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PubMed2006

Investigating the molecular basis of drug action and response: chemocentric genomics and proteomics.

The genomics and proteomics sciences have fundamentally changed the ways in which drug targets are being identified, characterized and validated. Here we review how genomics and proteomics research is improving our understanding of genetic determinants of drug susceptibility and response and, conversely, how organic small molecules mediate their pharmacological effects by modulating genome and proteome activities. We also examine the effect this improved understanding has on the drug discovery and development process.

Animals↗
PubMed2002

Chemical approaches for functionally probing the proteome.

With the availability of complete genome sequences, emphasis has shifted toward the understanding of protein function. We have developed a functional proteomic methodology that makes use of chemically reactive fluorescent probes to profile and identify enzymes in complex mixtures by virtue of their catalytic activity. This methodology allows a comparison of changes in activity of multiple enzymes under a variety of conditions using a single two-dimensional separation. The probes can also be used to localize active enzymes in intact cells using fluorescence microscopy. Furthermore, the probes enable screens for selective small molecule inhibitors of each enzyme family member within crude lysates or intact cells. Ultimately, this technology allows the rapid identification of potential drug targets and small molecule lead compounds targeted to them.

Affinity Labels↗
PubMed2004

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation.

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.

Amino Acid Sequence↗
PubMed2003

cDNA microarray and proteomic approaches in the study of brain diseases: focus on schizophrenia and Alzheimer's disease.

Recent advances in experimental genomics and proteomics, coupled with the wealth of sequence information available for a variety of organisms, have tremendous implications for how biomedical research is performed. Genomic techniques, such as complementary DNA (cDNA) microarrays, currently allow researchers to quickly and accurately quantify vast numbers of potential gene expression changes simultaneously. Modern proteomic techniques allow for the detection and elucidation of protein-protein interactions on a scale and at a speed never before possible. Although hurdles remain, together, these tools open the possibility of enormous change in our ability to analyze and interpret complex biological processes. The field of neuroscience is particularly well suited to analysis with these new techniques, given the complexity of neuronal signaling and the diversity of cellular responses. This review summarizes the major cDNA microarray and proteomic findings of relevance to schizophrenia and Alzheimer's disease (AD) as 2 representative areas of neuroscience research. The potential for these techniques to help unravel the underlying pathology of complex neurological and neuropsychiatric conditions is considerable and warrants continued investigation.

Alzheimer Disease↗
PubMed2005

Characterization of the secreted proteome of rat hepatocytes cultured in collagen sandwiches.

Analysis of proteins in biological samples opens up the possibility of discovering new markers of toxicity. The liver is one of the primary targets of drug-induced toxicity, and it also secretes many plasma proteins, which can be measured clinically. Most of the plasma proteins produced by the liver are secreted by hepatocytes, but there is little information regarding the protein profile secreted by these cells. The purpose of this study was to analyze the secreted proteome of primary rat hepatocytes in a collagen gel sandwich configuration by a gel-LC-MS/MS procedure. We identified over 600 peptides corresponding to more than 200 proteins. The protein profile included over 50 plasma proteins, suggesting that the cultured hepatocytes secrete many of the proteins that they produce in vivo. Our data also suggests that the hepatocytes are actively remodeling their environment, since we identified several structural extracellular matrix proteins as well as some proteins known to be secreted specifically during liver regeneration. We also identified two proteins, alpha1-antitrypsin and alpha2-macroglobulin, whose secretions appear to be down-regulated in cells exposed to aflatoxin B1. It was noted that a 15 nM dose of aflatoxin B1 led to substantially diminished levels of these proteins and that day 6 of incubation was the ideal time point for medium collection. These data suggest that proteins in the conditioned medium of hepatocyte sandwich culture might lead to the discovery of biomarkers for drug-induced chemical toxicity.

Aflatoxins↗
PubMed2003

PRISM, a generic large scale proteomic investigation strategy for mammals.

We have developed a systematic analytical approach, termed PRISM (Proteomic Investigation Strategy for Mammals), that permits routine, large scale protein expression profiling of mammalian cells and tissues. PRISM combines subcellular fractionation, multidimensional liquid chromatography-tandem mass spectrometry-based protein shotgun sequencing, and two newly developed computer algorithms, STATQUEST and GOClust, as a means to rapidly identify, annotate, and categorize thousands of expressed mammalian proteins. The application of PRISM to adult mouse lung and liver resulted in the high confidence identification of over 2,100 unique proteins including more than 100 integral membrane proteins, 400 nuclear proteins, and 500 uncharacterized proteins, the largest proteome study carried out to date on this important model organism. Automated clustering of the identified proteins into Gene Ontology annotation groups allowed for streamlined analysis of the large data set, revealing interesting and physiologically relevant patterns of tissue and organelle specificity. PRISM therefore offers an effective platform for in-depth investigation of complex mammalian proteomes.

