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Estrogen insufficiency at menopause cause accelerated bone loss due to unwarranted differentiation and function of osteoclasts. Unraveling the underlying mechanism/s may identify mediators of estrogen action which can be targeted for improved management of osteoporosis. Towards this, we analyzed the effect of 17β-estradiol on the proteomes of differentiating human osteoclasts. The major proteomic changes observed included upregulation of LYN by estrogen. We, therefore, investigated the effect of estrogen on osteoclast differentiation, survival, and function in control and LYN knockdown conditions. In control condition, estrogen treatment increased the apoptosis rate and suppressed the calcium signaling by reducing the intracellular Ca2+ levels as well as expression and activation of NFATc1 and c-Src during differentiation, resulting in reduced osteoclastogenesis. These osteoclasts were smaller in size with reduced extent of multinuclearity and produced significantly low levels of bone resorbing enzymes. They also exhibited disrupted sealing zone formation with low podosome density, impaired cell polarization and reduced resorption of dentine slices. Interestingly, in LYN knockdown condition, estrogen failed to induce apoptosis and inhibit activation of NFATc1 and c-Src. Compared to effect of estrogen on osteoclast in control condition, LYN knockdown osteoclasts did not show reduction in production of bone resorbing enzymes and had defined sealing zone formation with high podosome density with no impairment in cell polarization. They resorbed significant area on dentine slices. Thus, the inhibitory action of estrogen on osteoclast was severely restrained in LYN knockdown condition, demonstrating the importance of LYN as a key mediator of the effect of estrogen on osteoclastogenesis.

We have carried out proteogenomic analysis of the breast cancer transcriptomic and proteomic data, available at The Clinical Proteomic Tumor Analysis Consortium resource, to identify novel peptides arising from alternatively spliced events as well as other noncanonical expressions. We used a pipeline that consisted of de novo transcript assembly, six frame-translated custom database, and a combination of search engines to identify novel peptides. A portfolio of 4,387 novel peptide sequences initially identified was further screened through PepQuery validation tool (Clinical Proteomic Tumor Analysis Consortium), which yielded 1,558 novel peptides. We considered the dataset of 1,558 validated through PepQuery to understand their functional and clinical significance, leaving the rest to be further verified using other validation tools and approaches. The novel peptides mapped to the known gene sequences as well as to genomic regions yet undefined for translation, 580 novel peptides mapped to known protein-coding genes, 147 to non-protein-coding genes, and 831 belonged to novel translational sequences. The novel peptides belonging to protein-coding genes represented alternatively spliced events or 5' or 3' extensions, whereas others represented translation from pseudogenes, long noncoding RNAs, or novel peptides originating from uncharacterized protein-coding sequences-mostly from the intronic regions of known genes. Seventy-six of the 580 protein-coding genes were associated with cancer hallmark genes, which included key oncogenes, transcription factors, kinases, and cell surface receptors. Survival association analysis of the 76 novel peptide sequences revealed 10 of them to be significant, and we present a panel of six novel peptides, whose high expression was found to be strongly associated with poor survival of patients with human epidermal growth factor receptor 2-enriched subtype. Our analysis represents a landscape of novel peptides of different types that may be expressed in breast cancer tissues, whereas their presence in full-length functional proteins needs further investigations.

GPR56/ADGRG1 is a member of the adhesion G-protein coupled receptor (aGPCR) family and one of the important players in the normal development of the brain. It plays a pivotal role in the diverse neurobiological processes, including cortical formation, oligodendrocyte development, and myelination. Mutations in GPR56 are known to cause brain malformation, myelination defects and are also implied in many cancers, including brain tumors. Since its identification almost two decades ago, GPR56 has emerged from an orphaned and uncharacterized GPCR to an increasingly well studied receptor. Yet, much needs to be understood about GPR56, both in terms of its molecular interactions and biological functions that may be relevant in normal health and disease. The review is focussed on the recent available knowledge of GPR56, which would give useful insights into its known and potential roles in the human brain, neurological disorders, and brain tumors like glioblastoma.

