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Results for “targeted proteomics”

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At least 163 records · Page 9Linked to original sources
PubMed2006

Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach.

A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 muM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.

Amino Acids↗
PubMed2026

Natural Product Target Identification of Wheldone, a Fungal Metabolite, as a KIF11 Inhibitor in Ovarian Cancer Using the DiffPOP (Differential Protein Precipitation) Method.

Wheldone, a fungal metabolite, was identified as a cytotoxic compound in high-grade serous ovarian cancer (HGSOC). Wheldone induced caspase 3/7-dependent apoptosis and reduced migration, invasion, and spheroid growth. Wheldone stimulated apoptosis in chemoresistant HGSOC models. Wheldone treatment caused significant downregulation of HNRNPD, a DNA repair protein, and increased DNA damage that could be blocked by N-acetyl-L-cysteine. In vivo, wheldone displayed minimal toxicity but was rapidly cleared from circulation, despite in vitro metabolic stability. Wheldone treatment in vivo did not demonstrate significant reduction in tumor burden. Therefore, in order to overcome these liabilities, it was necessary to find the protein target of wheldone so that modifications can be made to improve the drug-like characteristics of the compound. Using the drug-target interaction proteomics method, differential precipitation of proteins, wheldone was found to act as an inhibitor of Kinesin superfamily protein 11 (KIF11), a motor protein essential for mitotic spindle formation. An ATPase biochemical cell-free assay confirmed direct binding and functional inhibition of KIF11. Wheldone resulted in G2/M arrest and downstream regulation of mitotic proteins such as TPX2, AURKA, and phospho-histone H3. Proteomics after treatment of wheldone in four different HGSOC cancer cell lines all supported changes consistent with mitotic spindle assembly disruption. Further, KIF11 was one of only 13 proteins upregulated in all 4 cell lines treated. Overall, wheldone was found to be a fungal metabolite that inhibits KIF11 in chemoresistant ovarian cancer, with future studies needed to improve its pharmacokinetics and delivery.

Female↗
PubMed2005

Sarcolemmal K(ATP) channels: what do we really know?

K(ATP) channels are present at an extremely high density in the heart, and we know from in vitro studies that channel activation causes dramatic action potential shortening and contractile failure. But, if and when this happens in vivo is still a matter of debate. Twenty one years of intense study have led to a well-developed understanding of the molecular basis of K(ATP) channel activity. Structure-function studies, together with cellular experiments probing regulatory molecules have told us much about the way the K(ATP) channel can activate, and gene-targeting and proteomic tools have further elucidated determinants of in vivo function. However, the true physiological determinants of sarcolemmal K(ATP) activity remain elusive, we still await full illumination of the role of the channel in the intact heart.

ATP-Binding Cassette Transporters↗
PubMed2004

Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains.

Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive membrane recruitment of their host proteins. We investigated phosphoinositide binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently membrane targeted. Our proteome-wide analysis argues that membrane targeting is important for only approximately 30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.

Binding Sites↗
PubMed2006

Phosphopeptide modification and enrichment by oxidation-reduction condensation.

Many cellular processes are regulated by the reversible phosphorylation of proteins. Despite the importance of monitoring protein phosphorylation, available methods to modify and enrich phosphopeptides from complex mixtures for subsequent mass spectrometric analysis are challenging. Here the oxidation-reduction condensation was shown for the first time to directly modify the phosphate of phosphopeptides and phosphoproteins. By coupling with a solid-phase resin, the oxidation-reduction condensation was validated for capture and recovery of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing peptides from a peptide mixture. In addition, full-length phosphoproteins or phosphopeptides from a protein digestion were captured and recovered using the oxidation-reduction condensation, demonstrating its compatibility with protein mixtures. The strategy modifies all phosphopeptides, maintains high chemical selectivity, requires only two steps, and relies on commercially available reagents, suggesting that the oxidation-reduction condensation has the potential to enhance phosphopeptide enrichment methods and encourage development of efficient biochemical and proteomics tools targeting phosphorylation.

