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A síndrome de vasoconstrição cerebral reversível é uma doença cerebrovascular que leva à constrição e dilatação arterial multifocal. A síndrome de vasoconstrição cerebral reversível é possivelmente causada pela desregulação transitória do tônus vascular cerebral. Relatamos um caso raro de uma paciente com queixa principal de cefaleia pós‐parto, posteriormente diagnosticada como um caso de síndrome de vasoconstrição cerebral reversível. A jovem primigesta a termo apresentando boa contração uterina foi internada em sala de parto. Mais tarde, a parturiente queixou‐se de perda de líquido pela vagina e, ao exame, líquido amniótico manchado foi observado. O parto cesariano sob raquianestesia foi realizado, e não houve intercorrência no período intraoperatório. Tanto a mãe quanto o bebê estavam normais e foram transferidos para a sala de recuperação pós‐operatória e berçário, respectivamente. Na sala de recuperação, a mãe queixou‐se de forte dor de cabeça após uma hora e depois desenvolveu convulsão. Midazolam foi administrado por via intravenosa, e a paciente foi intubada e transferida para uma unidade de terapia intensiva para posterior investigação e tratamento. A tomografia computadorizada sem contraste do cérebro mostrou hemorragia intracerebral occipital direita e subaracnoide. A angiotomografia mostrou estreitamento da artéria vertebral direita, sem qualquer outra malformação vascular. A paciente foi tratada em unidade de terapia intensiva por dois dias e, em seguida, foi extubada e transferida para a ala de alta dependência onde permaneceu um dia em observação, recebendo alta hospitalar três dias depois, após uma recuperação completa e sem intercorrências. Reversible cerebral vasoconstriction syndrome is a cerebrovascular disorder leading to multifocal arterial constriction and dilation. Reversible cerebral vasoconstriction syndrome is possibly caused by transient deregulation of cerebral vascular tone. We report a rare case of a patient with chief complain of postpartum headache, was later diagnosed as a case of reversible cerebral vasoconstriction syndrome. A young full term primigravida with good uterine contraction admitted to labour room. Later she complained of leaking per vagina and on examination meconium stained liquor was noted. Caesarean delivery under spinal anesthesia was done and intra‐operative period was uneventful. Both mother and baby were normal and shifted to postoperative ward and nursery respectively. In postoperative ward, mother complained of severe headache after one hour and later developed seizure. Midazolam was given intravenously and was intubated and transferred to critical care unit for further investigation and management. Non contrast computerized tomography scan of brain showed right occipital intracerebral as well as subarachnoid bleed. CT angiography showed right vertebral artery narrowing without any other vascular malformation. Patient was managed in critical care unit for 2 days and then extubated and shifted to high dependency ward after a day observation and discharged 3 days later after a full uneventful recovery. [ABSTRACT FROM AUTHOR]

Protein modification by ubiquitination plays a key evolutionarily conserved role in regulating membrane proteins. Nedd4-2, a ubiquitin ligase, targets membrane proteins such as ion channels and transporters for ubiquitination. This Nedd4-2-mediated ubiquitination provides a crucial step in controlling the membrane availability of these proteins, thus affecting their signaling and physiological outcomes. In one well-studied example, Nedd4-2 fine-tunes the physiological function of the epithelial sodium channel (ENaC), thus modulating Na + reabsorption by epithelia to maintain whole-body Na + homeostasis. This review summarizes the key signaling pathways regulated by Nedd4-2 and the possible implications of such regulation in various pathologies. [ABSTRACT FROM AUTHOR]

