A ubiquitin ligase HRD1 promotes the degradation of Pael receptor, a substrate of Parkin

It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R.     

Leaf beetle grazing does not induce willow trichome defence in the coppicing willow Salix viminalis

Abstract 1 Willows are frequently attacked and defoliated by adult leaf beetles (Phratora vulgatissima L.) early in the season and the plants are then attacked again when new larvae emerge. The native willow Salix cinerea has previously been shown to respond to adult grazing by producing new leaves with an increased trichome density. Subsequent larval feeding was reduced on new leaves. This type of induced plant response may reduce insect damage and could potentially be utilized for plant protection in agricultural systems. 2 Here, we investigated if the willow species most commonly used for biomass production in short rotation coppice, Salix viminalis, also responds to adult beetle grazing by increasing trichome density. Larval performance and feeding behaviour on plants previously exposed to adult beetles was compared with that on undefoliated control plants in a greenhouse. 3 We found an overall decrease in trichome density within all the plants (i.e. trichome density was lower on new leaves compared to that for older basal leaves on S. viminalis). However, leaves of beetle defoliated plants had a higher trichome density compared to control plants. Larval growth and feeding was not affected by this difference between treatments. Larvae appeared to remove trichomes when feeding on S. viminalis, a behaviour that might explain the lack of difference between treatments.     

Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.     

S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.     

Annokey: an annotation tool based on key term search of the NCBI Entrez Gene database

Background

The NCBI Entrez Gene and PubMed databases contain a wealth of high-quality information about genes for many different organisms. The NCBI Entrez online web-search interface is convenient for simple manual search for a small number of genes but impractical for the kinds of outputs seen in typical genomics projects.

Results

We have developed an efficient open source tool implemented in Python called Annokey, which annotates gene lists with the results of a keyword search of the NCBI Entrez Gene database and linked Pubmed article information. The user steers the search by specifying a ranked list of keywords (including multi-word phrases and regular expressions) that are correlated with their topic of interest. Rank information of matched terms allows the user to guide further investigation.We applied Annokey to the entire human Entrez Gene database using the key-term “DNA repair” and assessed its performance in identifying the 176 members of a published “gold standard” list of genes established to be involved in this pathway. For this test case we observed a sensitivity and specificity of 97% and 96%, respectively.

Conclusions

Annokey facilitates the identification of genes related to an area of interest, a task which can be onerous if performed manually on a large number of genes. Annokey provides a way to capitalize on the high quality information provided by the Entrez Gene database allowing both scalability and compatibility with automated analysis pipelines, thus offering the potential to significantly enhance research productivity.

     

De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line

It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in³⁵S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [³⁵S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

The Sec63p J-Domain Is Required for ERAD of Soluble Proteins in Yeast

How misfolded proteins are exported from the ER to the cytosol for degradation (ER-associated Degradation, ERAD) and which proteins are participating in this process is not understood. Several studies using a single, leaky mutant indicated that Sec63p might be involved in ERAD. More recently, Sec63p was also found strongly associated with proteasomes attached to the protein-conducting channel in the ER membrane which presumably form part of the export machinery. These observations prompted us to reinvestigate the role of Sec63p in ERAD by generating new mutants which were selected in a screen monitoring the intracellular accumulation of the ERAD substrate CPY*. We show that a mutation in the DnaJ-domain of Sec63p causes a defect in ERAD, whereas mutations in the Brl, acidic, and transmembrane domains only affect protein import into the ER. Unexpectedly, mutations in the acidic domain which mediates interaction of Sec63p with Sec62p also caused defects in cotranslational import. In contrast to mammalian cells where SEC63 expression levels affect steady-state levels of multi-spanning transmembrane proteins, the sec63 J-domain mutant was only defective in ERAD of soluble substrates.     

Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.