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PubMed2003

Proteomic identification of 14-3-3zeta as a mitogen-activated protein kinase-activated protein kinase 2 substrate: role in dimer formation and ligand binding.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3zeta. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3zeta in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3zeta at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3zeta to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3zeta dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3zeta functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.

14-3-3 Proteins↗
PubMed2000

Complementing genomics with proteomics: the membrane subproteome of Pseudomonas aeruginosa PAO1.

With the completion of many genome projects, a shift is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. The opportunistic pathogen Pseudomonas aeruginosa is one organism for which a genome sequence is now available, including the annotation of open reading frames (ORFs). However, approximately one third of the ORFs are as yet undefined in function. Proteomics can complement genomics, by characterising gene products and their response to a variety of biological and environmental influences. In this study we have established the first two-dimensional gel electrophoresis reference map of proteins from the membrane fraction of P. aeruginosa strain PA01. A total of 189 proteins have been identified and correlated with 104 genes from the P. aeruginosa genome. Annotated membrane proteins could be grouped into three distinct categories: (i) those with functions previously characterised in P. aeruginosa (38%); (ii) those with significant sequence similarity to proteins with assigned function or hypothetical proteins in other organisms (46%); and (iii) those with unknown function (16%). Transmembrane prediction algorithms showed that each identified protein sequence contained at least one membrane-spanning region. Furthermore, the current methodology used to isolate the membrane fraction was shown to be highly specific since no contaminating cytosolic proteins were characterised. Preliminary analysis showed that at least 15 gel spots may be glycosylated in vivo, including three proteins that have not previously been functionally characterised. The reference map of membrane proteins from this organism is now the basis for determining surface molecules associated with antibiotic resistance and efflux, cell-cell signalling and pathogen-host interactions in a variety of P. aeruginosa strains.

Bacterial Proteins↗
PubMed2004

Transcriptomic and proteomic characterization of the Fur modulon in the metal-reducing bacterium Shewanella oneidensis.

The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium. We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S. oneidensis Fur regulon. Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression. This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions. Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes. Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level. A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant. The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation.

Bacterial Proteins↗
PubMed2004

Reversible inhibitors of fatty acid amide hydrolase that promote analgesia: evidence for an unprecedented combination of potency and selectivity.

Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of alpha-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.

Amidohydrolases↗
PubMed2002

Formation of protein kinase C(epsilon)-Lck signaling modules confers cardioprotection.

The epsilon isoform of protein kinase C (PKCepsilon) is a member of the PKC family of serine/threonine kinases and plays a critical role in protection against ischemic injury in multiple organs. Functional proteomic analyses of PKCepsilon signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCepsilon orchestrates cardioprotection are poorly understood. Here we report that Lck, a member of the Src family of tyrosine kinases, forms a functional signaling module with PKCepsilon. In cardiac cells, PKCepsilon interacts with, phosphorylates, and activates Lck. In vivo studies showed that cardioprotection elicited either by cardiac-specific transgenic activation of PKCepsilon or by ischemic preconditioning enhances the formation of PKCepsilon-Lck modules. Disruption of these modules, via ablation of the Lck gene, abrogated the infarct-sparing effects of these two forms of cardioprotection, indicating that the formation of PKCepsilon-Lck signaling modules is required for the manifestation of a cardioprotective phenotype. These findings demonstrate, for the first time to our knowledge, that the assembly of a module (PKCepsilon-Lck) is an obligatory step in the signal transduction that results in a specific phenotype. Thus, PKCepsilon-Lck modules may serve as novel therapeutic targets for the prevention of ischemic injury.

Animals↗
PubMed2006

Proteomics in clinical trials and practice: present uses and future promise.

The study of clinical proteomics is a promising new field that has the potential to have many applications, including the identification of biomarkers and monitoring of disease, especially in the field of oncology. Expression proteomics evaluates the cellular production of proteins encoded by a particular gene and exploits the differential expression and post-translational modifications of proteins between healthy and diseased states. These biomarkers may be applied towards early diagnosis, prognosis, and prediction of response to therapy. Functional proteomics seeks to decipher protein-protein interactions and biochemical pathways involved in disease biology and targeted by newer molecular therapeutics. Advanced spectrometry technologies and new protein array formats have improved these analyses and are now being applied prospectively in clinical trials. Further advancement of proteomics technology could usher in an era of personalized molecular medicine, where diseases are diagnosed at earlier stages and where therapies are more effective because they are tailored to the protein expression of a patient's malignancy.