Splice variants are known to be important in the pathophysiology of tumors, including the brain cancers. We applied a proteogenomics pipeline to identify splice variants in glioblastoma (GBM, grade IV glioma), a highly malignant brain tumor, using in-house generated mass spectrometric proteomic data and public domain RNASeq dataset. Our analysis led to the identification of a novel exon that maps to the long isoform of Neural cell adhesion molecule 1 (NCAM1), expressed on the surface of glial cells and neurons, important for cell adhesion and cell signaling. The presence of the novel exon is supported with the identification of five peptides spanning it. Additional peptides were also detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separated proteins from GBM patient tissue, underscoring the presence of the novel peptides in the intact brain protein. The novel exon was detected in the RNASeq dataset in 18 of 25 GBM samples and separately validated in additional 10 GBM tumor tissues using quantitative real-time-polymerase chain reaction (qRT-PCR). Both transcriptomic and proteomic data indicate downregulation of NCAM1, including the novel variant, in GBM. Domain analysis of the novel NCAM1 sequence indicates that the insertion of the novel exon contributes extra low-complexity region in the protein that may be important for protein-protein interactions and hence for cell signaling associated with tumor development. Taken together, the novel NCAM1 variant reported in this study exemplifies the importance of future multiomics research and systems biology applications in GBM.

Glioblastoma multiforme (GBM) is one of the most lethal brain tumors with an inadequately understood pathophysiology. Biomarkers that guide accurate diagnosis and treatment decisions would greatly support precision medicine for GBM. Previous studies of GBM have focused on signaling pathways such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptors (PDGFRs), notch, wnt, and others, identified with single omics technology platforms (genomics, transcriptomics, or proteomics), but not with their integrated use. In this context, we report here a multi-omics pathway view, expanded through integration of the expression data at transcriptomic and proteomic levels, followed by selection of a functionally related group of proteins such as kinases deregulated in GBM. By using this strategy, we observed a highly significant enrichment of the gonadotropin-releasing hormone (GnRH) signaling pathway that was not deciphered with single omics datasets. The curation of the GnRH pathway with extensive literature analysis brought about a comprehensive annotation of the pathway, which included several additional pathway members that were not previously annotated. A targeted search resulted in identification of additional nonkinase members of the pathway in the GBM multi-omics datasets. We found evidence of GnRH receptor expression in GBM and other cancers. We offer here an updated generic pathway map of GnRH signaling, show its enrichment in the context of GBM, and discuss its plausible cross-connectivity with EGFR, wnt, calcium, and focal adhesion kinase signaling pathways that were earlier shown to be the top deregulated pathways in GBM. In conclusion, this study demonstrates the promise of multi-omics research and analyses to better understand complex cancers and suggests continued efforts and research in this direction in the field of integrative biology.

Glioblastoma multiforme (GBM) is one of the most aggressive and lethal forms of the primary brain tumors. With predominance of tumor heterogeneity and emergence of new subtypes, new approaches are needed to develop tissue-based markers for tumor typing or circulatory markers to serve as blood-based assays. Multi-omics data integration for GBM tissues would offer new insights on the molecular view of GBM pathogenesis useful to identify biomarker panels. On the other hand, mapping differentially expressed tissue proteins for their secretory potential through bioinformatics analysis or analysis of the tumor cell secretome or tumor exosomes would enhance our understanding of the tumor microenvironment and prospects for targeting circulatory biomarkers. In this review, the authors first present potential biomarker candidates for GBM that have been reported and then focus on plausible pipelines for multi-omic data integration to identify additional, high-confidence molecular panels for clinical applications in GBM.

