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1.  Phosphorylation by p38 Mitogen-Activated Protein Kinase Promotes Estrogen Receptor α Turnover and Functional Activity via the SCFSkp2 Proteasomal Complex 
Molecular and Cellular Biology  2012;32(10):1928-1943.
The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCFSkp2 proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers.
doi:10.1128/MCB.06561-11
PMCID: PMC3347406  PMID: 22431515
2.  Insight into the Salivary Transcriptome and Proteome of Dipetalogaster maxima 
Journal of proteome research  2011;10(2):669-679.
Dipetalogaster maximais a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications. Supplemental files can be downloaded from http://exon.niaid.nih.gov/transcriptome/D_maxima/Dm-S1-web.xls and http://exon.niaid.nih.gov/transcriptome/D_maxima/Dm-S2-web.xls.
doi:10.1021/pr100866h
PMCID: PMC3035102  PMID: 21058630
Saliva; hematophagy; transcriptome; proteome; triatomine; D. maxima
3.  Optimization of Protein Solubilization for the Analysis of the CD14 Human Monocyte Membrane Proteome Using LC-MS/MS 
Journal of proteomics  2009;73(1):112-122.
Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate) (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~98% positive for CD14 expression by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46 %) were mapped integral membrane proteins. However, employing PPS a total of 216 proteins of which 75 (35 %) were mapped integral membrane proteins. These results indicate that methanol unaided or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.
doi:10.1016/j.jprot.2009.08.008
PMCID: PMC3159575  PMID: 19709643
CD14 monocyte; Membrane proteins; Solubilization; Methanol; Detergents; LC-MS/MS
4.  A further insight into the sialome of the tropical bont tick, Amblyomma variegatum 
BMC Genomics  2011;12:136.
Background
Ticks--vectors of medical and veterinary importance--are themselves also significant pests. Tick salivary proteins are the result of adaptation to blood feeding and contain inhibitors of blood clotting, platelet aggregation, and angiogenesis, as well as vasodilators and immunomodulators. A previous analysis of the sialotranscriptome (from the Greek sialo, saliva) of Amblyomma variegatum is revisited in light of recent advances in tick sialomes and provides a database to perform a proteomic study.
Results
The clusterized data set has been expertly curated in light of recent reviews on tick salivary proteins, identifying many new families of tick-exclusive proteins. A proteome study using salivary gland homogenates identified 19 putative secreted proteins within a total of 211 matches.
Conclusions
The annotated sialome of A. variegatum allows its comparison to other tick sialomes, helping to consolidate an emerging pattern in the salivary composition of metastriate ticks; novel protein families were also identified. Because most of these proteins have no known function, the task of functional analysis of these proteins and the discovery of novel pharmacologically active compounds becomes possible.
doi:10.1186/1471-2164-12-136
PMCID: PMC3060141  PMID: 21362191
5.  An Insight into the Sialome of the Black Fly, Simulium vittatum 
Journal of proteome research  2009;8(3):1474-1488.
Adaptation to vertebrate blood feeding includes development of a salivary ‘magic potion’ that can disarm host hemostasis and inflammatory reactions. Within the lower Diptera, a vertebrate blood-sucking mode evolved in the Psychodidae (sand flies), Culicidae (mosquitoes), Ceratopogonidae (biting midges), Simuliidae (black flies), and in the frog-feeding Corethrellidae. Sialotranscriptome analyses from several species of mosquitoes and sand flies and from one biting midge indicate divergence in the evolution of the blood-sucking salivary potion, manifested in the finding of many unique proteins within each insect family, and even genus. Gene duplication and divergence events are highly prevalent, possibly driven by vertebrate host immune pressure. Within this framework, we describe the sialome (from Greek sialo, saliva) of the black fly Simulium vittatum and discuss the findings within the context of the protein families found in other blood-sucking Diptera. Sequences and results of Blast searches against several protein family databases are given in Supplemental Tables S1 and S2, which can be obtained from http://exon.niaid.nih.gov/transcriptome/S_vittatum/T1/SV-tb1.zip and http://exon.niaid.nih.gov/transcriptome/S_vittatum/T2/SV-tb2.zip.
doi:10.1021/pr8008429
PMCID: PMC2778207  PMID: 19166301
Simulium vittatum; black fly; sialotranscriptomes; salivary gland transcriptome; sialome; proteome; hematophagy; onchocerciasis
6.  An insight into the salivary transcriptome and proteome of the soft tick and vector of epizootic bovine abortion, Ornithodoros coriaceus 
Journal of proteomics  2008;71(5):493-512.