Animals↗
PubMed2005

Brain injury and proteomics/peptidomics: is it relevant? an overview.

Proteomics and peptidomics are different and supplemental to genomics, since in contrast to the basically constant genome - the proteome and peptidome are dynamic, constantly changing, and complex networks. Proteomics is traditionally linked to 2D-gel electrophoresis techniques. Concerning peptidomics, three different approaches are currently available, all using mass spectrometry as a key element. The use of proteomics or peptidomics in traumatic brain injury (TBI) research is demanding. From the technical point of view there are high-level requirements concerning the preanalytical phase, specific machinery, sophisticated software and skilled manpower/intellectual input. There are currently no bedside techniques and most methods are suitable for experimental TBI research in specialized laboratories. In screening experiments of CSF following controlled cortical impact in rats we identified several peptides, which, although previously known, were so far not reported in the TBI context or in CSF. Peptidomics and proteomics, as highly complex screening technologies, thus seem to carry a large potential to lead TBI science. Newly "discovered" peptide targets have to be validated with different methodology to establish a real diagnostic or therapeutic value.

Animals↗
PubMed2006

Inner ear proteomics of mouse models for deafness, a discovery strategy.

Inner ear dysfunction is often associated with defective hair cells. Therefore, hair cells are the focus of study in many of the mouse mutants showing auditory and vestibular deficits. However, harvesting sufficient numbers of hair cells from the tiny bony mouse inner ear for proteomic analysis is challenging. New approaches that would take advantage of mouse mutants and avoid processing steps, such as decalcification or microdissetion, would be more suitable for proteomic analysis. Here, we propose a novel approach called SSUMM-Subtractive Strategy Using Mouse Mutants. SSUMM takes advantage of the differences between control and affected or mutant samples. We predict that SSUMM would be a useful method in proteomics, especially in those cases in which the investigator must work with small numbers of diverse cell types from a tiny organ. Here, we discuss the potential utility of SSUMM to unravel the protein expression profiles of hair cells using the Pou4f3 mouse mutant as an example. Pou4f3 mutant mice exhibit a total loss of inner and outer hair cells, but supporting cells remain relatively intact in the cochlea, thus providing an excellent model for identifying proteins and transcripts that are specific to the hair cell at all life stages. SSUMM would maximize the sensitivity of the analyses while obviating the need for tedious sessions of microdissection and collection of hair cells. By comparing the mutant to control ears at specific time points, it is possible to identify direct targets of a gene product of interest. Further, SSUMM could be used to identify and analyze inner ear development markers and other known genes/proteins that are coexpressed in the ear. In this short technical report, we also discuss protein-profiling approaches suitable for SSUMM and briefly discuss other approaches used in the field of proteomics.

Animals↗
PubMed2004

Correlation between catalytic efficiency and the transcription read-out in chemical complementation: a general assay for enzyme catalysis.

High-throughput assays for enzyme catalysis that can be applied to a broad range of chemical reactions are key to advances in directed evolution and proteomics. Recently, we reported such a general assay, chemical complementation, which links enzyme catalysis to reporter gene transcription in vivo using the yeast three-hybrid assay. In this proof-of-principle experiment, it was shown that a wild-type beta-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen. Ideally, however, such an assay should be able to distinguish enzymes based on their catalytic activity. Thus, here, we set out to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level of transcription activation in the chemical complementation assay. First, the reaction mechanism for the cleavage of the beta-lactam substrate used in the chemical complementation proof-of-principle experiment was determined. Then a series of beta-lactamase variants was designed to span several orders of magnitude in k(cat)/K(m). The activity of each variant was determined both in vitro using purified enzyme and in vivo in the chemical complementation transcription assay. Beta-lactamase variants spanning three-orders of magnitude in k(cat)/K(m) could be distinguished in the assay, and the catalytic efficiency of each variant correlated with its level of transcription activation in vivo. These results establish the suitability of chemical complementation for the directed evolution of enzymes with improvements in catalytic activity and for profiling the relative substrate specificities of a group of enzymes in proteomics applications.

