A tale of two tails: Activation of DNA damage checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1

The DNA damage and replication checkpoint kinase Mec1/ATR is a member of the PI3-kinase related kinases that function in response to various genotoxic stresses. The checkpoint clamp 9-1-1 (Rad9-Rad1-Hus1 in S. pombe and mammals; Ddc1-Rad17-Mec3 in S. cerevisiae) executes two distinct checkpoint functions. In S. cerevisiae, DNA-bound 9-1-1 directly activates Mec1 kinase activity, a function that has not been demonstrated in other organisms. A second, conserved activity of 9-1-1 is that of TopBP1/Cut5/Dpb11 recruitment to stalled replication sites; subsequent activation of Mec1/ATR is carried out by TopBP1/Cut5/Dpb11. Biochemical studies indicate that the mode of Mec1/ATR activation by S. cerevisiae 9-1-1 is analogous to activation by S. cerevisiae Dpb11 or by vertebrate TopBP1: activation is mediated by the intrinsically disordered C-terminal tail of each activator. The relative contributions made by multiple activators of Mec1/ATR are discussed.     

Immune responsive resolvin D1 programs peritoneal macrophages and cardiac fibroblast phenotypes in diversified metabolic microenvironment

Bioactive lipid mediators derived from n-3 and n-6 fatty acids are known to modulate leukocytes. Metabolic transformation of essential fatty acids to endogenous bioactive molecules plays a major role in human health. Here we tested the potential of substrates; linoleic acid (LA) and docosahexaenoic acid (DHA) and their bioactive products; resolvin D1 (RvD1) and 12- S-hydroxyeicosatetraenoic acids (HETE) to modulate macrophage plasticity and cardiac fibroblast phenotype in presence or absence of lipid metabolizing enzyme 12/15-lipoxygenase (LOX). Peritoneal macrophages and cardiac fibroblasts were isolated from wild-type (C57BL/6J) and 12/15LOX ^−/− mice and treated with DHA, LA, 12(S)-HETE, and RvD1 for 4, 8, 12, and 24 hr. LA, DHA, 12(S)-HETE, and RvD1 elicited mRNA expression of proinflammatory markers; tumor necrosis factor-α ( Tnf-α), interleukin 6 ( IL-6), chemokine (C–C motif) ligand 2  (Ccl2), and IL-1β in wild type (WT) and in 12/15LOX ^−/− macrophages at early time point (4 hr). Bioactive immunoresolvent RvD1 lowered the levels of Tnf-α, IL-6, and IL-1β at 24 hr time point. Both DHA and RvD1 stimulated the proresolving markers such as arginase 1 ( Arg-1), chitinase-like protein 3 ( Ym-1), and mannose receptor C-type 1 in WT macrophage. RvD1 induced proresolving phenotype Arg-1 expression in both WT 12/15LOX ^−/− macrophages even in presence of 12(S)-HETE. RvD1 peaked 5LOX expression in both WT and 12/15LOX ^−/− at 24 hr time point compared with DHA. RvD1 diminished cyclooxygenase-2 but upregulated 5LOX expression in fibroblast compared with DHA. In summary, the feed-forward enzymatic interaction with fatty acids substrates and direct mediators (RvD1 and 12(S)-HETE) are responsive in determining macrophages phenotype and cardiac fibroblast plasticity. Particularly, macrophages and fibroblast phenotypes are responsive to milieu and RvD1 governs the milieu-dependent chemokine signaling in presence or absence of 12/15LOX enzyme to resolve inflammation.     

Pseudomonas putida CSV86: A Candidate Genome for Genetic Bioaugmentation

Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb) revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA^Gly, integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI) for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT) suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation.     

