Interplay between Np95 and Eme1 in the DNA damage response

Mus81 (methyl methansulfonate UV sensitive clone 81) and Eme1 (essential meiotic endonuclease 1, also known as MMS4) form a heterodimeric endonuclease that is critical for genomic stability and the response to DNA crosslink damage and replication blockade. However, relatively little is known as to how this endonuclease is regulated following DNA damage. Here, we report mammalian Eme1 interacts with Np95, an E3 ubiquitin ligase that participates in chromatin modification, replication-linked epigenetic maintenance and the DNA damage response. Np95 and Eme1 co-localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. Taken together, these findings link Mus81-Eme1 with the replication-associated chromatin modifier functions of Np95 in the cellular response to DNA damage.     

Local Cre-Mediated Gene Recombination in Vascular Smooth Muscle Cells in Mice

Here we describe a means to conditionally modify genes at a predefined and localized region of the vasculature using a perivascular drug delivery device (PDD). A 4-hydroxytamoxifen (4-OHT)-eluting PDD was applied around the carotid or femoral artery of a mouse strain carrying both the tamoxifen-inducible and smooth muscle cell (SMC)-specific Cre-recombinase (SM-Cre-ER^T2) transgene and a stop-floxed β-galactosidase gene in the Rosa26 locus: the SM-CreER^T2(ki)/rosa26 mouse. A dose and time curve of 0–10% (w/w) 4-OHT and 0–14 days application of the PDD in SM-CreER^T2(ki)/rosa26 mice showed optimal gene recombination at 1% (w/w) 4-OHT loading at 7 days post application (carotid artery 2.4±1.8%; femoral artery 4.0±3.8% of SMCs). The unique 4-OHT-eluting PDD allowed us to achieve SMC-specific recombination in the same order of magnitude as compared to systemic tamoxifen administration. In addition, recombination was completely confined to the PDD-treated vessel wall segment. Thus, local application of a 4-OHT-eluting PDD results in vascular SMC-specific Cre-mediated recombination in SM-CreER^T2(ki)/rosa26 mice without affecting additional SMCs. These authors contributed equally     

Local translation in primary afferent fibers regulates nociception

Recent studies have demonstrated the importance of local protein synthesis for neuronal plasticity. In particular, local mRNA translation through the mammalian target of rapamycin (mTOR) has been shown to play a key role in regulating dendrite excitability and modulating long-term synaptic plasticity associated with learning and memory. There is also increased evidence to suggest that intact adult mammalian axons have a functional requirement for local protein synthesis in vivo. Here we show that the translational machinery is present in some myelinated sensory fibers and that active mTOR-dependent pathways participate in maintaining the sensitivity of a subpopulation of fast-conducting nociceptors in vivo. Phosphorylated mTOR together with other downstream components of the translational machinery were localized to a subset of myelinated sensory fibers in rat cutaneous tissue. We then showed with electromyographic studies that the mTOR inhibitor rapamycin reduced the sensitivity of a population of myelinated nociceptors known to be important for the increased mechanical sensitivity that follows injury. Behavioural studies confirmed that local treatment with rapamycin significantly attenuated persistent pain that follows tissue injury, but not acute pain. Specifically, we found that rapamycin blunted the heightened response to mechanical stimulation that develops around a site of injury and reduced the long-term mechanical hypersensitivity that follows partial peripheral nerve damage--a widely used model of chronic pain. Our results show that the sensitivity of a subset of sensory fibers is maintained by ongoing mTOR-mediated local protein synthesis and uncover a novel target for the control of long-term pain states.     

Ca(2+) signaling occurs via second messenger release from intraorganelle synthesis sites

Cyclic ADP-ribose is an important Ca(2+)-mobilizing cytosolic messenger synthesized from beta-NAD(+) by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCbeta and ARCgamma are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a beta-NAD(+) transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca(2+) wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.     