Xylanases from bacterial sources have potential applications in various industrial processes. In the present study, the effect of enzymatic pre-treatment on pulp bleaching has been investigated using purified xylanase from alkaliphilic Bacillus licheniformis strain Alk-1. The xylanase produced by present isolate while growing on wheat bran was purified to homogeneity by 5.84-fold purification with ~15.97% recovery by anion exchange chromatography using DEAE-cellulose. The estimated molecular weight of the xylanase was ~46 kDa. The optimal pH and temperature for xylanase activity were pH 9.0 and 60 °C, respectively. The enzyme showed good activity retention (80%) after 1 h incubation at 60 °C and pH 9.0. Enzyme activity was stimulated by Ca 2+ , Fe 2+ and Mg 2+ and inhibited by Cd 2+ , Hg 2+ and Cu 2+ at 2 mM and 10 mM conc. For xylan, the enzyme gave a K m value of 5.26 mg/mL and V max value of 433 μM/min/mg proteins when the reaction was carried out at 60 °Cand pH 9.0. Pre-treatment of kraft pulp with xylanase showed 19% reduction in kappa number, compared to control pulp, after 2 h treatment. The SEM and FTIR analysis of xylanase-treated pulp revealed significant morphological and structural changes on pulp fibres. GC-MS analysis of filtrates from control and xylanase-treated pulps showed variation in the presence of different plant-derived organic compounds. The present work finds potential application in paper production by making it cleaner and ecofriendly process. [ABSTRACT FROM AUTHOR]

Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes and economic viability in terms of reusability. In the current study, purified xylanase from B. licheniformis Alk-1 was immobilized within glutaraldehyde activated calcium alginate beads and characterized in respect of free enzyme. Immobilization increases the optimum pH and temperature of entrapped and cross-linked enzyme from pH = 8.0 to 9.0 and 50–60 °C. The kinetics parameter of immobilized (cross-linked) enzyme showed an increase in K m (from 4.36 mg/mL to 5.38 mg/mL) and decrease in V max (from 383 IU/mg/min to 370 IU/mg/min). Immobilization increases the optimum reaction time for xylan degradation of immobilized xylanase from 15 to 30 min when compare to free form. The storage stability study suggested that the immobilized enzyme retains 80% of its original activity at 4 °C after 30 days compared to free enzyme (5%). Further, immobilization improved enzyme stability in presence of different additives. The immobilized (cross-linked) enzyme also exhibited adequate recycling efficiency up to five reaction cycles with 37% retention activity. The finding of this study suggests improvement of overall performance of immobilized xylanase in respect to free form and can be used to make a bioreactor for various applications such as poultry feed preparations. [ABSTRACT FROM AUTHOR]

A lignin peroxidases-producing Serratia liquefaciens was used for bioremediation of pulp and paper (P&P) mill effluent. The treatment led to reduction of chemical oxygen demand (COD), colour, lignin and phenolic content by 84%, 72%, 61% and 95%, respectively. The effluent detoxification was studied by genotoxicity assays using Allium cepa L. (onion) root tip cells. Genotoxicity studies included measuring mitotic index (MI), chromosomal aberrations (CA) and nuclear abnormalities (NA) in root tip cells following treatment with 25, 50, 75 and 100% (v/v) of effluent. The root tip cells grown in untreated effluent showed a significant decrease in MI from 69% (control) to 32%, 27%, 22% and 11% at 25%, 50%, 75% and 100% effluent concentration, respectively. This indicated that the untreated effluent was highly cytotoxic in nature. Further, root tip cells, when treated with different concentrations of effluent showed various CA and NA including c-mitosis, stickiness, chromosome loss, chromosome break, anaphase bridge, multipolar anaphase, vagrant chromosomes, micronucleated and binucleated cells. The MI observed in root tip cells grown in bacterial treated effluents at similar concentrations (25, 50, 75 and 100% v/v) showed an increase of 33%, 36%, 42% and 66%. CA showed a substantial decrease and in some instances, complete absence of CA was also observed. The findings suggest that S. liquefaciens culture could be a potential bacterial culture for bioremediation of P&P mill effluent, as it is effective in substantial lowering of pollutants load as well as reduces the cytotoxic and genotoxic effects of effluent. [ABSTRACT FROM AUTHOR]

