Phospho‐regulation,nucleotide binding and ion access control in potassium‐chloride cotransporters

Potassium‐coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho‐regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo‐EM structures of human KCC3b and KCC1, revealing structural determinants for phospho‐regulation in both N‐ and C‐termini. We show that phospho‐mimetic KCC3b is arrested in an inward‐facing state in which intracellular ion access is blocked by extensive contacts with the N‐terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho‐regulatory site in the KCC1 N‐terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP‐binding pocket in the large C‐terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.     

Identification of new human origins of DNA replication by an origin-trapping assay

Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr virus latent origin oriP. oriP contains two elements, an origin (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G(1) phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.     

Effect of temperature on developmental time and longevity of Psyttalia cosyrae (Hymenoptera: Braconidae)

Psyttalia cosyrae (Wilkinson) (Hymenoptera: Braconidae) is a koinobiont, solitary larval-pupal parasitoid of Ceratitis cosyra (Walker) (Diptera: Tephritidae), and possibly other tephritid fruit flies. The effect of temperature on developmental time and longevity of this parasitoid was investigated and the thermal requirement at six constant temperatures (15±0.5, 20±0.5, 25±0.5, 27±0.5, 30±0.5, and 33±0.05°C) and 60-70% R.H was determined. The developmental rate increased with an increase in temperature. Females took a longer time to complete development than males at all temperatures tested. Development from egg to adult emergence required 244 degree-days (DD) above a thermal threshold of 11.9°C for both sexes pooled, 233 DD above 12.0°C for males and 256 DD above 11.6°C for females. Adult longevity was affected by temperature, and females lived longer than males at all temperatures tested.     

ENaC proteins are required for NGF-induced neurite growth

Neurite growth is required for nervous system development and repair. Multiple signals, including neurotrophic factors and intact mechanosensing mechanisms, interact to regulate neurite growth. Degenerin/epithelial Na⁺ channel (DEG/ENaC) proteins have been identified as putative mechanosensors in sensory neurons. Recently, others have shown that the neurotrophic factor NGF stimulates expression of acid-sensing ion channel molecules, which are members of the DEG/ENaC family. However, it is unknown whether NGF regulates ENaC expression or whether ENaC expression is required for neurite formation. Therefore, the aims of the present study were to determine whether ENaC expression is 1) regulated by NGF and 2) required for NGF-induced neurite growth in pheochromocytoma PC-12 cells. We found NGF-induced expression of - and -subunits of ENaC, but not -ENaC. Tyrosine kinase A (TrkA) receptor blockade abolished NGF-induced - and -ENaC expression and neurite formation. NGF-induced neurite formation was inhibited by disruption of ENaC expression using 1) pharmacological blockade with benzamil, a specific ENaC inhibitor; 2) small interfering RNA; and 3) dominant-negative ENaC molecules. These data indicate NGF-TrkA regulation of ENaC expression may be required for neurite growth and may suggest a novel role for DEG/ENaC proteins in neuronal remodeling and differentiation. mechanosensation; degenerins; neurotrophins; tyrosine kinase A; pheochromocytoma cells     

Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier's disease

The mechanism(s) involved in regulation of store operated calcium entry in Darier's disease (DD) is not known. We investigated the distribution and function of transient receptor potential canonical (TRPC) in epidermal skin cells. DD patients demonstrated up-regulation of TRPC1, but not TRPC3, in the squamous layers. Ca2+ influx was significantly higher in keratinocytes obtained from DD patients and showed enhanced proliferation compared with normal keratinocytes. Similar up-regulation of TRPC1 was also detected in epidermal layers of SERCA2+/- mice. HaCaT cells expressed TRPC1 in the plasma membrane. Expression of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2 small interfering RNA (siRNA) in HaCaT cells increased TRPC1 levels and thapsigargin-stimulated Ca2+ influx, which was blocked by store-operated calcium entry inhibitors. Thapsigargin-stimulated intracellular Ca2+ release was decreased in DD cells. DD keratinocytes exhibited increased cell survival upon thapsigargin treatment. Alternatively, overexpression of TRPC1 or SERCA2-siRNA in HaCaT cells demonstrated resistance to thapsigargin-induced apoptosis. These effects were dependent on external Ca2+ and activation of nuclear factor-kappaB. Isotretinoin reduced Ca2+ entry in HaCaT cells and decreased survival of HaCaT and DD keratinocytes. These findings put forward a novel consequence of compromised SERCA2 function in DD wherein up-regulation of TRPC1 augments cell proliferation and restrict apoptosis. We suggest that the anti-apoptotic effect of TRPC1 could potentially contribute to abnormal keratosis in DD.     

Apc tumor suppressor gene is the "zonation-keeper" of mouse liver

The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.     

Deconstructing estradiol: Removal of B-ring generates compounds which are potent and subtype-selective estrogen receptor agonists

Estradiol and related estrogens have been widely used as supplements to relieve menopausal symptoms, but they lead to an increased risk of breast and endometrial cancer. Here we report the synthesis of a new family of compounds where we have removed the B-ring from the steroid ABCD structure, and functionalized the A-ring. These A-CD compounds show a preferential affinity for the estrogen receptor subtype ERβ. Some show binding affinities which are greater than estradiol. The presence of electron-withdrawing substituents on the A-ring should reduce the tendency of these compounds to form carcinogenic metabolites, so they might lead to a safer approach to hormone replacement therapy.     

Involvement of nitrate reduction in the tolerance of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) plants to prolonged root hypoxia

The putative role of nitrate and nitrate reductase in the tolerance to prolonged hypoxia was investigated in tomato plants. Nitrogen nutrition has been modified either by deprivation of nitrate or by addition of tungstate—an inhibitor of nitrate reductase (NR)—in the culture medium. In the absence of nitrate as well as in the presence of tungstate, plant growth was significantly disturbed. In the presence of nitrate, the growth of hypoxic plants maintained, nitrate absorption and NR activity increased and a significant release of nitrite into the medium was observed. This mechanism of nitrate reduction, called nitrate respiration, could be an alternative pathway to oxygen-dependent respiration during root hypoxia and a transient adaptation of tomato roots to hypoxic conditions.     

Exploiting yoeB-yefM toxin-antitoxin system of Streptococcus pneumoniae on the selective killing of miR-21 overexpressing breast cancer cell line (MCF-7)

Toxin-antitoxin (TA) systems are two-component genetic modules widespread in bacterial and archaeal genomes, in which the toxin module is rendered inactive under resting conditions by its antitoxin counterpart. Under stress conditions, however, the antitoxin is degraded, freeing the toxin to exert its lethal effects. Although not evolved to function in eukaryotes, some studies have established the lethal activity of these bacterial toxins by inducing apoptosis in mammalian cells, an effect that can be neutralized by its cognate antitoxin. Inspired by the way the toxin can become active in eukaryotes cells, we produced an engrained yoeB-yefM TA system to selectively kill human breast cancer cells expressing a high level of miR-21. Accordingly, we generated an engineered yefM antitoxin gene with eight miR-21 target sites placed in its 3′untranslated region. The resulting TA system acts autonomously in human cells, distinguishing those that overexpress miR-21, killed by YoeB, from those that do not, remaining protected by YefM. Thus, we indicated that microRNA-control of the antitoxin protein of bacterial TA systems constitutes a novel strategy to enhance the selective killing of human cancer cells by the toxin module. The present study provides significant insights for developing novel anticancer strategies avoiding off-target effects, a challenge that has been pursued by many investigators over the years.