Protein kinase C isoform-specific differences in the spatial-temporal regulation and decoding of metabotropic glutamate receptor1a-stimulated second messenger responses

Metabotropic glutamate receptors (mGluRs) coupled via Gq to the hydrolysis of phosphoinositides stimulate Ca(2+) and PKCbetaII oscillations in both excitable and non-excitable cells. In the present study, we show that mGluR1a activation stimulates the repetitive plasma membrane translocation of each of the conventional and novel, but not atypical, PKC isozymes. However, despite similarities in sequence and cofactor regulation by diacyglycerol and Ca(2+), conventional PKCs exhibit isoform-specific oscillation patterns. PKCalpha and PKCbetaI display three distinct patterns of activity: (1) agonist-independent oscillations, (2) agonist-stimulated oscillations, and (3) persistent plasma membrane localization in response to mGluR1a activation. In contrast, only agonist-stimulated PKCbetaII translocation responses are observed in mGluR1a-expressing cells. PKCbetaI expression also promotes persistent increases in intracellular diacyglycerol concentrations in response to mGluR1a stimulation without affecting PKCbetaII oscillation patterns in the same cell. PKCbetaII isoform-specific translocation patterns are regulated by specific amino acid residues localized within the C-terminal PKC V5 domain. Specifically, Asn-625 and Lys-668 localized within the V5 domain of PKCbetaII cooperatively suppress PKCbetaI-like response patterns for PKCbetaII. Thus, redundancy in PKC isoform expression and differential decoding of second messenger response provides a novel mechanism for generating cell type-specific responses to the same signal.     

Discovery of a new peptide natural product by Streptomyces coelicolor genome mining

Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding proteins typically involved in complex natural product biosynthesis but not associated with the production of known natural products. In Streptomyces coelicolor M145 there are several gene clusters encoding new nonribosomal peptide synthetase (NRPS) systems not associated with known metabolites. Application of structure-based models for substrate recognition by NRPS adenylation domains predicts the amino acids incorporated into the putative peptide products of these systems, but the accuracy of these predictions is untested. Here we report the isolation and structure determination of the new tris-hydroxamate tetrapeptide iron chelator coelichelin from S. coelicolor using a genome mining approach guided by substrate predictions for the trimodular NRPS CchH, and we show that this enzyme, which lacks a C-terminal thioesterase domain, together with a homolog of enterobactin esterase (CchJ), are required for coelichelin biosynthesis. These results demonstrate that accurate prediction of adenylation domain substrate selectivity is possible and raise intriguing mechanistic questions regarding the assembly of a tetrapeptide by a trimodular NRPS.     

WEE1 inhibition and genomic instability in cancer

One of the hallmarks of cancer is genomic instability controlled by cell cycle checkpoints. The G₁ and G₂ checkpoints allow DNA damage responses, whereas the mitotic checkpoint enables correct seggregation of the sister chromosomes to prevent aneuploidy. Cancer cells often lack a functional G₁ arrest and rely on G₂ arrest for DNA damage responses. WEE1 kinase is an important regulator of the G₂ checkpoint and is overexpressed in various cancer types. Inhibition of WEE1 is a promising strategy in cancer therapy in combination with DNA-damaging agents, especially when cancer cells harbor p53 mutations, as it causes mitotic catastrophy when DNA is not repaired during G₂ arrest. Cancer cell response to WEE1 inhibition monotherapy has also been demonstrated in various types of cancer, including p53 wild-type cancers. We postulate that chromosomal instability can explain tumor response to WEE1 monotherapy. Therefore, chromosomal instability may need to be taken into account when determining the most effective strategy for the use of WEE1 inhibitors in cancer therapy.     

Persistence and Change in Community Composition of Reef Corals through Present,Past, and Future Climates

The reduction in coral cover on many contemporary tropical reefs suggests a different set of coral community assemblages will dominate future reefs. To evaluate the capacity of reef corals to persist over various time scales, we examined coral community dynamics in contemporary, fossil, and simulated future coral reef ecosystems. Based on studies between 1987 and 2012 at two locations in the Caribbean, and between 1981 and 2013 at five locations in the Indo-Pacific, we show that many coral genera declined in abundance, some showed no change in abundance, and a few coral genera increased in abundance. Whether the abundance of a genus declined, increased, or was conserved, was independent of coral family. An analysis of fossil-reef communities in the Caribbean revealed changes in numerical dominance and relative abundances of coral genera, and demonstrated that neither dominance nor taxon was associated with persistence. As coral family was a poor predictor of performance on contemporary reefs, a trait-based, dynamic, multi-patch model was developed to explore the phenotypic basis of ecological performance in a warmer future. Sensitivity analyses revealed that upon exposure to thermal stress, thermal tolerance, growth rate, and longevity were the most important predictors of coral persistence. Together, our results underscore the high variation in the rates and direction of change in coral abundances on contemporary and fossil reefs. Given this variation, it remains possible that coral reefs will be populated by a subset of the present coral fauna in a future that is warmer than the recent past.