Catalysis↗
PubMed2005

Clinical-scale high-throughput human plasma proteome analysis: lung adenocarcinoma.

Clinical proteomics requires the stable and reproducible analysis of a large number of human samples. We report a high-throughput comprehensive protein profiling system comprising a fully automated, on-line, two-dimensional microflow liquid chromatography/tandem mass spectrometry (2-D microLC-MS/MS) system for use in clinical proteomics. A linear ion-trap mass spectrometer (ITMS) also known as a 2-D ITMS instrument, which is characterized by high scan speed, was incorporated into the microLC-MS/MS system in order to obtain highly improved sensitivity and resolution in MS/MS acquisition. This system was used to evaluate bovine serum albumin and human 26S proteasome. Application of these high-throughput microLC conditions and the 2-D ITMS resulted in a 10-fold increase in sensitivity in protein identification. Additionally, peptide fragments from the 26S proteasome were identified three-fold more efficiently than by the conventional 3-D ITMS instrument. In this study, the 2-D microLC-MS/MS system that uses linear 2-D ITMS has been applied for the plasma proteome analysis of a few samples from healthy individuals and lung adenocarcinoma patients. Using the 2-D and 1-D microLC-MS/MS analyses, approximately 250 and 100 different proteins were detected, respectively, in each HSA- and IgG-depleted sample, which corresponds to only 0.4 microL of blood plasma. Automatic operation enabled the completion of a single run of the entire 1-D and 2-D microLC-MS/MS analyses within 11 h. Investigation of the data extracted from the protein identification datasets of both healthy and adenocarcinoma groups revealed that several of the group-specific proteins could be candidate protein disease markers expressed in the human blood plasma. Consequently, it was demonstrated that this high-throughput microLC-MS/MS protein profiling system would be practically applicable to the discovery of protein disease markers, which is the primary objective in clinical plasma proteome projects.

Adenocarcinoma↗
PubMed2005

Identification of coexpressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues.

A major advantage of the mouse model lies in the increasing information on its genome, transcriptome, and proteome, as well as in the availability of a fast growing number of targeted and induced mutant alleles. However, data from comparative transcriptome and proteome analyses in this model organism are very limited. We use DNA chip-based RNA expression profiling and 2D gel electrophoresis, combined with peptide mass fingerprinting of liver and kidney, to explore the feasibility of such comprehensive gene expression analyses. Although protein analyses mostly identify known metabolic enzymes and structural proteins, transcriptome analyses reveal the differential expression of functionally diverse and not yet described genes. The comparative analysis suggests correlation between transcriptional and translational expression for the majority of genes. Significant exceptions from this correlation confirm the complementarities of both approaches. Based on RNA expression data from the 200 most differentially expressed genes, we identify chromosomal colocalization of known, as well as not yet described, gene clusters. The determination of 29 such clusters may suggest that coexpression of colocalizing genes is probably rather common.

Animals↗
PubMed2005

Gene profiling of laser-microdissected brain regions and sub-regions.

The application of transcriptomics and proteomics approaches to accurately dissected anatomically-defined brain regions and sub-regions remains a central focus of current neurobiological investigations as well as a necessary step towards single-neuron neurogenomics and neuroproteomics. A protocol is described for the simple, rapid, and reproducible laser microdissection of brain regions and sub-regions for microarray-based gene expression analyses from individual rats or mice using two rounds of in vitro transcription (IVT). The results presented also demonstrate that the current Affymetrix GeneChip arrays are well suited for this experimental design with high reproducibility and limited effects of the shortening of target RNA caused by the double IVT approach.

Animals↗
PubMed2005

Proteomic analysis of colonic myofibroblasts and effect on colon cancer cell proliferation.