The salivary glands of blood sucking arthropods contain a redundant ‘magic potion’ that counteracts their vertebrate host’s hemostasis, inflammation, and immunity. We here describe the salivary transcriptome and proteomics (sialome) of the soft tick Ornithodoros coriaceus. The resulting analysis helps to consolidate the classification of common proteins found in both soft and hard ticks, such as the lipocalins, Kunitz, cystatin, basic tail, hebraein, defensin, TIL domain, metalloprotease, 5′-nucleotidase/apyrase, and phospholipase families, and also to identify protein families uniquely found in the Argasidae, such as the adrenomedullin/CGRP peptides, 7DB, 7 kDa, and the RGD containing single Kunitz proteins. Additionally, we found a protein belonging to the cytotoxin protein family that has so far only been identified in hard ticks. Three other unique families common only to the Ornithodoros genus were discovered. Edman degradation, 2D and 1D PAGE of salivary gland homogenates followed by tryptic digestion and HPLC MS/MS of results confirms the presence of several proteins. These results indicate that each genus of hematophagous arthropods studied to date evolved unique protein families that assist blood feeding, thus characterizing potentially new pharmacologically active components or antimicrobial agents.
doi:10.1016/j.jprot.2008.07.006
PMCID: PMC2617759  PMID: 18725333
Ornithodoros coriaceus; Ixodidae; Argasidae; Sialotranscriptome; Salivary gland transcriptome; Sialome; Tick salivary gland; Ixolaris
7.  Casein kinase 1α governs antigen receptor-induced NF-κB and human lymphoma cell survival 
Nature  2008;458(7234):92-96.
The transcription factor NF-κB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types1, 2. Antigen receptor stimulation assembles an NF-κB activating platform containing the scaffold protein CARMA1/CARD11, the adaptor BCL10, and the paracaspase MALT1 (CBM complex), linked to the inhibitor of NF-κB kinase (IKK) complex3–12, but signal transduction is not fully understood1. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNAi screen for growth inhibition of the CBM-dependent “activated B cell-like” (ABC) subtype of diffuse large B-cell lymphoma (DLBCL)12. Here, we report that both screens identified casein kinase 1α (CK1α) as a bifunctional regulator of NF-κB. CK1α dynamically associates with the CBM complex upon T cell receptor (TCR) engagement to augment cytokine production and lymphocyte proliferation. However, CK1α kinase activity plays a counterposing role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1α has thus a dual “gating” function which first promotes and then terminates receptor-induced NF-κB. ABC DLBCL cells required CK1α for constitutive NF-κB activity indicating that CK1α functions as a “conditionally essential malignancy” (CEMal) gene - a member of a new class of potential cancer therapeutic targets.
doi:10.1038/nature07613
PMCID: PMC2688735  PMID: 19118383
8.  A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis 
Science (New York, N.Y.)  2009;323(5914):644-648.
Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, including the telomerase reverse transcriptase (TERT), the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein1), uniquely enriched in Cajal bodies, nuclear sites of RNP processing important for telomerase function. TCAB1 associates with active telomerase enzyme, with established telomerase components, and with small Cajal body RNAs involved in modifying splicing RNAs. Depletion of TCAB1 using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.
doi:10.1126/science.1165357
PMCID: PMC2728071  PMID: 19179534
9.  Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility 
Cancer research  2008;68(3):639-644.
The role of Gemin5 in alternative mRNA splicing, tumor cell motility, and proteomic instability was investigated. Isotope Capture Affinity Tag proteomic analysis was conducted on MDA-MB-435 tumor cells transfected with either a control vector (C-100) or the Nm23-H1 metastasis suppressor (H1-177). Ingenuity pathway analysis revealed that RNA posttranscriptional processing was the most prominent class of differentially expressed proteins. Within this category, overexpression of Acinus1, Poly(a) binding protein, HNRPA2B1, Bop1, and Gemin5 was confirmed in less metastatic H1-177 cells. Overexpression of the latter four proteins was also observed in the lower metastatic antisense Ezrin transfectant of a murine osteosarcoma model system, confirming the general relevance of the trends. Gemin5, a component of the spliceosomal complex, was chosen for further study. Analysis of global mRNA splicing by SpliceArray chips revealed that 16 genes were differentially spliced in C-100 compared with H1-177 cells; transient transfection of gemin5 into C-100 cells restored the splice pattern to that of H1-177 cells. Alternative splicing patterns for the engulfment and cell motility 1 and thrombospondin 4 genes were confirmed by semiquantitative reverse transcription-PCR. Gemin5 overexpression coordinately reduced C-100 cell motility by 50%, and siRNA-mediated reduction of Gemin5 expression increased the motility of H1-177 cells by 2-fold (P < 0.004). The data provide the first demonstration that alterations in the expression of a spliceosome protein can effect both specific splicing events and tumor cell motility. The data also show that changes in mRNA splicing patterns accompany metastatic progression, which may contribute to proteome instability.
doi:10.1158/0008-5472.CAN-07-2632
PMCID: PMC2678556  PMID: 18245461
10.  Brugia malayi Excreted/Secreted Proteins at the Host/Parasite Interface: Stage- and Gender-Specific Proteomic Profiling 
Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES) products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf), L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs) in the available databases. Moreover, this analysis was able to confirm the presence of 274 “hypothetical” proteins inferred from gene prediction algorithms applied to the B. malayi (Bm) genome. Not surprisingly, the majority (160/274) of these “hypothetical” proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase), MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females) compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host–parasite interaction.
