Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H 2O2) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30 -90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H 2O2 or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redoxsensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.chaperone ͉ proteomics ͉ thiol modification

The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

We carried out a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in LC-MS-based proteomics. We distributed a test sample consisting of an equimolar mix of 20 highly purified recombinant human proteins, to 27 laboratories for identification. Each protein contained one or more unique tryptic peptides of 1250 Da to also test for ion selection and sampling in the mass spectrometer. Of the 27 labs, initially only 7 labs reported all 20 proteins correctly, and only 1 lab reported all the tryptic peptides of 1250 Da. Nevertheless, a subsequent centralized analysis of the raw data revealed that all 20 proteins and most of the 1250 Da peptides had in fact been detected by all 27 labs. The centralized analysis allowed us to determine sources of problems encountered in the study, which include missed identifications (false negatives), environmental contamination, database matching, and curation of protein identifications. Improved search engines and databases are likely to increase the fidelity of mass spectrometry-based proteomics.

Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-beta induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC-MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down-regulated. Down-regulated proteins were predominantly enzymes involved in regulating nutrient or drug metabolism. The majority of the TGF-beta-induced proteins (such as tropomyosins, filamin A, B, & C, integrin-beta1, heat shock protein27, transglutaminase2, cofilin, 14-3-3 zeta, ezrin-radixin-moesin) are involved in the regulation of cell migration, adhesion and invasion, suggesting the acquisition of a invasive phenotype.

The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA E , in late steps of large ribosomal subunit biogenesis. CgtA E belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA E cause both polysome and rRNA processing defects; small-and large-subunit precursor rRNAs accumulate in a cgtA E mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA E is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA E . We also show that purified CgtA E associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA E cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.Although assembly of prokaryotic ribosomes from purified ribosomal proteins (r-proteins) and rRNAs can occur independently in vitro (51, 52, 75), accumulating evidence suggests that, as in eukaryotes, in vivo prokaryotic ribosome biogenesis depends on the aid of nonribosomal factors. The higher temperature, increased Mg 2ϩ concentration, and longer incubation times necessary for in vitro relative to in vivo conditions (51) imply that the likely role of accessory factors is to expedite ribosome maturation by reducing the activation energies for the rate-limiting reactions. Although not complicated by the involvement of different cellular compartments, the prokaryotic ribosome assembly process is far from simple, requiring coordinated synthesis of 3 rRNAs (5S, 16S, and 23S) and 55 r-proteins, processing and modification of these components, and their appropriate sequential unification to produce mature ribosomes. The details of how this process is controlled temporally, even spatially, in the small bacterial cell are incompletely understood.More than 170 nonribosomal proteins that transiently associate with different preribosomal particles have been identified in Saccharomyces cerevisiae (19,22,38,62), largely due to progress in combining biochemical affinity purification methods with newly developed proteomic techniques (24,25,29,54,58,61). By contrast, only a few such assembly factors have been found in bacteria, and most were identified via conventional genetic methods. These proteins consist of RNA-modifying enzymes such as methyltransferases and pseudouridine synthases, RNA-remodeling proteins such as RNA helicases, chaperones, GTPases, and proteins with unknown functions (1,

The hormone ghrelin stimulates eating and helps maintain blood glucose upon caloric restriction. While previous studies have demonstrated that hypothalamic arcuate AgRP neurons are targets of ghrelin, the overall relevance of ghrelin signaling within intact AgRP neurons is unclear. Here, we tested the functional significance of ghrelin action on AgRP neurons using a new, tamoxifen-inducible AgRP-CreERT2 transgenic mouse model that allows spatiotemporally-controlled re-expression of physiological levels of ghrelin receptors (GHSRs) specifically in AgRP neurons of adult GHSR-null mice that otherwise lack GHSR expression. AgRP neuron-selective GHSR re-expression partially restored the orexigenic response to administered ghrelin and fully restored the lowered blood glucose levels observed upon caloric restriction. The normalizing glucoregulatory effect of AgRP neuron-selective GHSR expression was linked to glucagon rises and hepatic gluconeogenesis induction. Thus, our data indicate that GHSR-containing AgRP neurons are not solely responsible for ghrelin's orexigenic effects but are sufficient to mediate ghrelin's effects on glycemia.

Growth hormone secretagogue receptor (GHSR) 1a is the only molecularly identified receptor for ghrelin, mediating ghrelin-related effects on eating, body weight and blood glucose control, among others. The expression pattern of GHSR within the brain has been assessed previously using several neuroanatomical techniques. However, inherent limitations to these techniques and the lack of reliable anti-GHSR antibodies and reporter rodent models that identify GHSR-containing neurons have prevented a more comprehensive functional characterization of ghrelin-responsive neurons. Here, we have systematically characterized the brain expression of an enhanced green fluorescence protein (eGFP) transgene controlled by the Ghsr promoter in a recently-reported GHSR reporter mouse. Expression of eGFP in coronal brain sections was compared with GHSR mRNA expression detected in the same sections by in situ hybridization histochemistry. eGFP-immunoreactivity was detected in several areas including the prefrontal cortex, insular cortex, olfactory bulb, amygdala and hippocampus, which showed no or low GHSR mRNA expression. In contrast, eGFP expression was low in several midbrain regions and in several hypothalamic nuclei – particularly the arcuate nucleus– where robust GHSR mRNA expression has been well-characterized. eGFP expression in several brainstem nuclei showed high to moderate degrees of co-localization with GHSR mRNA labeling. Further quantitative PCR and electrophysiological analyses of eGFP-labeled hippocampal cells confirmed faithful expression of eGFP within GHSR-containing, ghrelin-responsive neurons. In summary, the GHSR-eGFP reporter mouse model may be a useful tool to study GHSR function – particularly within the brainstem and hippocampus– however, it underrepresents GHSR expression in nuclei within the hypothalamus and midbrain.

Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders1–6 and are among the most prevalent in autism spectrum disorders1,2,7. Of many 16p11.2 genes, Kctd13 has been implicated as a major driver of neurodevelopmental phenotypes8,9. The function of KCTD13 in the mammalian brain, however, remains unknown. Here we delete the Kctd13 gene in mice and demonstrate reduced synaptic transmission. Reduced synaptic transmission correlates with increased levels of Ras homolog gene family, member A (RhoA), a KCTD13/CUL3 ubiquitin ligase substrate, and is reversed by RhoA inhibition, suggesting increased RhoA as an important mechanism. In contrast to a previous knockdown study8, deletion of Kctd13 or kctd13 does not increase brain size or neurogenesis in mice or zebrafish, respectively. These findings implicate Kctd13 in the regulation of neuronal function relevant to neuropsychiatric disorders and clarify the role of Kctd13 in neurogenesis and brain size. Our data also reveal a potential role for RhoA as a therapeutic target in disorders associated with KCTD13 deletion.