Amino Acid Sequence↗
PubMed2006

The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases.

How lipid transmitters move within and between cells to communicate signals remains an important and largely unanswered question. Integral membrane transporters, soluble lipid-binding proteins, and metabolic enzymes have all been proposed to collaboratively regulate lipid signaling dynamics in vivo. Assignment of the relative contributions made by each of these classes of proteins requires selective pharmacological agents to perturb their individual functions. Recently, LY2183240, a heterocyclic urea inhibitor of the putative endocannabinoid (EC) transporter, was shown to disrupt the cellular uptake of the lipid EC anandamide and promote analgesia in vivo. Here, we show that LY2183240 is a potent, covalent inhibitor of the EC-degrading enzyme fatty acid amide hydrolase (FAAH). LY2183240 inactivates FAAH by carbamylation of the enzyme's serine nucleophile. More global screens using activity-based proteomic probes identified several additional serine hydrolases that are also inhibited by LY2183240. These results indicate that the blockade of anandamide uptake observed with LY2183240 may be due primarily to the inactivation of FAAH, providing further evidence that this enzyme serves as a metabolic driving force that promotes the diffusion of anandamide into cells. More generally, the proteome-wide target promiscuity of LY2183240 designates the heterocyclic urea as a chemotype with potentially excessive protein reactivity for drug design.

Amidohydrolases↗
PubMed2006

Multiplexed mass spectrometric immunoassay in biomarker research: a novel approach to the determination of a myocardial infarct.

Reported here is the development of a multiplexed mass spectrometric immunoassay (MSIA) for the detection of myocardial infarction (MI). The assay is the product of a study that systematically progresses from biomarker discovery--to identification and verification--to assay design, data analysis, and statistical challenge. During targeted population proteomics investigations, two novel biomarkers, serum amyloid A1alpha and S-sulfated transthyretin, were found to be responsive to MI. These putative markers were subsequently screened in larger cohorts of individuals to verify their responsiveness toward MI. Upon verification, a multiplexed assay was designed that was capable of simultaneously monitoring the new markers plus a previously established MI-marker (myoglobin). The multiplexed MSIA was applied to two 96-sample sets comprised of 48-MI/48-healthy and 19-MI/77-healthy, which served as training and case cohorts, respectively. Data evaluation using either preset reference levels or multivariate analysis exhibited sensitivities and specificities of >97%. These findings illustrate the importance of using systematic approaches in clinical proteomics to discover biomarkers and produce high-performance assays relevant to disease.

Biomarkers↗
PubMed2002

Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach.

Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.

Animals↗
PubMed2006

EBP, a program for protein identification using multiple tandem mass spectrometry datasets.

MS/MS combined with database search methods can identify the proteins present in complex mixtures. High throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.

Algorithms↗
PubMed2026

Phosphorylation of BigH1 regulates its expression pattern and promotes embryonic development.

Metazoan genomes typically encode several linker histone variants, often expressed in a tissue- or developmental stage-specific manner. The Drosophila melanogaster genome contains only two linker histone variants: H1 is present in somatic cells, while BigH1 substitutes H1 in the germline and early embryos. In the early stages of embryogenesis, BigH1 is replaced by H1 in the chromatin of somatic cells, contributing to the initiation and maintenance of the zygotic gene expression program. Nevertheless, the molecular mechanism of this exchange and the possible functions of post-translational modifications of BigH1 in this process remain elusive. Here, we identify phosphorylation as a key post-translational regulator of BigH1 dynamics. Using proteomics and targeted mutagenesis of the endogenous BigH1 locus, we show that the loss of N-terminal phosphorylation results in persistent retention of BigH1 in somatic nuclei throughout embryogenesis, indicating a failure in BigH1 turnover. In contrast, disruption of C-terminal phosphorylation does not markedly affect BigH1 clearance but increases defects during early nuclear divisions and compromises embryonic development, particularly under suboptimal conditions. Together, these findings demonstrate that domain-specific phosphorylation differentially regulates BigH1 function, coordinating its early embryonic role with its subsequent removal from the chromatin.

