A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated 6-(4-hydroxyphenyl)-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.     

The Arabidopsis AtOPT3 protein functions in metal homeostasis and movement of iron to developing seeds

The Arabidopsis thaliana AtOPT3 belongs to the oligopeptide transporter (OPT) family, a relatively poorly characterized family of peptide/modified peptide transporters found in archebacteria, bacteria, fungi, and plants. A null mutation in AtOPT3 resulted in embryo lethality, indicating an essential role for AtOPT3 in embryo development. In this article, we report on the isolation and phenotypic characterization of a second AtOPT3 mutant line, opt3-2, harboring a T-DNA insertion in the 5' untranslated region of AtOPT3. The T-DNA insertion in the AtOPT3 promoter resulted in reduced but sufficient AtOPT3 expression to allow embryo formation in opt3-2 homozygous seeds. Phenotypic analyses of opt3-2 plants revealed three interesting loss-of-function phenotypes associated with iron metabolism. First, reduced AtOPT3 expression in opt3-2 plants resulted in the constitutive expression of root iron deficiency responses regardless of exogenous iron supply. Second, deregulation of root iron uptake processes in opt3-2 roots resulted in the accumulation of very high levels of iron in opt3-2 tissues. Hyperaccumulation of iron in opt3-2 resulted in the formation of brown necrotic areas in opt3-2 leaves and was more pronounced during the seed-filling stage. Third, reduced AtOPT3 expression resulted in decreased accumulation of iron in opt3-2 seeds. The reduced accumulation of iron in opt3-2 seeds is especially noteworthy considering the excessively high levels of accumulated iron in other opt3-2 tissues. AtOPT3, therefore, plays a critical role in two important aspects of iron metabolism, namely, maintenance of whole-plant iron homeostasis and iron nutrition of developing seeds.     

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs

Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG. Previously, we showed that cleavage by I-CreII leaves ends (2-nt 3′ overhangs) that are characteristic of GIY-YIG endonucleases, yet it has a relaxed metal requirement like H-N-H enzymes. Here we show that I-CreII can bind DNA without an added metal ion, and that it binds as a monomer, akin to GIY-YIG enzymes. Moreover, cleavage of supercoiled DNA, and estimates of strand-specific cleavage rates, suggest that I-CreII uses a sequential cleavage mechanism. Alanine substitution of a number of residues in the GIY-YIG motif, however, did not block cleavage activity, although DNA binding was substantially reduced in several variants. Substitution of conserved histidines in the H-N-H motif resulted in variants that did not promote DNA cleavage, but retained high-affinity DNA binding—thus identifying it as the catalytic motif. Unlike the non-specific H-N-H colicins, however; substitution of the conserved asparagine substantially reduced DNA binding (though not the ability to promote cleavage). These results indicate that, in I-CreII, two catalytic motifs have evolved to play important roles in specific DNA binding. The data also indicate that only the H-N-H motif has retained catalytic ability.     

Neutralizing antibody against severe acute respiratory syndrome (SARS)-coronavirus spike is highly effective for the protection of mice in the murine SARS model

We evaluated the efficacy of three SARS vaccine candidates in a murine SARS model utilizing low-virulence Pp and SARS-CoV coinfection. Vaccinated mice were protected from severe respiratory disease in parallel with a low virus titer in the lungs and a high neutralizing antibody titer in the plasma. Importantly, the administration of spike protein-specific neutralizing monoclonal antibody protected mice from the disease, indicating that the neutralization is sufficient for protection. Moreover, a high level of IL-6 and MCP-1 production, but not other 18 cytokines tested, on days 2 and 3 after SARS-CoV infection was closely linked to the virus replication and disease severity, suggesting the importance of these cytokines in the lung pathogenicity of SARS-CoV infection.     

Protein kinase C epsilon activation delays neuronal depolarization during cardiac arrest in the euthermic arctic ground squirrel

During the pre-hibernation season, arctic ground squirrels (AGS) can tolerate 8 min of asphyxial cardiac arrest (CA) without detectable brain pathology. Better understanding of the mechanisms regulating innate ischemia tolerance in AGS has the potential to facilitate the development of novel prophylactic agents to induce ischemic tolerance in patients at risk of stroke or CA. We hypothesized that neuroprotection in AGS involves robust maintenance of ion homeostasis similar to anoxia-tolerant turtles. Ion homeostasis was assessed by monitoring ischemic depolarization (ID) in cerebral cortex during CA in vivo and during oxygen glucose deprivation in vitro in acutely prepared hippocampal slices. In both models, the onset of ID was significantly delayed in AGS compared with rats. The epsilon protein kinase C (εPKC) is a key mediator of neuroprotection and inhibits both Na⁺/K⁺-ATPase and voltage-gated sodium channels, primary mediators of the collapse of ion homeostasis during ischemia. The selective peptide inhibitor of εPKC (εV1-2) shortened the time to ID in brain slices from AGS but not in rats despite evidence that εV1-2 decreased activation of εPKC in brain slices from both rats and AGS. These results support the hypothesis that εPKC activation delays the collapse of ion homeostasis during ischemia in AGS.     

Carotenoids provide the major antioxidant defence in the globally significant N2-fixing marine cyanobacterium Trichodesmium

Photosynthetic oxygen-evolving microorganisms contend with continuous self-production of molecular oxygen and reactive oxygen species. The deleterious effects of reactive oxygen species are exacerbated for cyanobacterial nitrogen-fixers (diazotrophs) due to the innate sensitivity of nitrogenase to oxygen. This renders incompatible the processes of oxygen-evolving photosynthesis and N-fixation. We examined total antioxidative potential of various diazotrophic and non-diazotrophic cyanobacteria. We focused on Trichodesmium spp., a bloom-forming marine diazotroph that contributes significantly to global nitrogen fixation. Among the species tested, Trichodesmium possessed the highest antioxidant activity. Moreover, while proteins constituted the dominant antioxidative component of all other cyanobacteria tested, Trichodesmium was unique in that small-molecule natural products provided the majority of antioxidant activity, while proteins constituted only 13% of total antioxidant activity. Bioassay-guided fractionation followed by high-performance liquid chromatography profiling of antioxidant purified fractions identified the highly potent antioxidant all- trans -β-carotene, and small amounts of 9- cis -β-carotene and retinyl palmitate. Search of the Trichodesmium genome identified protein sequences homologous to key enzymes in the β-carotene to retinyl palmitate biosynthetic pathway, including 33–37% identity to lecithin retinol acyltransferase. The present study demonstrates the importance of carotenoids in Trichodesmium 's arsenal of defensive compounds against oxidative damage and protection of nitrogenase from oxygen and its radicals.     

Hsp90 recognizes a common surface on client kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.     

Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein-protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD.     

E3 ligases determine ubiquitination site and conjugate type by enforcing specificity on E2 enzymes

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono- and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.