Ubiquitination plays a crucial role in regulating proteins post-translationally. The focus of this review is on NEDD4, the founding member of the NEDD4 family of ubiquitin ligases that is evolutionarily conserved in eukaryotes. Many potential substrates of NEDD4 have been identified and NEDD4 has been shown to play a critical role in the regulation of a number of membrane receptors, endocytic machinery components and the tumour suppressor PTEN. In this review we will discuss the diverse pathways in which NEDD4 is involved, and the patho-physiological significance of this important ubiquitin ligase. [ABSTRACT FROM AUTHOR]

The present study deals with the bioremediation of potentially hazardous pulp and paper mill effluent by a laccase producing Paenibacillus sp. strain LD-1 (JX499920) isolated from contaminated soil sample by lignin enrichment method. The bacterium effectively reduced pollution parameters (colour 68%, lignin 54%, phenol 86%, BOD 83% and COD 78%) after 144 h of treatment at 34 ± 1 °C and 120 rpm. GC–MS analysis of control and treated samples showed that concentration of most of the low molecular weight phenolic compounds like 2-methoxyphenol, 2,6-dimethoxy phenol, 2-methoxy-4-ethyl-phenol, 3-allyl-6-methoxyphenol, ethanone 1-(-4-hydroxy-3,5-dimethoxyphenyl), benzoic acid, 2-methoxy-4-(1-propenyl) phenol and 4-methoxycinnamic acid present in control untreated effluent were reduced after bacterial treatment. The toxicity assessments were carried out with treated and untreated effluent by studying the growth and germination of seeds of mung bean ( Vigna radiata L.). The mung bean bioassay confirmed the detoxification of effluent after bacterial treatment. [ABSTRACT FROM AUTHOR]

Abstract: Members of the HECT family of E3 ubiquitin-protein ligases are characterized by a C-terminal HECT domain that catalyzes the covalent attachment of ubiquitin to substrate proteins and by N-terminal extensions of variable length and domain architecture that determine the substrate spectrum of a respective HECT E3. Since their discovery in 1995, it has become clear that deregulation of distinct HECT E3s plays an eminent role in human disease or disease-related processes including cancer, cardiovascular and neurological disorders, viral infections, and immune response. Thus, a detailed understanding of the structure–function aspects of HECT E3s as well as the identification and characterization of the substrates and regulators of HECT E3s is critical in developing new approaches in the treatment of respective diseases. In this review, we summarize what is currently known about mammalian HECT E3s, with a focus on their biological functions and roles in pathophysiology.This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. [Copyright &y& Elsevier]

Abstract: In the present work the performance of a solar air heater duct provided with artificial roughness in the form of thin circular wire in arc shaped geometry has been analysed using Computational Fluid Dynamics (CFD). The effect of arc shaped geometry on heat transfer coefficient, friction factor and performance enhancement was investigated covering the range of roughness parameter (relative roughness height (e/D) from 0.0299 to 0.0426 and relative roughness angle (α/90) from 0.333 to 0.666) and working parameter (Reynolds number, Re from 6000 to 18,000 and solar radiation of 1000W/m2). Different turbulent models have been used for the analysis and their results are compared. Renormalization-group (RNG) k-ɛ model based results have been found in good agreement and accordingly this model is used to predict heat transfer and friction factor in the duct. The overall enhancement ratio has been calculated in order to discuss the overall effect of the roughness and working parameters. A maximum value of overall enhancement ratio has been found to be as 1.7 for the range of parameters investigated. [Copyright &y& Elsevier]

Abstract: Drosophila melanogaster is a highly amenable model system for examining programmed cell death during animal development, offering sophisticated genetic techniques and in vivo cell biological analyses. The reproducible pattern of apoptosis, as well as the apoptotic response to genotoxic stress, has been well characterized during Drosophila development. The main cellular components required for cell death are highly conserved throughout evolution. Central to the regulation of apoptosis is the caspase family of cysteine proteases, and studies in Drosophila have revealed insights into their regulation and function. This chapter describes protocols for detecting apoptotic cells during Drosophila development, as well as the use of Drosophila cell lines. Commonly used methods for detecting apoptosis are described, including TUNEL, acridine orange, and immunostaining with specific components of the apoptotic pathway such as active caspases. A crucial step in the induction of apoptosis is caspase activation and cleavage, which can be measured by use of fluorogenic peptide substrates or detection of cleaved protein products by immunoblotting, respectively. In addition, one of the advantages of the use of Drosophila as model is the ability to examine genetic interactions with various components of the cell death pathway. [Copyright &y& Elsevier]