Sample processing protocols that enable compatible recovery of differentially expressed (DE) transcripts and proteins are necessary for integration of the multiomics data applied in the analysis of tumors. In this pilot study, we compared two different isolation methods for extracting RNA and protein from laryngo-pharyngeal tumor tissues and the corresponding adjacent normal sections. In Method 1, RNA and protein were isolated from a single tissue section sequentially and in Method 2, the extraction was carried out using two different sections and two independent and parallel protocols for RNA and protein. RNA and protein from both methods were subjected to RNA-seq and iTRAQ-based LC-MS/MS analysis, respectively. Analysis of data revealed that a higher number of differentially expressed transcripts and proteins were concordant in their regulation trends in Method 1 as compared to Method 2. Cross method comparison of concordant entities revealed that RNA and protein extraction from the same tissue section (Method 1) recovered more concordant entities that are missed in the other extraction method (Method 2) indicating heterogeneity in distribution of these entities in different tissue sections. Method 1 could thus be the method of choice for integrated analysis of transcriptome and proteome data. This article is protected by copyright. All rights reserved

Tobacco-related oral cancer is the most common cancer among Indian males, gingivo-buccal complex (GBC) being the most affected subsite due to the habit of chewing tobacco. Proteins from the lysates of microdissected normal and transformed epithelium from clinically well-characterized tissue samples of the GBC were separated by two-dimensional gel electrophoresis to identify differentially expressed proteins. Eleven protein spots showed differential expression, which could withstand the stringency of statistical evaluation. The observations were confirmed with additional tissues. Nine of these differentiators were identified by MS as lactate dehydrogenase B, α-enolase, prohibitin, cathepsin D, apolipoprotein A-I, tumor protein translationally controlled-1, an SFN family protein, 14-3-3σ and tropomyosin. Cluster analysis indicated that these proteins, as a coexpressed set, could distinguish normal and transformed epithelium. Functionally, these differentiator molecules are relevant to the pathways and processes that have been previously implicated in oral carcinogenesis and could therefore be investigated further as a panel of markers for management of cancer of the GBC.

High throughput molecular profiling and integrated data analysis with tumor tissues require overcoming challenges like tumor heterogeneity and tissue paucity. This study is an attempt to understand and optimize various steps during tissue processing and in establishing pipelines essential for integrated analysis. Towards this effort, we subjected laryngo-pharyngeal primary tumors and the corresponding adjacent normal tissues (n=2) to two RNA and protein isolation methods, one wherein RNA and protein were isolated from the same tissue sequentially (Method 1) and second, wherein the extraction was carried out using two independent methods (Method 2). RNA and protein from both methods were subjected to RNA-seq and iTRAQ based LC-MS/MS analysis. Transcript and peptide identification and quantification was followed by both individual -ome and integrated data analysis. As a result of this analysis, we identified a higher number of total, as well as differentially expressed (DE) transcripts (1329 vs 1134) and proteins (799 vs 408) with fold change ≥ 2.0, in Method 1. Among these, 173 and 86 entities were identified by both transcriptome and proteome analysis in Method 1 and 2, respectively, with higher concordance in the regulation trends observed in the former. The significant cancer related pathways enriched with the individual DE transcript or protein data were similar in both the methods. However, the entities mapping to them were different, allowing enhanced view of the pathways identified after integration of the data and subsequent mapping. The concordant DE transcripts and proteins also revealed key molecules of the pathways with important roles in cancer development. This study thus demonstrates that sequential extraction of the RNA and proteins from the same tissue allows for better profiling of differentially expressed entities and a more accurate integrated data analysis.

