Syntaxin 5 is required for copper homeostasis in Drosophila and mammals

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.     

An automatable screen for the rapid identification of proteins amenable to refolding

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.     

Pharmacological inhibition of the MEK5/ERK5 and PI3K/Akt signaling pathways synergistically reduces viability in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

Demethylation by DNA glycosylases is important for endosperm development, but only a subset of the affected loci are imprinted, suggesting demethylation may have additional functions.

IN A NUTSHELL Background: In 1970, Jerry Kermicle reported that maize kernels could have dramatically different pigmentation depending on which parent the r1 gene is inherited from. This was the first discovery of many genomically imprinted genes that are selectively expressed from the maternal genome in endosperm. Later, Kermicle also discovered a mutant with poor maternal r1 expression. He hypothesized that the normal function of the mutated gene would be to derepress maternal r1; hence the name maternal depression of r1 (mdr1). The identify of mdr1 has remained unknown since then, but studies using Arabidopsis thaliana have revealed that DNA demethylation by enzymes called DNA glycosylases is important for expression of some maternally inherited genes in endosperm. Question: We wanted to identify the mdr1 gene. We hypothesized that mdr1 would reveal insights into molecular mechanisms of genomic imprinting in maize. Findings: We discovered that mdr1 encodes one of two DNA glycosylases with high expression in endosperm. We found that at least one of the two must be functional for endosperm to develop normally, but the one encoded by mdr1 is expressed higher. Surprisingly, most of the genes the mdr1 DNA glycosylase demethylates do not appear to be genomically imprinted, and about half the DNA it demethylates is not even near genes. These findings suggest that DNA glycosylases also have an undiscovered function unrelated to genomic imprinting in endosperm. Next steps: We want to know how specific regions in the genome are targeted for demethylation. What distinguishes these regions from other regions in endosperm? And what keeps them from being demethylated in other tissues? On the flip side, little is known about the effect of demethylation in endosperm, other than genomic imprinting. We want to know what effect DNA demethylation by DNA glycosylases has on chromatin structure and why it is important.     

Interval timing in rats: tracking unsignaled changes in the fixed interval schedule requirement

The present experiment examined interval timing in rats under dynamic conditions. A session began with FI60 s intervals, changed to a FI20 s, FI30 s, or FI40 s schedule at an unpredictable point, and then returned to a FI60 s schedule after the rats received 1, 8, or 24 successive short FI intervals. Variations in the duration and number of shorter intervals occurred across sessions and conditions. We observed rapid control of wait time duration by the FI duration of the preceding interval (one-back tracking), and changes in wait time depended on the number and duration of the shorter intervals. Furthermore, we observed proportional and scalar timing effects in overall wait time duration. The results provide information about the relation between interval timing under dynamic and steady state conditions.     

A Eukaryotic Translation Initiation Factor 4E-Binding Protein Promotes mRNA Decapping and Is Required for PUF Repression

PUF proteins are eukaryotic RNA-binding proteins that repress specific mRNAs. The mechanisms and corepressors involved in PUF repression remain to be fully identified. Here, we investigated the mode of repression by Saccharomyces cerevisiae Puf5p and Puf4p and found that Puf5p specifically requires Eap1p to repress mRNAs, whereas Puf4p does not. Surprisingly, we observed that Eap1p, which is a member of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) class of translational inhibitors, does not inhibit the efficient polyribosome association of a Puf5p target mRNA. Rather, we found that Eap1p accelerates mRNA degradation by promoting decapping, and the ability of Eap1p to interact with eIF4E facilitates this activity. Deletion of EAP1 dramatically reduces decapping, resulting in accumulation of deadenylated, capped mRNA. In support of this phenotype, Eap1p associates both with Puf5p and the Dhh1p decapping factor. Furthermore, recruitment of Eap1p to downregulated mRNA is mediated by Puf5p. On the basis of these results, we propose that Puf5p promotes decapping by recruiting Eap1p and associated decapping factors to mRNAs. The implication of these findings is that a 4E-BP can repress protein expression by promoting specific mRNA degradation steps in addition to or in lieu of inhibiting translation initiation.