A Conserved Di‐Basic Motif of Drosophila Crumbs Contributes to Efficient ER Export

The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico‐basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse‐chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di‐basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene‐encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di‐basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism.      

Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca²⁺-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of ³²[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca²⁺-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.     

Characterization of the Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative Standard using SDS-PAGE shotgun proteomics

Although there are now multiple methods for the analysis of membrane proteomes, there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative (MPI) using a large range of workflows. Here, we describe the characterization of the MPI mouse liver microsomal membrane Standard using SDS-PAGE prior to in-gel tryptic digestion and LC-ESI-MS/MS. The Na(2) CO(3) wash followed by SDS-PAGE prior to in-gel tryptic digestion and LC-MS/MS strategy was effective for the detection of membrane proteins with 47.1% of the identified proteins being transmembrane proteins. Gene Ontology term enrichment analysis showed that biological processes involving transport, lipid metabolism, cell communication, cell adhesion, and cellular component organization were significantly enriched. Comparison of the present data with the previously published reports on mouse liver proteomes confirmed that the MPI Standard provides an excellent resource for the analysis of membrane proteins in the AOHUPO MPI.     

Targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice

Use of the chemotherapeutic agent doxorubicin (Dox) is limited by dose-dependent cardiotoxic effects. The molecular mechanism underlying these toxicities are incompletely understood, but previous results have demonstrated that Dox induces p53 expression. Because p53 is an important regulator of the cell birth and death we hypothesized that targeted disruption of the p53 gene would attenuate Dox-induced cardiotoxicity. To test this, female 6–8 wk old C57BL wild-type (WT) or p53 knockout (p53 KO) mice were randomized to either saline or Dox 20 mg/kg via intraperitoneal injection. Animals were serially imaged with high-frequency (14 MHz) two-dimensional echocardiography. Measurements of left ventricle (LV) systolic function as assessed by fractional shortening (FS) demonstrated a decline in WT mice as early as 4 days after Dox injection and by 2 wk demonstrated a reduction of 31± 16% (P < 0.05) from the baseline. In contrast, in p53 KO mice, LV FS was unchanged over the 2 wk period following Dox injection. Apoptosis of cardiac myocytes as measured by the TUNEL and ligase reactions were significantly increased at 24 h after Dox treatment in WT mice but not in p53 KO mice. After Dox injection, levels of myocardial glutathione and Cu/Zn superoxide dismutase were preserved in p53 KO mice, but not in WT animals. These observations suggest that p53 mediated signals are likely to play a significant role in Dox-induced cardiac toxicity and that they may modulate Dox-induced oxidative stress.These two authors equally contributed to this study.     

An evaluation, comparison, and accurate benchmarking of several publicly available MS/MS search algorithms: sensitivity and specificity analysis

MS/MS and associated database search algorithms are essential proteomic tools for identifying peptides. Due to their widespread use, it is now time to perform a systematic analysis of the various algorithms currently in use. Using blood specimens used in the HUPO Plasma Proteome Project, we have evaluated five search algorithms with respect to their sensitivity and specificity, and have also accurately benchmarked them based on specified false-positive (FP) rates. Spectrum Mill and SEQUEST performed well in terms of sensitivity, but were inferior to MASCOT, X!Tandem, and Sonar in terms of specificity. Overall, MASCOT, a probabilistic search algorithm, correctly identified most peptides based on a specified FP rate. The rescoring algorithm, PeptideProphet, enhanced the overall performance of the SEQUEST algorithm, as well as provided predictable FP error rates. Ideally, score thresholds should be calculated for each peptide spectrum or minimally, derived from a reversed-sequence search as demonstrated in this study based on a validated data set. The availability of open-source search algorithms, such as X!Tandem, makes it feasible to further improve the validation process (manual or automatic) on the basis of "consensus scoring", i.e., the use of multiple (at least two) search algorithms to reduce the number of FPs. complement.     

Activation and migration of allo-peptide specific TCR transgenic T cells in cardiac allograft rejection

T cells from TCR transgenic mice, expressing receptors specific for an allogeneic MHC class I peptide, were used to track T cell activation and migration in normal adoptive recipients that were subsequently transplanted with heterotopic hearts that were syngeneic except for a transgenic MHC class I antigen. T cells rapidly disappeared from the blood into the lymphoid tissues where they were activated within one day after transplantation. T cells initially formed discrete clusters in the spleen and lymph nodes. After proliferating for 2-4 days in lymphoid tissues, T cells reappeared in the blood and migrated to the heart and the intestines. The T cells underwent another round of proliferation in the heart, but not the intestines, and induced cardiac rejection uniformly on 6 day.     

E6AP ubiquitin ligase mediates ubiquitin‐dependent degradation of peroxiredoxin 1

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high‐risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin‐dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP‐binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25‐kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull‐down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi‐mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin‐dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1‐dependent intracellular oxidative signal transduction pathways. J. Cell. Biochem. 111: 676–685, 2010. © 2010 Wiley‐Liss, Inc.     

Ameliorating role of caffeic acid phenethyl ester (CAPE) against isoniazid-induced oxidative damage in red blood cells

Isoniazid (INH) still remains a first-line drug both for treatment and prophylaxis of tuberculosis, but various organs toxicity frequently develops in patients receiving this drug. We aimed to investigate possible toxic effects of INH on rat red blood cells (RBCs), and to elucidate whether Caffeic acid phenethyl ester (CAPE) prevents a possible toxic effect of INH. Experimental groups were designed as follows: control group, INH group, INH + CAPE group. Compared with the control, the INH caused a significant increase in superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels, and a decrease in glutathione peroxidase (GSH-Px) and catalase (CAT), which are recently used to monitor the development and extent of damage due to oxidative stresses. CAPE administration to INH group ameliorated above changes due to INH.