BigH1↗
PubMed2006

Chemical and structural diversity of siRNA molecules.

Short interfering RNAs (siRNAs) are 21-23 nt long double-stranded oligoribonucleotides which in mammalian cells exhibit a potency for sequence-specific gene silencing via an RNA interference (RNAi) pathway. It has been already proven that exogenous, chemically synthesized siRNA molecules are effective inhibitors of gene expression and are widely applied for analysis of protein function and proteomics-based target identification. Moreover, since their discovery siRNA molecules have been implemented as potential candidates for therapeutic applications. Variously modified siRNA molecules containing sugar modifications (2'-OMe, -F, -O-allyl, -amino, orthoesters and LNA analogues), internucleotide phospodiester bond modifications (phosphorothioates, boranophosphates), base modifications (s(2)U) as well as 3'-terminal cholesterol-conjugated constructs were investigated as potential candidates for effective inhibition of gene expression. This chapter reviews an impact of chemical and structural modifications of siRNA molecules on their serum and thermal stability, cellular and in vivo activity, cellular uptake, biodistribution and cytotoxicity. Functional analysis of chemically modified siRNA molecules allows for better understanding of the mechanism of the RNA interference process as well as demonstrates immense efforts in optimizing in vivo potency of siRNA molecules for RNAi-based drug design.

Animals↗
PubMed2005

Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils.

The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.

Amino Acid Sequence↗
PubMed2004

Targeted degradation of proteins by small molecules: a novel tool for functional proteomics.

A novel strategy that targets protein for degradation has recently been developed by exploiting a protein-targeting chimeric molecule ('Protac'). Typically, the chimeric Protac is composed of a small-molecule ligand ('bait') on one end and a synthetic octapeptide on the other. This octapeptide is recognized by E3 ubiquitin ligase pVHL (von Hippel Lindau tumor suppressor protein), thereby recruiting a small molecule-bound protein ('prey') to pVHL for ubiquitination and degradation. Since selective degradation of a cellular protein generates a "loss of function" mutation, this protein knock-out strategy may be useful to study the function of a given protein or to evaluate whether a cellular protein is a potential target for drug intervention, in a manner reminiscent of gene knock-out or siRNA approaches. Herein, we show that a synthetic pentapeptide is sufficient to interact with pVHL E3 ligase, and that the pentapeptide-based Protac efficiently induces ubiquitination and degradation of target protein. Our results also demonstrate that the pentapeptide-based Protac can enter cells efficiently to exerts its biological activity effectively. These results suggest that the synthetic pentapeptide can be used either directly in the preparation of cell-permeable Protacs or as a template to develop peptidomimetic or non-peptide Protacs.

Blotting, Western↗
PubMedDate not supplied

Ovarian cancer in the proteomics era: diagnosis, prognosis, and therapeutics targets.

Ovarian cancer is clinically quiet as it plants seeds of metastases in the peritoneal cavity, even during early stages when there is highest potential for cure. The only available biomarker is CA125, which has an unacceptably low sensitivity and specificity for diagnostic use. Highly sensitive and specific tools to further optimize early diagnosis and treatment are needed. We propose that proteomic technologies have an important role to play in the development of these tools. Mass spectrometry platforms, such as surface-enhanced laser desorption/ionization time-of-flight, may be used to mine patient's serum for proteomic signatures that are shed by tumor and stroma. Such signatures could serve as a diagnostic tool during early-stage disease and as a remission-monitoring tool in later-stage disease. Reverse-phase protein microarrays are a new microproteomic tool to profile signaling pathways in ovarian cancer, thus identifying therapeutic targets while simultaneously suggesting prognostic indicators. Proteomic technologies have the capacity to build upon our genomic and clinical understanding of ovarian cancer by moving the focal point to the tumor and its microenvironment. This unique proteomic vantage point allows the creation of tools that will aid clinicians in making rational decisions in the diagnosis and treatment of women with ovarian cancer.

Biomarkers, Tumor↗
PubMed2005

Mass spectrometry-based quantitative proteomic profiling.

