α4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)‐binding protein

Mammalian α4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target‐of‐rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell‐cycle progression. α4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using α4 as bait in yeast two‐hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of α4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)‐binding protein (PABP), suggesting that PABP may also interact with α4. PABP recruits translation factors to the poly(A)‐tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between α4 and EDD in rat Nb2 T‐lymphoma and human MCF‐7 breast cancer cell lines. α4 also interacted with PABP in Nb2, MCF‐7 and the human Jurkat T‐leukemic and K562 myeloma cell lines. COS‐1 cells, transfected with Flag‐tagged‐pSG5‐EDD, gave a (Flag)‐EDD–α4 immunocomplex. Furthermore, deletion mutants of α4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C‐terminal region of α4, independent of the α4‐PP2Ac binding site. Therefore, in addition to PP2Ac, α4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell‐cycle progression. J. Cell. Biochem. 110: 1123–1129, 2010. Published 2010 Wiley‐Liss, Inc.     

Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis

Ubiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly specific manner. Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three 'core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts specificity for the ubiquitination of selected target proteins. Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways. In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins. Analysis of the protein interaction profiles involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more specifically with members of the F-box protein family. Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins. Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis. These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hip-pel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBCVHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBCVHL complex, and is probably one of the new substrates of the CBCVHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor- binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

The single‐subunit RING‐type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling

Membrane‐delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single‐subunit RING‐type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C‐terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1–PYL4 and RSL1–PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half‐life, protein interactions or trafficking.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBC^VHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBC^VHL complex, and is probably one of the new substrates of the CBC^VHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor-binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

TRIP12 functions as an E3 ubiquitin ligase of APP-BP1

The NEDD8 pathway plays an essential role in various physiological processes, such as cell cycle progression and signal transduction. The conjugation of NEDD8 to target proteins is initiated by the NEDD8-activating enzyme composed of APP-BP1 and Uba3. In the present study, we show that APP-BP1 is degraded by ubiquitin-dependent proteolysis. To study biological functions of TRIP12, a HECT domain-containing E3 ubiquitin ligase, we used the yeast two-hybrid system and identified APP-BP1 as its binding partner. Immunoprecipitation analysis showed that TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhanced the degradation of APP-BP1, whereas knockdown of TRIP12 stabilized it. In vitro ubiquitination assays revealed that TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. Moreover, neddylation of endogenous CUL1 was increased in TRIP12 knockdown cells, while complementation of the knockdown cells with TRIP12 lowered neddylated CUL1. Our data suggest that that TRIP12 promotes degradation of APP-BP1 by catalyzing its ubiquitination, which in turn modulates the neddylation pathway.     

In vitro and in vivo digestion of octenyl succinic starch

This study aimed to understand effects of octenyl succinic anhydride (OSA) modification of normal corn (NCS) and high-amylose corn (HA7) starch on their enzymatic hydrolysis rates. After modification with 3% and 10% OSA, resistant starch (RS) contents of the cooked OS-NCS increased from 0.8% of the control starch to 6.8% and 13.2% (Englyst Method), respectively, whereas that of the cooked OS-HA7 decreased from 24.1% to 23.7% and 20.9%, respectively. When the cooked NCS, HA7 and OS (10%)-HA7 were used to prepare diets for rats at 55% (w/w) starch, RS contents of the diets were 1.1%, 13.2% and 14.6%, respectively. After feeding to the rats, 20.2–31.1% of the starch in the OS (10%)-HA7-diet was not utilized in vivo and was found in rat feces, which was substantially larger than that of the HA7-diet (≤4.9%) and NCS-diet (≤0.2%). The body weights of the rats, however, remained similar between different groups.     

Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8)

Arabidopsis COP1 is a negative regulator of photomorphogenesis, which targets HY5, a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the absence of light. COP1 and its interactive partner CIP8 both possess RING finger motifs, characteristic of some E3 ubiquitin ligases. Here we show that CIP8 promotes ubiquitin attachment to HY5 in E2-dependent fashion in vitro. CIP8 exhibits a strong interaction with the E2 enzyme AtUBC8 through its N-terminal domain. Phosphorylation of HY5 by casein kinase II requires the beta subunit 2, but does not affect HY5's susceptibility to ubiquitination. The RING domain of CIP8 is required but is not sufficient for ubiquitin ligase activity. Although the RING domain of CIP8 interacts with the RING domain of COP1, addition of recombinant COP1 fails to affect CIP8's ubiquitin ligase activity towards HY5 in vitro. However, recombinant COP1 can pull-down native CIP8 from the extract of dark-grown seedlings, but not from the extract of light-grown seedlings in a column-binding assay, implying a requirement for light-regulated modification in vivo. Our data suggest that CIP8 can form a minimal ubiquitin ligase in co-operation with the E2 enzyme AtUBC8. It is possible that the AtUBC8-CIP8 module might interact with COP1 in vivo, thereby participating in proteasome-mediated degradation of HY5.     

The E3 ubiquitin ligase WVIP2 highlights the versatility of protein ubiquitination

Plant cells regulate many cellular processes controlling the half-life of critical proteins through ubiquitination. Previously, we characterized two interacting RING-type E3 ubiquitin ligases of Triticum durum, TdRF1 and WVIP2. We revealed their role in tolerance to dehydration, and existing knowledge about their partners also indicated their involvement in the regulation of some aspects of plant development. Here we located WVIP2 in the regulation of the ABA signaling, based on sequence similarities. Further we acquired general evidence about the versatility of ubiquitination in plant cells. A protein can be target of different E3 ligases for a perfect tuning of its abundance as well as the same E3 ligase can ubiquitinate different and unrelated proteins, thus representing a cross-connections between different signaling pathways for a global coordination of cellular processes.     

In vitro and in vivo screening of essential oils for the control of wet bubble disease of Agaricus bisporus

Proliferation of fungal pathogens, such as Mycogone perniciosa, can severely affect the yields of cultivated mushrooms, including that of the button mushroom, Agaricus bisporus. A reduction in the number of fungicidal products approved for commercial application is currently providing new challenges to the mushroom industry. Forty essential oils, seven pure terpenoids and one phenylpropanoid were screened in vitro to determine the abilities of these substances to inhibit the growth of M. perniciosa. The fungal growth medium of both A. bisporus and M. perniciosa was supplemented with each test substance at a concentration of 50 μL/L. Ten essential oils were further investigated at lower concentrations ranging from 5 to 40 μL/L. The main components of these oils were determined by GC–FID and GC–MS. Lemon verbena (Lippia citriodora), lemongrass (Cymbopogon citratus) and thyme (Thymus vulgaris) oils were found to substantially inhibit the growth of the pathogen, while demonstrating lower toxicity towards A. bisporus than any of the other oils tested. A preliminary in vivo trial using M. perniciosa-inoculated casings revealed that the preventative use of lemon verbena or thyme oils was able to control the development of the disease. A commercial trial using these oils, as well as two of their main components (nerol and thymol), at a concentration of 40 μL/L, revealed that none of these treatments were detrimental to the growth of the A. bisporus and an overall yield similar to that following application of a commercial fungicide (Chronos 450 SC) was obtained. These results suggest that essential oils or mixtures of selected pure components of essential oils may in future find application in button mushroom production, either as a substitute for synthetic fungicides or as an additional protective measure.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

Activation of the E3 ligase function of the BRCA1/BARD1 complex by polyubiquitin chains

Loss of the tumour suppressor BRCA1 results in profound chromosomal instability. The fundamental defect underlying this catastrophic phenotype is not yet known. In vivo, BRCA1 forms a heterodimeric complex with BARD1. Both proteins contain an N-terminal zinc RING-finger domain which confers E3 ubiquitin ligase activity. We have isolated full-length human BRCA1/BARD1 complex and have shown that it has a dual E3 ubiquitin ligase activity. First, it mediates the monoubiquitylation of nucleosome core histones in vitro, including the variant histone H2AX that co-localizes with BRCA1 at sites of DNA damage. Secondly, BRCA1/BARD1 catalyses the formation of multiple polyubiquitin chains on itself. Remarkably, this auto-polyubiquitylation potentiates the E3 ubiquitin ligase activity of the BRCA1/BARD1 complex >20-fold. Even though BRCA1 has been reported to associate with a C-terminal ubiquitin hydrolase, BAP1, this enzyme does not appear to function in the deubiquitylation of the BRCA1/BARD1 complex.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesiabovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination.     

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.