Abstract: Nedd1 was originally identified as a developmentally regulated gene in the mouse central nervous system. NEDD1 has homologues across a range of species, being particularly conserved in a region of WD40 repeats contained in the amino-terminal half of the protein. Human NEDD1 was recently found to localise to the centrosome and mitotic spindle. It binds to the components of the γ-tubulin ring complex and target this complex to the centrosome and spindle. Depletion of NEDD1 causes loss of the γ-tubulin ring complex from the centrosome and results in the failure of microtubule nucleation and spindle assembly. In addition, phosphorylation of NEDD1 during mitosis is critical for targeting γ-tubulin to the spindle, but not the centrosome. There is still much unknown about the function of this protein and how it may be involved in development and disease. This short review summarises some of the recent work on NEDD1 and discusses how this interesting protein may have additional yet unexplored functions. [Copyright &y& Elsevier]

Developmental and physiological cues, as well as signals in response to cell damage, initiate a cell suicide program that is often referred to as apoptosis or programmed cell death (PCD). PCD is mediated by the core cell-death machinery consisting of proteins that mediate and control the activation of caspases – enzymes that dismantle cellular architecture during apoptosis. Because components of the caspase-activation apparatus are constitutively present in most cells, most studies during the last decade have focused on the posttranslational regulation of this preexisting machinery. However, accumulating evidence suggests that, in many contexts, transcriptional regulation also plays a crucial role in the activation of the physiological cell-death program. [Copyright &y& Elsevier]

Programmed cell death (PCD) is essential for the removal of unwanted cells and is critical for both restricting cell numbers and for tissue patterning during development. Components of the cell death machinery are remarkably conserved through evolution, from worms to mammals. Central to the PCD process is the family of cysteine proteases, known as caspases, which are activated by death-inducing signals. Comparisons between C. elegans and mammalian PCD have shown that there is additional complexity in the regulation of PCD in mammals. The fruitfly, Drosophila melanogaster, is proving an ideal genetically tractable model organism, of intermediary complexity between C. elegans and mammals, in which to study the intricacies of PCD. Here, we review the literature on PCD during Drosophila development, highlighting the methods used in these studies. [Copyright &y& Elsevier]

Investigates the mechanisms of caspase activation through oligomerization. Classes of caspases; Types of protein-protein-interaction domains that are present in apoptotic molecules; Review of related studies; Events that lead to activation of class I caspases.