Clinical Trials as Topic↗
PubMed2003

Proteomic analysis of cancer-cell mitochondria.

Mitochondrial dysfunction and mutations in mitochondrial DNA have been frequently reported in cancer cells. Mitochondrial gene-expression signatures of transformed cells have been identified; however, the phenotypic effects of these genetic alterations remain to be established. Identification of mitochondrial proteins that are aberrantly expressed in cancer cells has been made possible by the recent development of mitochondrial functional proteomics and could identify new markers for early detection and risk assessment, as well as targets for therapeutic intervention.

Humans↗
PubMed2006

An initial proteomic analysis of human preterm labor: placental membranes.

Human preterm labor (PL) is the single most significant problem in modern Obstetrics and Gynecology, affecting approximately 10% of pregnancies worldwide, constituting the leading cause of perinatal mortality and morbidity, and contributing significantly to chronic childhood disease. Currently, our molecular understanding of PL remains staggeringly inadequate to reliably diagnose or rationally intervene in PL events; several molecular alterations have been implicated in PL, but these have proven of limited value as diagnostic/prognostic markers. The majority of PL events remain spontaneous and unpredictable: critical care emergencies. Here, we apply functional proteomics to dissect molecular mechanisms of human PL. Human placental tissue was collected in clearly differentiated cases of preterm and term labor. Highly refined two-dimensional gel electrophoresis (2DE) was used for protein separation, coupled with automated differential gel image analysis to compare the resulting proteomic maps. For this initial study, only the most important protein differences were selected for further analysis, that is, proteins that were unique to one sample, and absent from the other, with 100% reproducibility across the sample population. In total, 11 such proteins were identified by tandem mass spectrometry, falling into three distinct functional classes: structural/cytoskeletal components, ER lumenal proteins with enzymatic or chaperone functions, and proteins with anticoagulant properties. These expression changes form the groundwork for further molecular investigation of this devastating medical condition. This approach therefore holds the potential not only to define the underlying molecular components, but also to identify novel diagnostic tools and targets for rational drug intervention.

Anticoagulants↗
PubMed2006

Identification of apolipoprotein A-I as a "STOP" signal for myopia.

Good visual acuity requires that the axial length of the ocular globe is matched to the refractive power of the cornea and lens to focus the images of distant objects onto the retina. During the growth of the juvenile eye, this is achieved through the emmetropization process that adjusts the ocular axial length to compensate for the refractive changes that occur in the anterior segment. A failure of the emmetropization process can result in either excessive or insufficient axial growth, leading to myopia or hyperopia, respectively. Emmetropization is mainly regulated by the retina, which generates two opposite signals: "GO/GROW" signals to increase axial growth and "STOP" signals to block it. The presence of GO/GROW and STOP signals was investigated by a proteomics analysis of the retinas from chicken with experimental myopia and hyperopia. Of 18 differentially expressed proteins that were identified, five displayed an expression profile corresponding to GO/GROW signals, and two corresponded to STOP signals. Western blotting confirmed that apolipoprotein A-I (apoA-I) has the characteristics of a STOP signal both in the retina as well as in the fibrous sclera. In accordance with this, intraocular application of the peroxisome proliferator-activated receptor alpha agonist GW7647 resulted in up-regulation of apoA-I levels and in a significant reduction of experimental myopia. In conclusion, using a comprehensive functional proteomics analysis of chicken ocular growth models we identified targets for ocular growth control. The correlation of elevated apoA-I levels with reduced ocular axial growth points toward a functional relationship with the observed morphological changes of the eye.

Animals↗
PubMed2006

Proteome analysis of combined effects of androgen and estrogen on the mouse mammary gland.