A human-derived probe,p82H,hybridizes to the centromeres of gorilla,chimpanzee, and orangutan

A human-derived centromeric sequence, p82H, hybridizes to DNA from gorilla, chimpanzee, pygmy chimpanzee, and orangutan. On DNA blots, multimeric ladders based on 170 or 340 bp repeat units are seen. In metaphase chromosome preparations from these species, p82H hybridizes to the centromeric region of every chromosome. p82H forms less stable hybrids with DNA from the lion-tailed macaque and does not hybridize to DNA or chromosomes of the owl monkey or the mouse.     

Expression of a novel tropomyosin isoform in axolotl heart and skeletal muscle

TPM1κ is an alternatively spliced isoform of the TPM1 gene whose specific role in cardiac development and disease is yet to be elucidated. Although mRNA studies have shown TPM1κ expression in axolotl heart and skeletal muscle, it has not been quantified. Also the presence of TPM1κ protein in axolotl heart and skeletal muscle has not been demonstrated. In this study, we quantified TPM1κ mRNA expression in axolotl heart and skeletal muscle. Using a newly developed TPM1κ specific antibody, we demonstrated the expression and incorporation of TPM1κ protein in myofibrils of axolotl heart and skeletal muscle. The results support the potential role of TPM1κ in myofibrillogenesis and sarcomeric function. J. Cell. Biochem. 110: 875–881, 2010. © 2010 Wiley‐Liss, Inc.     

Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.     

Transmembrane domain 9 of presenilin determines the dynamic conformation of the catalytic site of gamma-secretase

One of the most prominent drug targets for the treatment of Alzheimer disease is gamma-secretase, a multi-protein complex responsible for the generation of the amyloid-beta peptide. The catalytic core of the complex lies on presenilin, a multi-spanning membrane protease, the activity of which depends on two aspartate residues located in transmembrane domains 6 and 7. We have recently shown by cysteine-scanning mutagenesis that these aspartates are facing a water-filled cavity in the lipid bilayer, demonstrating how proteolytic cleavage of the substrates can be taking place within the membrane. Here, we demonstrate that transmembrane domain 9 and hydrophobic domain VII in the large cytoplasmic loop of presenilin are dynamic structural parts of this cavity. Hydrophobic domain VII is associated with transmembrane domain 7 in the membrane, probably facilitating the entrance of water molecules in the catalytic site. Transmembrane domain 9, on the other hand, exhibits a highly flexible structure, potentially involved in the transport of substrates to the catalytic site, as well as in the binding of gamma-secretase inhibitors. The conserved proline-alanine-leucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp257 and is crucial for conformational changes leading to the activation of the catalytic site. We, also, identify a unique mutant in this domain (I437C) that specifically blocks amyloid-beta peptide production without affecting the processing of the physiologically indispensable Notch substrate. Our data are finally combined to propose a model for the architectural organization and activation of the catalytic site of presenilin.     

Human Rad52 facilitates a three-stranded pairing that follows no strand exchange: a novel pairing function of the protein

Human Rad52 protein, by analogy with the genetics of yeast Rad52, is believed to mediate a pathway of homologous recombination even independent of Rad51. Current study is focused on unraveling the molecular properties of hRad52 that endow the protein such an ability. We show here that the hRad52 protein binds single-stranded DNA (ssDNA) as well as 3'- and 5'-tailed duplexes severalfold better than blunt-ended duplexes, altering the sensitivity of the bound DNA to the action of DNase I. Protein binding is sensitive to the length of the ssDNA: targets as short as a 33mer poorly bind the protein, whereas that of a 61mer and above bind the protein stably well. Such stable ssDNA-hRad52 complexes are highly competent in mediating not only the annealing of two complementary strands but also three-stranded pairing. The latter involves homologous recognition of linear duplex DNA by the ssDNA-hRad52 complex. We show that the hRad52 protein facilitates homologous recognition between ssDNA and duplex-DNA through a process that involves unwinding or transient unpairing of the interacting duplex via a novel three-stranded intermediate that does not lead to strand exchange. The results enable us to visualize a novel role for hRad52 that may model its function in a pathway requiring no hRad51.