Because of higher energy absorbing capacity of porous metals, these materials are used in car crash protection, armours, etc. However, to the best of our knowledge the porous metals are not used in solid particle erosion (SPE) resistant coatings. Nanotechnology allowed development of effective erosion resistant coatings by reducing the grain size of coatings to ≤ 10 nm. As a further development to it, here the porous metal layers, sandwiched between blocks of Ti/TiN multilayers to develop the next generation SPE resistant coatings. Two different ultra-thin Ti/TiN (bi-layer ~7.5 nm, 373 bilayers) multi-layered coatings (each ~9 μm) with dense (Ti/TIN-D) and porous (Ti/TIN-P) Ti layers (320 nm) were deposited on Ti6Al4V substrates using magnetron sputtering system. The erosion tests were conducted with respect to erodent speed (30 to 100 m/s), angle (30 to 90°), and temperature (25 to 700 °C). The average erosion resistance performance of Ti/TIN-P coating is 44 times better than Ti6Al4V substrate and 3.3 times better than Ti/TIN-D coating for 100 m/s erodent speed. Finite element simulations were used to understand the superior performance of Ti/TIN-P over Ti/TIN-D coating for different speeds (20 to 100 m/s). The simulation results are in agreement with the experimental results. Unlabelled Image • Energy absorption capacity of porous metals and nano-technology was used to develop the advanced solid particle erosion resistant coatings. • Two different ultra-thin multi-layered coatings with dense (Ti/TIN-D) and porous (Ti/TIN-P) Ti layers were deposited on Ti6Al4V substrates. • Erosion resistance (@ 100 m/s) of Ti/TIN-P coating is 44 and 3.3 times better than Ti6Al4V and Ti/TIN-D coating, respectively. • Finite element simulations were used to understand the erosion phenomena and the results are in agreement with the experiment. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Autophagy-dependent cell death is a distinct mode of regulated cell death required in a context specific manner. One of the best validated genetic models of autophagy-dependent cell death is the removal of the Drosophila larval midgut during larval-pupal transition. We have previously shown that down-regulation of growth signaling is essential for autophagy induction and larval midgut degradation. Sustained growth signaling through Ras and PI3K blocks autophagy and consequently inhibits midgut degradation. In addition, the morphogen Dpp plays an important role in regulating the correct timing of midgut degradation. Here we explore the potential roles of Hh and Wg signaling in autophagy-dependent midgut cell death. We demonstrate that Hh and Wg signaling are not involved in the regulation of autophagy-dependent cell death. However, surprisingly we found that one key component of these pathways, the Drosophila Glycogen Synthase Kinase 3, Shaggy (Sgg), may regulate midgut cell size independent of Hh and Wg signaling. [ABSTRACT FROM AUTHOR]

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the in vivo functions of the arrestin domain–containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver. [ABSTRACT FROM AUTHOR]

NEDD4-2 (also known as NEDD4L, neural precursor cell expressed developmentally down-regulated 4-like) is a ubiquitin protein ligase of the Nedd4 family which is known to bind and regulate a number of membrane proteins to aid in their internalization and turnover. Several of the NEDD4-2 substrates include ion channels, such as the epithelial and voltage-gated sodium channels. Given the critical function of NEDD4-2 in regulating membrane proteins, this ligase is essential for the maintenance of cellular homeostasis. In this article we review the biology and function of this important ubiquitin-protein ligase and discuss its pathophysiological significance. [ABSTRACT FROM AUTHOR]

Highlights: [•] Age-dependent gait abnormalities are evident in Nedd4 heterozygous mice. [•] Nedd4 is highly expressed in the Purkinje neurons of the cerebellum. [•] Gait abnormalities in Nedd4 heterozygous mice may be associated with subtle changes in distribution of GluR1 in Purkinje neurons. [ABSTRACT FROM AUTHOR]

Abstract: During the development of metazoans, programmed cell death (PCD) is essential for tissue patterning, removal of unwanted cells and maintaining homeostasis. In the past 20years Drosophila melanogaster has been one of the systems of choice for studies involving developmental cell death, providing an ideal genetically tractable model of intermediary complexity between Caenorhabditis elegans and mammals. The lessons learned from studies using Drosophila indicate both the conserved nature of the many cell death pathways as well as novel and unexpected mechanisms. In this article we review the understanding of PCD during Drosophila development, highlighting the key mechanisms that are evolutionarily conserved as well as apparently unusual pathways, which indicate divergence, but provide evidence of complexity acquired during organismic evolution. This article is part of a Special Section entitled: Cell Death Pathways. Guest Editors: Frank Madeo and Slaven Stekovic. [Copyright &y& Elsevier]

Abstract: Nedd4 and Nedd4-2 are closely related HECT-type ubiquitin-protein ligases (E3) implicated in the regulation of a number of proteins and pathways. Given the close homology between these E3 enzymes it would be predicted that a conserved ubiquitin-conjugating enzyme (E2) specificity exists between the two proteins. However, E2 specificities for Nedd4 and Nedd4-2 are not well established. In the present studies we aimed at clarifying the E2-specificities of Nedd4 and Nedd4-2 using in vitro ubiquitination assays. We demonstrate strong substrate ubiquitination in the presence of UbcH5b by both Nedd4 and Nedd4-2. We also found that Ube2e3, an E2 previously shown to be used by Nedd4-2, is used less efficiently than UbcH5b. Our results suggest that for optimal ubiquitination Nedd4 and Nedd4-2 require the same E2 enzymes. [Copyright &y& Elsevier]