We and other investigators have previously shown that estrogen and androgen have synergistic effects on the growth of mammary epithelial ducts and alveoli in the Noble mouse. However, the molecular mechanisms behind the synergy are unknown. In the present study, we treated female FVB mice with 17-estrodial (E2) and 5-dihydrotestosterone-bezonate (DHT-B) using slow-releasing hormone pellets for 7 months. Dissection showed that hormone treatment caused atypical hyperplasia of mammary ducts and alveoli. A functional proteomic approach was used to study the holistic protein changes in mammary glands. 2-DE was used to separate proteins. Twenty-five protein spots that were differentially expressed in hormone-treated tissues compared to the control were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF-MS, which include some proteins that are correlative with response to estrogen and androgen stimulation, cells differentiation and growth, signal transduction, metabolism, etc. Real-time RT-PCR was carried out to verify the different expression. These results offered some clues to understand the function of E2 and DHT-B.

Androgens↗
PubMed2005

Ultra-high sensitivity multi-photon detection imaging in proteomics analyses.

We report on the use of 125I and 131I labeling and of new, multicolor, multi-photon detection (MPD) methods to routinely and quantitatively detect protein spots on two-dimensional gel electrophoresis plates in the zeptomole to attomole range. We demonstrate that the MPD methodology can be used to detect radioactive labels on two-dimensional gels and has several characteristics that are advantageous for functional proteomics. First, by using single particle detectors, the sensitivity for detection of radiolabels can be improved dramatically. Second, because single particle detectors can differentiate the particle energies produced by different decay processes, it is possible to choose combinations of radioisotopes that can be detected and quantified individually on the same 2-D gel. Third, the MPD technology is essentially linear over six to seven orders of magnitude, i.e., it is possible to accurately quantify radiolabeled proteins over a range from at least 60 zeptomoles to 60 femtomoles. Finally for radionuclides that decay by electron capture, e.g., with emission of both beta and gamma rays, co-incident detection of two particles/photons can be used to detect such radionuclides well below background radiation levels. These methods are used to monitor acidic/phosphorylated proteins in as little as 60 ng of HeLa cells proteins.

Color↗
PubMed2005

Proteomic characterization of skin and epidermis in response to environmental agents.

The skin and its outer epidermis layer in particular, prevent access of various environmental agents including potential allergens, irritants, carcinogens, ultraviolet radiation and microbes. Cells in the epidermis make a significant contribution to innate as well as adaptive immune reactions in skin. The skin immunity thus provides a biologic defense in response to hazardous environmental agents. Although proteomics has been utilized to establish skin proteomes and investigate skin responses to some environmental agents, it has not been extensively used to address the complexity of skin responses to various environments. This review summarizes cutaneous genes and proteins that have been characterized as related to skin exposure to environmental agents. In parallel, this review emphasizes functional proteomics and systems biology, which are believed to be an important future direction toward characterizing the skin proteome-environmental interaction and developing successful therapeutic strategies for skin diseases caused by environmental insults.

Animals↗
PubMed2006

Enrichment of integral membrane proteins from small amounts of brain tissue.

Subcellular fractionation represents an essential technique for functional proteome analysis. Recently, we provided a subcellular fractionation protocol for minute amounts of tissue that yielded a nuclear fraction, a membrane and organelle fraction, and a cytosolic fraction. In the current study, we attempted to improve the protocol for the isolation of integral membrane proteins, as these are particularly important for brain function. In the membrane and organelle fraction, we increased the yield of membranes and organelles by about 50% by introducing a single re-extraction step. We then tested two protocols towards their capacity to enrich membrane proteins present in the membrane and organelle fraction. One protocol is based on sequential solubilization using subsequent increases of chaotropic conditions such as urea, thereby partitioning hydrophobic proteins from hydrophilic ones. The alternative protocol applies high-salt and high-pH washes to remove non-membrane proteins. The enrichment of membrane proteins by these procedures, as compared to the original membrane and organelle fraction, was evaluated by 16-BAC-SDS-PAGE followed by mass spectrometry of randomly selected spots. In the original membrane and organelle fraction, 7 of 50 (14%) identified proteins represented integral membrane proteins, and 15 (30%) were peripheral membrane proteins. In the urea-soluble fraction, 4 of 33 (12%) identified proteins represented integral membrane proteins, and 10 (30%) were peripheral membrane proteins. In the high-salt/high-pH resistant sediment, 12 of 45 (27%) identified proteins were integral membrane proteins and 13 (29%) represented peripheral membrane proteins. During the analysis, several proteins involved in neuroexocytosis were detected, including syntaxin, NSF, and Rab3-interaction protein 2. Taken together, differential centrifugation in combination with high-salt and high-pH washes resulted in the highest enrichment of integral membrane proteins and, therefore, represents an adequate technique for region-specific profiling of membrane proteins in the brain.