Peripheral monocytes, precursors of osteoclasts, have emerged as important candidates for identifying proteins relevant to osteoporosis, a condition characterized by low Bone Mineral Density (BMD) and increased susceptibility for fractures. We employed 4-plex iTRAQ (isobaric tags for relative and absolute quantification) coupled with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) to identify differentially expressed monocyte proteins from premenopausal and postmenopausal women with low versus high BMD. Of 1801 proteins identified, 45 were differentially abundant in low versus high BMD, with heat shock protein 27 (HSP27) distinctly upregulated in low BMD condition in both premenopausal and postmenopausal categories. Validation in individual samples (n=80) using intracellular ELISA confirmed that total HSP27 (tHSP27) as well as phosphorylated HSP27 (pHSP27) was elevated in low BMD condition in both categories (P<0.05). Further, using transwell assays, pHSP27, when placed in the upper chamber, could increase monocyte migration (P<0.0001) and this was additive in combination with RANKL (receptor activator ofNFkBligand) placed in the lower chamber (P=0.05). Effect of pHSP27 in monocyte migration towards bone milieu can result in increased osteoclast formation and thus contribute to pathogenesis of osteoporosis. Overall, this study reveals for the first time a novel link between monocyte HSP27 and BMD.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

Gliomas are the most common of the primary intracranial tumors with astrocytomas constituting about 40%. Using clinically and histologically assessed astrocytomas, we have studied their protein profiles using a two-dimensional gel electrophoresis-mass spectrometry approach and identified differentially expressed proteins which may be useful molecular indicators to understand these tumors. Examination of the protein profiles of 27 astrocytoma samples of different grades revealed 72 distinct, differentially expressed proteins belonging to various functional groups such as cytoskeleton and intermediate filament proteins, heat shock proteins (HSPs), enzymes and regulatory proteins. Based on the consistency of their differential expression, 29 distinct proteins could be short-listed and may have a role in the pathology of astrocytomas. Some were found to be differentially expressed in both Grade III and IV astrocytomas while others were associated with a particular grade. A notable observation was underexpression of Prohibitin, a potential tumor suppressor protein, Rho-GDP dissociation inhibitor, Rho-GDI, a regulator of Rho GTPases and HSPs as well as destabilization of glial fibrillary acidic protein, GFAP, major protein of the glial filaments, in Grade III malignant tumors. We attempt to explain glioma malignancy and progression in terms of their combined role.

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary malignant tumor of the central nervous system. We have carried out a deep analysis of the secretome of a rapidly proliferating and tumorigenic cell line HNGC-2, representing GBM, in an effort to identify proteins, which may be targeted in the plasma of GBM patients as markers for diagnosis and disease surveillance. Prefractionation of the proteins from the conditioned medium of HNGC-2 cells in SDS gels followed by LC-MS/MS analysis using an ESI-IT mass spectrometer (LTQ) led to a total of 996 protein identifications with ≥2 peptides each. Of them, 664 proteins were observed in the transcriptome of HNGC-2 cells. The dataset of 996 proteins was mapped to important functional groups, such as cellular assembly and organisation, DNA recombination and repair, and other classes. Actin cytoskeleton signalling, phosphatidyl inositol 3 kinase (PI3K/AKT) and integrin linked kinase (ILK) signalling pathways were seen as enriched pathways. Comparisons with the published secretome of cell lines from 12 different cancers, including GBM, revealed that 348 proteins shared a commonality with a secretome of at least one other cell line, 321 of which were found to contain signal sequences or transmembrane domains and 335 could be linked to a plasma membrane or extracellular localization. Through intergration of this data we arrived at a non-redundant list of 597 protein identifications with the potential for secretion either by classical secretory pathways or by non-secretory processes; 233 of them have been detected in cerebrospinal fluid or plasma as per the published literature, and 172 have been implicated in GBM or other cancers. The HNGC-2 secretome dataset could serve as a useful resource for designing a targeted investigation of GBM biomarkers in plasma.