The Drosophila steroid hormone ecdysone mediates cell death during metamorphosis by regulating the transcription of a number of cell death genes. The apical caspase DRONC is known to be transcriptionally regulated by ecdysone during development. Here we demonstrate that ecdysone also regulates the transcription of DRICE, a major effector caspase and a downstream target for DRONC in the fly. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that drice up-regulation is essential for apoptosis induced by ecdysone. We also show that drice expression is specifically controlled by the ecdysone-regulated transcription factor BR-C. Combined with previous observations, our results indicate that transcriptional regulation of the components of the core apoptotic machinery plays a key role in hormone-regulated programmed cell death during Drosophila development. [ABSTRACT FROM AUTHOR]

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC. [Copyright &y& Elsevier]

The consolidation of short-term memory into long-term memory involves changing protein level and activity for the synaptic plasticity required for long-term potentiation (LTP). AMPA receptor trafficking is a key determinant of LTP and recently ubiquitination by Nedd4 has been shown to play an important role via direct action on the GluA1 subunit, although the physiological relevance of these findings are yet to be determined. We therefore investigated learning and memory in Nedd4 +/− mice that have a 50% reduction in levels of Nedd4. These mice showed decreased long-term spatial memory as evidenced by significant increases in the time taken to learn the location of and subsequently find a platform in the Morris water maze. In contrast, there were no significant differences between Nedd4 +/+ and Nedd4 +/− mice in terms of short-term spatial memory in a Y-maze test. Nedd4 +/− mice also displayed a significant reduction in post-synaptic LTP measured in hippocampal brain slices. Immunofluorescence of Nedd4 in the hippocampus confirmed its expression in hippocampal neurons of the CA1 region. These findings indicate that reducing Nedd4 protein by 50% significantly impairs LTP and long-term memory thereby demonstrating an important role for Nedd4 in these processes. [ABSTRACT FROM AUTHOR]

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with a-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na+ absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant (K220RGRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRHGRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 (D110AGRI(2) increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the a-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against GaqIll increases ENaC activity. The effect of Gaq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC. [ABSTRACT FROM AUTHOR]

Summary: The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ∼6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has “lost” a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution. [ABSTRACT FROM AUTHOR]

It has recently been shown that the epithelial Na+ channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the β- and γ-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel. [ABSTRACT FROM AUTHOR]

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50-90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress. [ABSTRACT FROM AUTHOR]

The Tweety proteins comprise a family of chloride ion channels with three members identified in humans (TTYH1-3) and orthologues in fly and murine species. In humans, increased TTYH2 expression is associated with cancer progression, whereas fly Tweety is associated with developmental processes. Structurally, Tweety proteins are characterized by five membrane-spanning domains and N-glycan modifications important for trafficking to the plasma membrane, where these proteins are oriented with the amino terminus located extracellularly and the carboxyl terminus cytoplasmically. In addition to N-glycosylation, ubiquitination mediated by the HECT type E3 ubiquitin ligase Nedd4-2 is a post-translation modification important in regulating membrane proteins. In the present study, we performed a comprehensive analysis of the ability of each of TTYH1-3 to interact with Nedd4-2 and to be ubiquitinated and regulated by this ligase. Our data indicate that Nedd4-2 binds to two family members, TTYH2 and TTYH3, which contain consensus PY ((L/P)PXY) binding sites for HECT type E3 ubiquitin ligases, but not to TTYH1, which lacks this motif. Consistently, Nedd4-2 ubiquitinates both TTYH2 and TTYH3. Importantly, we have shown that endogenous TTYH2 and Nedd4-2 are binding partners and demonstrated that the TTYH2 PY motif is essential for these interactions. We have also shown that Nedd4-2-mediated ubiquitination of TTYH2 is a critical regulator of cell surface and total cellular levels of this protein. These data, indicating that Nedd4-2 differentially interacts with and regulates TTYH1-3, will be important for understanding mechanisms controlling Tweety proteins in physiology and disease. [ABSTRACT FROM AUTHOR]