Animals↗
PubMed2006

Tissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infection.

BACKGROUND: Chlamydophila pneumoniae infection has been implicated as a potential risk factor for atherosclerosis, however the mechanism leading to persistent infection and its role in the disease process remains to be elucidated. METHODS: We validated the use of tissue microarray (TMA) technology, in combination with immunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raised against differentially expressed proteins under an interferon-gamma (IFN-gamma) induced model of chlamydial persistence. RESULTS: In the cell pellet array, we were able to identify differences in protein expression patterns between untreated and IFN-gamma treated samples. Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples. The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis. Subsequently, it was shown in a second TMA including archival atheromatous heart tissues from 12 patients undergoing heart transplantation, that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well, and were detectable morphologically within lesions by IHC. CONCLUSION: TMA technology proved useful in documenting functional proteomics data with the morphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may be useful markers for the detection of persistent infection in vitro and in vivo.

Cell Line↗
PubMed2005

Cell cycle: proteomics gives it a spin.

The eukaryotic cell division cycle has been studied at the molecular level for over 30 years, most fruitfully in model organisms. In the past 5 years, developments in mass spectrometry-based proteomics have been applied to the study of protein interactions and post-translational modifications involving key cell cycle regulators such as cyclin-dependent kinases and the anaphase-promoting complex, as well as effectors such as centrosomes, the kinetochore and DNA replication forks. In addition, innovations in chemical biology, functional proteomics and bioinformatics have been employed to study the cell cycle at the proteome level. This review surveys the contributions of proteomics to cell cycle research. The near future should see the application of more quantitative proteomic approaches to probe the dynamic aspects of the molecular system that underlie the cell cycle in model organisms and in human cells.

Animals↗
PubMed2006

Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts.

Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures.

Antigens, CD↗
PubMed2003

Genome-based bioinformatic selection of chromosomal Bacillus anthracis putative vaccine candidates coupled with proteomic identification of surface-associated antigens.

Bacillus anthracis (Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins, were selected as B. anthracis-specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version, 460 contigs, The Institute for Genomic Research, Rockville, Md.). The selection procedure combined preliminary annotation (sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen and additional filtering criteria (size, number of paralogs). The reductive strategy, combined with manual curation, resulted in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function. Proteomic analysis of two-dimensional gels of a B. anthracis membrane fraction, verified the expression of some gene products. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera from B. anthracis immunized animals. These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose expression and immunogenicity was not reported before. Five of these 8 immunogens were preselected by the bioinformatic analysis (EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates. This study demonstrates that a combination of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine.

Adhesins, Bacterial↗
PubMed2003

The German cDNA network: cDNAs, functional genomics and proteomics.

Among the greatest challenges facing biology today is the exploitation of huge amounts of genomic data, and their conversion into functional information about the proteins encoded. For example, the large-scale cDNA sequencing project of the German cDNA Consortium is providing vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterization we have tagged over 500 of these with the green fluorescent protein (GFP), and examined the subcellular localizations of these fusion proteins in living cells. These data have allowed us to classify the proteins into subcellular groups which determines the next step towards a detailed functional characterization. To make further use of these GFP-tagged constructs, a series of functional assays have been designed and implemented to assess the effect of these novel proteins on processes such as cell growth, cell death, and protein transport. Functional assays with such a large set of molecules is only possible by automation. Therefore, we have developed, and adapted, functional assays for use by robotic liquid handling stations and reading stations. A transport assay allows to identify proteins which localize to distinct organelles of the secretory pathway and have the potential to be new regulators in protein transport, a proliferation assay helps identifying proteins that stimulate or repress mitosis. Further assays to monitor the effects of the proteins in apoptosis and signal transduction pathways are in progress. Integrating the functional information that is generated in the assays with data from expression profiling and further functional genomics and proteomics approaches, will ultimately allow us to identify functional networks of proteins in a morphological context, and will greatly contribute to our understanding of cell function.

Cloning, Molecular↗