Quantitative proteomics involves the identification and quantitation of protein components in various biological systems. Stable isotope labelling technology, by both metabolic and chemical methods, has been the most commonly used approach for global proteome-wide profiling. Recently, its capability has been extended from labelled pairs to multiple labels, allowing for the simultaneous quantification of multiplex samples. The ion intensity-based quantitative approach has progressively gained more popularity as mass spectrometry performance has improved significantly. Although some success has been reported, it remains difficult comprehensively to characterise the global proteome, due to its enormous complexity and dynamic range. The use of sub-proteome fractionation techniques permits a simplification of the proteome and provides a practical step towards the ultimate dissection of the entire proteome. Further development of the technology for targeting sub-proteomes on a functional basis - such as selecting proteins with differential expression profiles from mass spectrometric analyses, for further mass spectrometric sequencing in an intelligent manner--is expected in the near future.

Biotin↗
PubMed2006

Proteomic analyses to identify novel therapeutic targets for the treatment of advanced prostate cancer.

At present there is no cure for advanced prostate cancer once it progresses to an androgen independent stage. Hormonal therapy, radiotherapy, and chemotherapy all have limited durations of efficacy for men diagnosed with androgen independent disease and patients will succumb over a period of several months to two years. The androgen receptor (AR) has been suspected to play an important role in the mechanism of progression to androgen independence. This is because the AR is a transcription factor that 'normally' mediates the effects of androgen to regulate expression of genes involved in proliferation and survival of prostate cells. Thus identifying and characterizing the proteins that interact with the AR to facilitate an activated receptor is of critical importance. Proteomic approaches such as isotope-coded affinity tags (ICAT), isotope Tags for Relative and Absolute Quantification (iTRAQ)(TM), Stable Isotope Labeling with Amino acids in Cell culture (SILAC), Tandem Affinity Purification (TAP) of tagged proteins (TAP-tag) and Multidimensional Protein Identification Technology (MudPIT) provide large scale unbiased strategies and have not been previously applied to identify proteins that interact with the AR. Here an example of the power of these proteomic approaches to identify potential therapeutic targets for prostate cancer is provided. Application of MudPIT identified 82 peptides in endogenous complexes immunoprecipitated with the AR from prostate cancer cells. Identification of these novel proteins may ultimately lead to the development of better therapies for the treatment or prevention of advanced prostate cancer.

Journal Article↗
PubMed2003

An efficient proteomics method to identify the cellular targets of protein kinase inhibitors.

Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38alpha whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis reveals hyperactivation of the mammalian target of rapamycin pathway in neurofibromatosis 1-associated human and mouse brain tumors.

Individuals with the tumor predisposition syndrome, neurofibromatosis 1 (NF1), are prone to development of nervous system tumors, including neurofibromas and pilocytic astrocytomas. Based on the ability of the NF1 gene product (neurofibromin) to function as a GTPase activating protein for RAS, initial biologically based therapies for NF1-associated tumors focused on the use of RAS inhibitors, but with limited clinical success. In an effort to identify additional targets for therapeutic drug design in NF1, we used an unbiased proteomic approach to uncover unanticipated intracellular signaling pathways dysregulated in Nf1-deficient astrocytes. We found that the expression of proteins involved in promoting ribosome biogenesis was increased in the absence of neurofibromin. In addition, Nf1-deficient astrocytes exhibit high levels of mammalian target of rapamycin (mTOR) pathway activation, which was inhibited by blocking K-RAS or phosphatidylinositol 3-kinase activation. This mTOR pathway hyperactivation was reflected by high levels of ribosomal S6 activation in both Nf1 mutant mouse optic nerve gliomas and in human NF1-associated pilocytic astrocytoma tumors. Moreover, inhibition of mTOR signaling in Nf1-/- astrocytes abrogated their growth advantage in culture, restoring normal proliferative rates. These results suggest that mTOR pathway inhibition may represent a logical and tractable biologically based therapy for brain tumors in NF1.

Animals↗