Author Summary
Human lymphatic filariasis caused by the nematode parasites Brugia malayi and Wuchereria bancrofti are a major cause of concern in tropical countries. Studies over several decades have identified various proteins of these parasites that have highlighted their role in host–parasite interactions and possible chemotherapeutic and prophylactic interventions. The availability of the parasite genome facilitates the identification of all of the proteins of the parasite that could interact with the host. In this study, we have attempted to identify the excretory-secretory proteins of the various stages of the parasite that could be maintained in vitro for a limited period utilizing a high-throughput proteomics approach. We observe and report that the parasites expend resources to secrete out various molecules that they utilize to evade the host immune system and modulate its responses. Further, this study also provides information on the predicted hypothetical proteins to be bonafide proteins and thus a catalogue of the excretory-secretory proteins towards a better understanding of the host–parasite interactions.
doi:10.1371/journal.pntd.0000410
PMCID: PMC2659452  PMID: 19352421
11.  Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly 
Cell  2008;132(6):945-957.
Telomerase is a multi-subunit ribonucleoprotein (RNP) complex that adds telomere repeats to the ends of linear chromosomes. Three essential telomerase components have been identified thus far: the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC), and the TERC-binding protein dyskerin. Few other proteins are known to be required for human telomerase function, significantly limiting our understanding of both telomerase regulation and mechanisms of telomerase action. Here, we identify the ATPases pontin and reptin as telomerase components through affinity purification of TERT from human cells. Pontin interacts with both TERT and dyskerin, and the amount of TERT bound to pontin and reptin peaks in S phase, evidence for dynamic cell cycle-dependent regulation of TERT. Depletion of pontin and reptin markedly impairs accumulation of the telomerase RNP, indicating an essential role in telomerase assembly. These findings reveal an unanticipated requirement for additional enzymes in telomerase RNP biogenesis and suggest new approaches for inhibiting telomerase in human cancer.
doi:10.1016/j.cell.2008.01.019
PMCID: PMC2291539  PMID: 18358808
12.  An insight into the sialome of the soft tick, Ornithodorus parkeri 
While hard ticks (Ixodidae) take several days to feed on their hosts, soft ticks (Argasidae) feed faster, usually taking less than one hour per meal. Saliva assists in the feeding process by providing a cocktail of anti-hemostatic, anti-inflammatory and immunomodullatory compounds. Saliva of hard ticks has been shown to contain several families of genes each having multiple members, while those of soft ticks are relatively unexplored.
Analysis of the salivary transcriptome of the soft tick Ornithodorus parkeri, the vector of the relapsing fever agent Borrelia parkeri, indicates that gene duplication events have led to a large expansion of the lipocalin family, as well as of several genes containing Kunitz domains indicative of serine protease inhibitors, and several other gene families also found in hard ticks. Novel protein families with sequence homology to insulin growth factor-binding protein (prostacyclin-stimulating factor), adrenomedulin, serum amyloid A protein precursor and similar to HIV envelope protein were also characterized for the first time in the salivary gland of a blood-sucking arthropod.
The sialotranscriptome of O. parkeri confirms that gene duplication events are an important driving force in the creation of salivary cocktails of blood-feeding arthropods, as was observed with hard ticks and mosquitoes. Most of the genes coding for expanded families are homologous to those found in hard ticks, indicating a strong common evolutionary path between the two families. As happens to all genera of blood-sucking arthropods, several new proteins were also found, indicating the process of adaptation to blood feeding still continues to recent times.
doi:10.1016/j.ibmb.2007.09.009
PMCID: PMC2233652  PMID: 18070662
Ornithodorus parkeri; Ixodidae; Argasidae; Sialotranscriptomes; salivary gland transcriptome; sialome; Tick salivary glands
13.  MASS SPECTROMETRY OF RNA 
Ribonucleic acids (RNAs) are continuing to attract increased attention as they are found to play pivotal roles in biological system. Just as genomics and proteomics have been enabled by the development of effective analytical techniques and instrumentation, the large-scale analysis of non-protein coding (nc)RNAs will benefit as new analytical methodologies are developed which are appropriate to RNA analysis. Mass spectrometry offers a number of advantageous for RNA analysis arising from its ability to provide mass and sequence information starting with limited amounts of sample. This Briefings will highlight recent developments in the field that enable the characterization of RNA modification status, RNA tertiary structures, and ncRNA expression levels. These developments will also be placed in perspective of how mass spectrometry of RNAs can help elucidate the link between the genome and proteome.
doi:10.1093/bfgp/ell012
PMCID: PMC2442014  PMID: 16769684
Ribonucleic acid; posttranscriptional modifications; Quantitation; mass spectrometry

Results 1-13 (13)