Corticotropin-releasing hormone (CRH), also known as corticotropin-releasing factor (CRF), is a 41-amino acid neuropeptide, expressed abundantly by CRH neurons present in the paraventricular nucleus (PVN) of the hypothalamus, and other parts of the brain (Aguilera and Liu 2012; Vale et al. 1981). CRH is also expressed in adrenal gland, immune cells, placenta, testis, spleen, gut, thymus and skin (Dautzenberg and Hauger 2002; Aguilera and Liu 2012). The activity of the hypothalamic CRH neurons varies in resting and stress conditions. Circadian variations have an effect on CRH neuron activity. On stressful stimuli, sensory information is either transmitted directly to the PVN or integrated by the limbic system and transmitted to the CRH neurons via complex monoaminergic and peptidergic neural pathways. This is dependent on the nature, intensity and duration of the stressor. Systemic and metabolic stressors including blood loss, pain, immune challenge and hypoglycemia, which require immediate response, utilize monosynaptic pathways, whereas, psychogenic stressors utilize complex multisynaptic pathways. These neural pathways trigger signaling in CRH neurons, increasing CRH gene transcription and rapid CRH secretion (Aguilera and Liu 2012). The actions of CRH are transduced through CRH receptors, which belong to the class II/secretin-like family of the G-protein coupled receptor (GPCR) superfamily (Martin et al. 2005). There are three types of CRH receptors – type 1 (CRHR1), type 2 (CRHR2) and type 3 (CRHR3). Among these, CRHR3 has not been identified in mammals. CRHR1 and CRHR2 are encoded by different genes. CRHR1 mRNA has several splice variants encoding different isoforms – R1α, R1β, R1c, R1d, R1e, R1f, R1g and R1h, predominant of which is CRHR1α (Chen et al. 1993; Ross et al. 1994; Grammatopoulos et al. 1999; Pisarchik and Slominski 2001, 2004). CRHR1 is expressed predominantly in the CNS, pituitary, heart (Chen et al. 1993) and also in adrenal gland, ovary, and placenta (Grammatopoulos et al. 1999; Seres et al. 2004; Asakura et al. 1997). The gene encoding the human CRHR2 has three mRNA splice variants which encodes 3 isoforms - R2α, R2β and R2γ and is expressed predominantly in brain and heart (Kostich et al. 1998; Yang et al. 2010). CRH is a high-affinity ligand of CRHR1. It also binds to CRHR2, but with lower affinity (Hsu and Hsueh 2001; Grammatopoulos et al. 1999; Wille et al. 1999). CRH receptors do not have any intrinsic kinase activity and the signal is transduced via the heterotrimeric G-proteins (Freissmuth et al. 1989). Biological activity of CRH is regulated by CRH binding protein (CRHBP), which by binding to the former with high affinity, makes it unavailable for binding to CRH receptors (Potter et al. 1991; Behan et al. 1995; Seasholtz et al. 2002). CRHBP is expressed in high levels in the brain, heart, lungs, and placenta (Potter et al. 1991; Behan et al. 1995; Binder and Nemeroff 2010). CRH exerts its functions through activation of several signaling pathways including adenylate cyclase/protein kinase A (PKA), phospholipase C (PLC)/protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) pathways. CRH, as a principal mediator of endocrine stress response, activates the HPA axis (Hypothalamic–pituitary–adrenal axis) by binding to the CRHR1 in the anterior pituitary. This, through a cascade of reactions, increases the expression of proopiomelanocortin (POMC) gene and the subsequent release of POMC-derived peptides, adrenocorticotropic hormone (ACTH) and β-endorphin. ACTH, in turn, stimulates the secretion of glucocorticoids from adrenal cortex (Vale et al. 1981). CRH is involved in the etiologies of several stress-related physiological responses and behavioral responses, such as altered blood pressure, increased arousal, enhanced learning, thermogenesis, anxiogenesis, anhendonia, reduced sleep, psychomotor alterations, decreased appetite and libido (Binder and Nemeroff 2010). CRH, upon binding to hippocampal CRHR1, mediates stress-induced enhancement of fear conditioning and learning. In contrast, CRH by binding to the lateral septal CRHR2 mediates stress-induced anxiety and impairs fear conditioning and learning (Radulovic et al. 1999). Pro-inflammatory and anti-inflammatory responses are exerted through peripheral and central CRH, respectively (Karalis et al. 1991; Friedman and Irwin 2001). Binding of peripheral and central CRH to their receptors were implicated in hemodynamic actions (Yang et al. 2010). Endometrial CRH was found to be involved in stromal cell decidualization during estrus cycle (Zoumakis et al. 2000) and also in implantation of blastocyst (Athanassakis et al. 1999). Placental CRH is involved in the maintenance of pregnancy and onset of labor (McLean et al. 1995). Excess secretion of CRH during severe depression and its association with increased levels of cortisol have been observed (Widerlov et al. 1988). CRH was also reported to be involved in anxiety disorders (Roy-Byrne et al. 1986), and anorexia nervosa (Connan et al. 2007). Decrease in cortical CRH content was observed in Alzheimer’s disease (Heilig et al. 1995) and Parkinson’s disease (Suemaru et al. 1995). Diverse aspects of CRH signaling have been well-studied. Owing to its high biological significance, a detailed documentation of all molecular reactions in a centralized resource and depiction of these reactions into a pathway map is desired in the public domain. Therefore, we have curated each pathway reaction reported downstream to CRH-CRHR interaction and submitted a detailed CRH signaling pathway data to the NetPath (http://www.netpath.org) (Kandasamy et al. 2010). Several such ligand-receptor signaling pathways including Leptin (Nanjappa et al. 2011), TWEAK (Bhattacharjee et al. 2012) and Prolactin (Radhakrishnan et al. 2012), had been developed by our group and submitted to NetPath. Here, we describe the generation of an integrated pathway map of CRH signaling by manual curation.