The epithelial sodium channel (ENaC) plays an important role in transepithelial Na+ absorption; hence its function is essential for maintaining Na+ and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1K179M, decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser342 and Ser428, completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na+ absorption by upregulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2. [ABSTRACT FROM AUTHOR]

The muscarine-sensitive K+ current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K+ currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system. [ABSTRACT FROM AUTHOR]

Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, we produced a null mutation in dronc. Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark, and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis. [Copyright &y& Elsevier]

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na+ channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na+ (Nav) channels contain typical PY motifs (PPXY), and a further Nav contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Nav channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na+ channel (Nav1.2 and Nav1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Navs (Nav1.2, Nav1.7, and Nav1.8) tested. Interestingly, Nedd4 suppressed the activity of Nav1.2 and Nav1.7 but was a poor inhibitor of Nav1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Nav channels in vivo. [ABSTRACT FROM AUTHOR]

One of the cellular mechanisms used to prevent continuous and enhanced activation in response to growth factors is the internalization and degradation of their receptors. Little is known about the molecular mechanisms involved in vascular endothelial growth factor receptor-2 (VEGF-R2) degradation. In a previous work, we have shown that the adaptor protein Grb10 is a positive regulator of the VEGF signaling pathway. Indeed, VEGF stimulates Grb10 expression, and Grb10 overexpression induces an increase in the amount and the tyrosine phosphorylation of VEGF-R2. In the present manuscript, we demonstrate that Grb10 stimulates VEGF-R2 expression by inhibiting the Nedd4-mediated VEGF-R2 degradation. First, we show that proteasome inhibition by MG132 induces an increase in VEGF-R2 amount, and that VEGF-R2 is ubiquitinated in response to VEGF. Expression of Nedd4, a HECT domain-containing ubiquitin ligase, induces the disappearance of VEGF-R2 in cells, suggesting that Nedd4 is involved in VEGF-R2 degradation. To determine whether Nedd4 directly ubiquitinates VEGF-R2, we expressed a ubiquitin ligase-deficient mutant Nedd4C854S. In the presence of Nedd4C854S, VEGF-R2 is expressed and ubiquitinated. These results suggest that VEGF-R2 is ubiquitinated but that Nedd4 is not involved in this process. Finally, we show that Grb10 constitutively associates with Nedd4. Co-expression of Nedd4 and Grb10 restores the expression of VEGF-R2, suggesting that Grb10 inhibits the Nedd4-mediated degradation of VEGF-R2. In this study, we show that Grb10 acts as a positive regulator in VEGF-R2 signaling and protects VEGF-R2 from degradation by interacting with Nedd4, a component of the endocytic machinery. [ABSTRACT FROM AUTHOR]

Developmentally programmed cell death is regulated by a balance between pro- and anti-death signaling. During Drosophila metamorphosis, the removal of larval tissues is dependent on the steroid hormone ecdysone, which controls the levels of pro- and anti-death molecules. Ecdysone binds to its heterodimeric receptor ecdysone receptor/ultraspiracle to mediate transcription of primary response genes. Here we show that CARMER, an arginine-histone methyltransferase, is critical in coordinating ecdysone-induced expression of Drosophila cell death genes. Ablation of CARMER blocks ecdysone-induced tell death in Drosophila cells, but not apoptosis induced by cell stress. We demonstrate that CARMER associates with the ecdysone receptor complex and modulates the ecdysone-induced transcription of a number of apoptotic genes. Thus, the chromatin-modifying protein, CARMER, modulates cell death by controlling the hormone-dependent expression of the core cell death machinery. [ABSTRACT FROM AUTHOR]

Studies the functional regions of the caspase-2 prodomain that modulate nuclear transport and enzyme activation. Lys residue critical to the function of prodomain; Single point mutation that abolishes binding; Regulation of the formation of dots/filaments correlating with the cell killing ability of caspase-2.