DNA-PKcs brings the ends together, activating its kinase activity (15). DNA-PKcs then phosphorylates itself and its target proteins (16). The ends are processed if necessary, and XL catalyzes the final ligation step (17).Important questions remain unanswered. NHEJ requires DNA-PKcs kinase activity (18), but it is not known whether the kinase activity aff...

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Dual inhibition of angiotensin-converting enzyme (ACE) and neprilysin (NEP) by drugs such as omapatrilat produces superior antihypertensive efficacy relative to ACE inhibitors but is associated with a higher risk of life-threatening angioedema due to bradykinin elevations. We hypothesized that dual AT 1 (angiotensin II type 1 receptor) blockade and NEP inhibition with a single molecule would produce similar antihypertensive efficacy to omapatrilat without the risk of angioedema since ACE (the rate limiting enzyme in bradykinin metabolism) would remain uninhibited. Merging the structures of losartan (an AT 1 antagonist) and thiorphan (a NEP inhibitor) led to the discovery of a novel series of orally active, dual AT 1 antagonist/ NEP inhibitors (ARNIs) exemplified by compound 35 (TD-0212). In models of renin-dependent and-independent hypertension, 35 produced blood pressure reductions similar to omapatrilat and combinations of AT 1 receptor antagonists and NEP inhibitors. Upper airway angioedema risk was assessed in a rat tracheal plasma extravasation (TPE) model. Unlike omapatrilat, 35 did not increase TPE at antihypertensive doses. Compound 35 therefore provides the enhanced activity of dual AT 1 /NEP inhibition with a potentially lower risk of angioedema relative to dual ACE/NEP inhibition.

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