Studying proteins expressed in placenta is important to understand its function in pregnancy and fetal growth. Here, we present protein expression profiling from normal human placenta by 2-D gel – MS/MS approach that resulted in identification of 117 unique proteins. Integration with earlier analyses resulted in a profile of 423 non-redundant proteins, 75 of them being new identifications unique to this study including their isoforms. We present a compilation of placental protein expressions identified by proteomic approaches, their functions and known clinical implications. We believe that our dataset would be a useful resource for studies related to placental dysfunction.

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate-washing step enriches for integral and lipid-anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.

Onconase, a cytotoxic and antitumor RNase inhibits viral replication in chronically HIV-1-infected human cells under sub lethal concentrations. Cellular tRNA has been implicated as the target for onconase action. We have recently shown that onconase cleaves selectively at GG residues in the UGG context in the variable loop and D-arm of the tRNA substrates. We therefore examined onconase cleavage specificity in in vitro transcribed tRNA Lys3 , which is the primer for HIV-1 reverse transcription but does not have UGG anywhere in its sequence. Onconase was found to cleave tRNA Lys3 predominantly at the GG residues in the GGG triplet present in the variable loop. Mutations at this site did not effect onconase cleavages. Interestingly thus, onconase seems to cleave predominantly in the variable loop of tRNA Lys3 regardless of the sequence context implying possible contribution of even structural determinants for its selective cleavages.

Onconase, a protein from amphibian eggs and a homologue of pancreatic ribonuclease (RNase) superfamily, is cytotoxic, exhibits antitumor and antiviral activity, and is in phase III clinical trials. It has been shown to predominantly target cellular tRNA on its entry into mammalian cells (Saxena, S. K., Sirdeshmukh, R., Ardelt, W., Mikulski, S. M., Shogen, K., and Youle, R. J. (2002) J. Biol. Chem. 277, 15142-15146). Cleavage site mapping using natural tRNA substrates, in vitro, revealed predominant cleavage sites at UG and GG residues. Cleavages at UG or the less intense cleavages at CG sites are consistent with the known base specificity of onconase. However, predominance of cleavages at selected G-G bonds is unusual for a homologue of pancreatic RNases. Interestingly, in at least three of the four tRNA substrates studied, the predominant cleavages mapped in the triplet UGG located in the context of the variable loop or the D-arm of the tRNA. The cleavage specificity of onconase observed by us thus indicates another special feature of this enzyme, which may be relevant to its cellular actions.

Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.

Membrane proteins play key roles in the development and progression of cancer. We have studied differentially expressed membrane proteins in glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, by high resolution LC-MS/MS mass spectrometry and quantitation by iTRAQ. A total of 1834 membrane proteins were identified with high confidence, of which 356 proteins were found to be altered by 2-fold change or more (198 up- and 158 down-regulated); 56% of them are known membrane proteins associated with major cellular processes. Mass spectrometry results were confirmed for representative proteins on individual specimens by immunohistochemistry. On mapping of the differentially expressed proteins to cellular pathways and functional networks, we notably observed many calcium-binding proteins to be altered, implicating deregulation of calcium signaling and homeostasis in GBM, a pathway also found to be enriched in the report (Dong, H., Luo, L., Hong, S., Siu, H., Xiao, Y., Jin, L., Chen, R., and Xiong, M. (2010) Integrated analysis of mutations, miRNA and mRNA expression in glioblastoma. BMC Syst. Biol. 4, 163) based on The Cancer Genome Atlas analysis of GBMs. Annotations of the 356 proteins identified by us with The Cancer Genome Atlas transcriptome data set indicated overlap with 295 corresponding transcripts, which included 49 potential miRNA targets; many transcripts correlated with proteins in their expression status. Nearly 50% of the differentially expressed proteins could be classified as transmembrane domain or signal sequence-containing proteins (159 of 356) with potential of appearance in cerebrospinal fluid or plasma. Interestingly, 75 of them have been already reported in normal cerebrospinal fluid or plasma along with other proteins. This first, in-depth analysis of the differentially expressed membrane proteome of GBM confirms genes/proteins that have been implicated in earlier studies, as well as reveals novel candidates that are being reported for the first time in GBM or any other cancer that could be investigated further for clinical applications.

To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed.

The cytosolic, alkaline RNase in rat vaginal epithelial cells (VEC) from normal, immature rats was found to be present largely in the free, active form unlike in many other mammalian tissues where it is known to be present in a latent form as a complex with RNase inhibitor (RNasin). Estradiol (E2) administration induced expression of RNasin activity in the VEC from such animals and caused virtually total inhibition of cytosolic RNase activity in these cells by 12 h after the hormone injection. These changes may have metabolic implications in relation to other biochemical events stimulated by estradiol in rat VEC.

In a strain of E. coli deficient in RNase III (ABL1), 23S rRNA has been shown to be present in incompletely processed form with extra nucleotides at both the 5' and 3' ends (King et al., 1984, Proc. Natl. Acad. Sci. U.S. 81, 185-188). RNA molecules with four different termini at the 5' end are observed in vivo, and are all found in polysomes. The shortest of these ("C3") is four nucleotides shorter than the accepted mature terminus. In growing cells of both wild-type and mutant strains up to 10% of the 23S rRNA chains contain the 5' C3 terminus. In stationary phase cells, the proportion of C3 termini remains the same in the wild-type cells; but C3 becomes the dominant terminus in the mutant. Species C3 is also one of the 5' termini of 23S rRNA generated in vitro from larger precursors by the action of purified RNase III. We therefore suggest that some form of RNase III may still exist in the mutant; and since no cleavage is detectable at any other RNase III-specific site, the remaining enzyme would have a particular affinity for the C3 cleavage site, especially in stationary phase cells. We raise the question whether the C3 terminus has a special role in cellular metabolism.

In an RNase III-deficient mutant of Escherichia coli, all 23 S ribosomal RNA in ribosomes is present in an unprocessed form with a double-stranded stem at the base of the molecule stable enough to be detected by electron microscopy under conditions where all other secondary structure is denatured. Molecules with variable stem lengths enter freely into polysomes, consistent with the existence of a similar but much shorter stem in mature 23 S rRNA in wild-type ribosomes.