Identification of novel ERF transcription factor genes in papaya and analysis of their expression in different tissues and in response to the plant defense inducer benzothiadiazole (BTH)
Contributors: Miguel A. Vallejo-Reyna, Jorge M. Santamaría, Luis C. Rodríguez-Zapata, Virginia A. Herrera-Valencia, Santy Peraza-Echeverria
... In the present study, we isolated and characterized four novel papaya ERF transcription factor cDNAs (CpERF5–CpERF8) with homology to ERF genes involved in disease resistance, and analyzed their expression in different tissues and in response to the plant defense inducer benzothiadiazole (BTH). Interestingly, CpERF7 was the most responsive to BTH. Further examination revealed that it was also up-regulated by the bacterial pathogen Pseudomonas syringae and encoded a nuclear protein with transcription factor activity. These novel insights on CpERF7 have provided a valuable platform to continue dissecting the BTH signal transduction pathway that may lead to engineering disease resistance in papaya.
A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation
Contributors: Brenna Osborne, Tsun-Wen Yao, Xin Maggie Wang, Yiqian Chen, L. Damla Kotan, Naveed A. Nadvi, Mustafa Herdem, Geoffrey W. McCaughan, John D. Allen, Denise M.T. Yu
... Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells.
Single molecule approaches to monitor the recognition and resection of double-stranded DNA breaks during homologous recombination
Contributors: Carolina Carrasco, Mark S. Dillingham, Fernando Moreno-Herrero
... The fate of a cell depends on its ability to repair the many double-stranded DNA breaks (DSBs) that occur during normal metabolism. Improper DSB repair may result in genomic instability, cancer, or other genetic diseases. The repair of a DSB can be initiated by the recognition and resection of a duplex DNA end to form a 3′-terminated single-stranded DNA overhang. This task is carried out by different single-strand exonucleases, endonucleases, and helicases that work in a coordinated manner. This manuscript reviews the different single-molecule approaches that have been employed to characterize the structural features of these molecular machines, as well as the intermediates and products formed during the process of DSB repair. Imaging techniques have unveiled the structural organization of complexes involved in the tethering and recognition of DSBs. In addition to that static picture, single molecule studies on the dynamics of helicase-nuclease complexes responsible for the processive resection of DSBs have provided detailed mechanistic insights into their function.
Contributors: Moshe Ben-David, Grzegorz Wieczorek, Mikael Elias, Israel Silman, Joel L. Sussman, Dan S. Tawfik
... Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca2+. We describe the crystal structures of active-site mutants bearing mutations at position 115. The histidine at this position acts as a base to activate the lactone-hydrolyzing water molecule. Mutations to Trp or Gln indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-Å upward displacement towards the enzyme's surface of the catalytic Ca2+ in the His115 mutants and configurational changes in the ligating side chains and water molecules, relative to the wild-type enzyme. Biochemical analysis and molecular dynamics simulations suggest that this alternative, upward metal mode mediates the promiscuous hydrolysis of organophosphates. The upward Ca2+ mode observed in the His115 mutants also appears to mediate the wild type's paraoxonase activity. However, whereas the upward mode dominates in the Trp115 mutant, it is scarcely populated in wild type. Thus, the plasticity of active-site metal ions may permit alternative, latent, promiscuous activities and also provide the basis for the divergence of new enzymatic functions.