BACKGROUND: The stromal microenvironment influences many steps of tumor progression through the elaboration of signals from myofibroblasts. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in colonic epithelial proliferation. The purpose of this study was to determine (1) the influence of myofibroblasts on colon cancer cell proliferation and PI3K activity, and (2) the protein alterations associated with myofibroblasts derived from polyp versus normal margins. METHODS: Myofibroblasts were derived from polyps and corresponding normal mucosa. Myofibroblasts were cocultured with colon cancer cells HT29 stably transfected with green fluorescent protein and KM20 cells. Proliferation was quantitated by green fluorescent protein count and cytokeratin enzyme-linked immunosorbent assay. HT29 cells were incubated with conditioned medium from myofibroblasts, and the effect on proliferation and PI3K activity was determined by 5-bromo 2-deoxyuridine incorporation and Akt kinase assay, respectively. Protein profiles were obtained by SELDI-TOF MS analysis. RESULTS: In coculture experiments, all myofibroblasts significantly enhanced HT29 and KM20 cell proliferation. However, polyp myofibroblasts enhanced proliferation of the cancer cells to a greater extent than normal myofibroblasts. Conditioned medium from all myofibroblasts stimulated Akt kinase activity. SELDI-TOF MS profiles showed more than 40 protein peaks for each isolate. One protein was differentially expressed in polyps versus normal cells. CONCLUSIONS: Utilizing a novel proteomic approach, we identify distinct protein profiles in myofibroblasts of polyps compared with stromal cells of normal mucosa. Moreover, myofibroblasts can stimulate indirectly PI3K activity and enhance colon cancer cell proliferation. These findings suggest that targeted therapy to signaling pathways in myofibroblasts may be useful in colorectal cancer chemoprevention and possible treatment.

Adenomatous Polyps↗
PubMed2004

Proteomic approaches to antigen discovery.

Proteomics has been widely applied to develop two-dimensional polyacrylamide gel electrophoresis maps and databases, evaluate gene expression profiles under different environmental conditions, assess global changes associated with specific mutations, and define drug targets of bacterial pathogens. When coupled to immunological assays, proteomics may also be used to identify B-cell and T-cell antigens within complex protein mixtures. This chapter describes the proteomic approaches developed by our laboratories to accelerate the antigen discovery program for Mycobacterium tuberculosis. As presented or with minor modifications, these techniques may be universally applied to other bacterial pathogens or used to identify bacterial proteins possessing other immunological properties.

Animals↗
PubMed2004

Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry.

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic ras(V12) allele. Of approximately 2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

Antioxidants↗
PubMed2006

DNA microarray and proteomic strategies for understanding alcohol action.

This article summarizes the proceedings of a symposium presented at the 2005 annual meeting of the Research Society on Alcoholism in Santa Barbara, California. The organizer was James M. Sikela, and he and Michael F. Miles were chairs. The presentations were (1) Genomewide Surveys of Gene Copy Number Variation in Human and Mouse: Implications for the Genetics of Alcohol Action, by James M. Sikela; (2) Regional Differences in the Regulation of Brain Gene Expression: Relevance to the Detection of Genes Associated with Alcohol-Related Traits, by Robert Hitzemann; (3) Identification of Ethanol Quantitative Trait Loci Candidate Genes by Expression Profiling in Inbred Long Sleep/Inbred Short Sleep Congenic Mice, by Robnet T. Kerns; and (4) Quantitative Proteomic Analysis of AC7-Modified Mice, by Kathleen J. Grant.

Alcoholism↗
PubMed2004

Action and reaction: Chlamydophila pneumoniae proteome alteration in a persistent infection induced by iron deficiency.

Chlamydophila pneumoniae is an obligate intracellular pathogen implicated in a variety of acute and chronic diseases. Long-term infections are associated with a persistent life stage, in which bacteria can stay for years. They are less accessible to antibiotic treatment but still prone to sustain an inflammatory response. Different in vitro models have been established to mimic and characterize chlamydial persistency. For C. pneumoniae and Chlamydia trachomatis, altered metabolic activities and changed antigenic profiles compared to acute infections have been reported. Most studies including transcriptome and proteome analyses describe persistency induced by IFNgamma treatment. Here, we use iron depletion of the infected cell culture that also leads into persistent infection. We describe differently regulated proteins found by subtractive proteome analysis comparing two early stages of infection with and without addition of the iron chelator deferoxamine-mesylate. While only one bacterial protein was up-regulated during iron deficiency up to 24 h post infection (p.i.), 11 were found to be up-regulated and eight to be down-regulated from 24-48 h p.i. Two down-regulated proteins could be identified by peptide mass fingerprinting as thioredoxin reductase and chromosome partitioning protein (ParB). The latter is involved in chromosome segregation. Thus, using a comparative approach we identified on a proteome level down-regulation of ParB in persistent chlamydial forms, which is in agreement with previous results describing changes in cell division and atypical altered morphology of persistent Chlamydiae.

Amino Acid Sequence↗