Structural modifications of Methanococcus jannaschii flagellin proteins revealed by proteome analysis.

Methanococcus jannaschii is an autotrophic archaeon originally isolated from an oceanic thermal vent. The primary metabolic pathway for energy production in this hyperthermophilic microbe is methanogenesis from H2 and CO2. As an autotroph, M. jannaschii requires only CO2 as a carbon source for synthesizing all necessary biomolecules. Changes in the environmental availability of these molecules can be expected to activate regulatory mechanisms manifested as the up and down regulation of specific genes and the concomitant increase and decrease in abundance of the corresponding proteins. In our analysis of the proteome of M. jannaschii, we have observed significant changes in the abundance of a common subset of predominant proteins in response to reduced H2 concentration, limited ammonium availability, and the stage of cell growth (exponential compared with stationary). The masses of tryptic peptides from these proteins match those predicted by M. jannaschii genome open reading frames annotated as flagellin B1 (MJ0891) and flagellin B2 (MJ0892). Multiple proteins with different isoelectric points and molecular weights match each of these proteins, and the abundance of these protein variants changes with growth conditions. These data indicate that structural modifications altering both the isoelectric point and size of the M. jannaschii flagellin B1 and B2 proteins occur in response to growth conditions and growth stage of M. jannaschii and further suggest the regulation of M. jannaschii motility through structural modifications of the building blocks of the flagella.

Identification of membrane proteins in the hyperthermophilic archaeon pyrococcus furiosus using proteomics and prediction programs.

Cell-free extracts from the hyperthermophilic archaeon Pyrococcus furiosus were separated into membrane and cytoplasmic fractions and each was analyzed by 2D-gel electrophoresis. A total of 66 proteins were identified, 32 in the membrane fraction and 34 in the cytoplasmic fraction. Six prediction programs were used to predict the subcellular locations of these proteins. Three were based on signal-peptides (SignalP, TargetP, and SOSUISignal) and three on transmembrane-spanning alpha-helices (TSEG, SOSUI, and PRED-TMR2). A consensus of the six programs predicted that 23 of the 32 proteins (72%) from the membrane fraction should be in the membrane and that all of the proteins from the cytoplasmic fraction should be in the cytoplasm. Two membrane-associated proteins predicted to be cytoplasmic by the programs are also predicted to consist primarily of transmembrane-spanning beta-sheets using porin protein models, suggesting that they are, in fact, membrane components. An ATPase subunit homolog found in the membrane fraction, although predicted to be cytoplasmic, is most likely complexed with other ATPase subunits in the membrane fraction. An additional three proteins predicted to be cytoplasmic but found in the membrane fraction, may be cytoplasmic contaminants. These include a chaperone homolog that may have attached to denatured membrane proteins during cell fractionation. Omitting these three proteins would boost the membrane-protein predictability of the models to near 80%. A consensus prediction using all six programs for all 2242 ORFs in the P. furiosus genome estimates that 24% of the ORF products are found in the membrane. However, this is likely to be a minimum value due to the programs' inability to recognize certain membrane-related proteins, such as subunits associated with membrane complexes and porin-type proteins.

Mouse liver selenium-binding protein decreased in abundance by peroxisome proliferators.

Several studies with two-dimensional gel electrophoresis (2-DE) have shown that the abundance of numerous mouse liver proteins is altered in response to treatment with chemicals known to cause peroxisome proliferation. The peptide masses from tryptic digests of two liver proteins showing dramatic decreases in abundance in response to numerous peroxisome proliferators were used to search sequence databases. The selenium-binding protein 2 (SBP2 formerly 56 kDa acetaminophen-binding protein, AP 56) and selenium-binding protein 1 (SBP1 formerly 56 kDa selenium-binding protein, SP 56) in mouse liver, proteins with a high degree of sequence similarity, were the highest ranked identities obtained. Identity with SBP2 was subsequently confirmed by immunodetection with specific antiserum. Treatment of mice with 0.025% ciprofibrate resulted in the more basic of this pair of proteins being decreased to 30% of control abundance while the acidic protein was decreased to 7% of the control amount. Dexamethasone treatment, in contrast, caused increases of 80% and 20% in the abundance of the acidic and basic forms, respectively. Administration of dexamethasone to mice in combination with ciprofibrate produced expression of the acidic SBP2 at 23% of the control level and the basic SBP2 at 36%, a slightly moderated reduction compared with the decrease that occurred with ciprofibrate alone. These data suggest that peroxisome proliferators such as ciprofibrate cause a decrease in the abundance of the SBP2, which leads to increased cell proliferation, even in the presence of an inhibitor such as dexamethasone. Such a decrease in SBP, thought to serve as cell growth regulation factors, could be central to the nongenotoxic carcinogenicity of the peroxisome proliferators observed in rodents.

A comparison of liver protein changes in mice and hamsters treated with the peroxisome proliferator Wy-14,643.

Interspecies differences in the liver response to Wy-14,643, a potent peroxisome proliferator in rats and mice, have been demonstrated. While both rats and mice show dramatic increases in the number of peroxisomes, the activity of peroxisomal enzymes involved in the beta-oxidation of fatty acids, and heptocyte replication, Syrian hamsters have a more moderate peroxisome proliferation response and no sustained increase in cell replication. Rats and mice, but not hamsters, develop hepatocellular carcinoma after prolonged exposure to Wy-14,643. To further characterize this species difference, two-dimensional gel electrophoresis (2-DE) has been used to compare the effect of 14-day exposure to various dietary concentrations of Wy-14,643 on liver protein expression in male mice and hamsters. Digitized images of the 2-DE protein maps were searched for significant changes. The peroxisome bifunctional enzyme (PBE) enoyl CoA hydratase/3-hydroxyacyl dehydrogenase, which migrates to the same position in mouse and hamster liver protein 2-DE patterns, increased in abundance by more than three times the control level in both mice and hamsters. In addition to the quantitative change in PBE, significant quantitative changes (P < 0.001) were found in 49 mouse liver proteins (47 decreasing and 2 increasing) and in 35 hamster liver proteins (27 decreasing and 8 increasing). There was little overlap in the mouse and hamster proteins showing quantitative changes in response to Wy-14,643, with the exception of PBE and one unidentified liver protein with an approximate molecular weight of 50,000. These results show that although peroxisome proliferation occurs in the livers of both mice and hamsters exposed to Wy-14,643, other species-specific changes in proteins occur that are independent of the peroxisome proliferation response and that could be related to species-specific susceptibility or resistance to liver tumor induction.

A two-dimensional electrophoresis database of human breast epithelial cell proteins.

As sequencing of the human genome progresses, attention is turning to when and where specific genes are being expressed and how that expression is regulated. The human breast, with the highly specific, but transient, function of milk production (lactation), exemplifies human gene regulation. The molecular mechanisms for the dramatic structural and functional changes involved in shifting from lactation-capable to lactation-incapable tissue are poorly understood, as are the mechanisms that result in deviation from normal breast cell growth into different types of breast neoplasms. We are using quantitative two-dimensional electrophoresis (2-DE) to determine which proteins are present in different types of human breast cells (milk-producing and -nonproducing, estrogen-receptor-positive and -negative, normal and malignant) and which proteins change in abundance in response to stimuli that trigger cell differentiation, growth, or death. A composite map of proteins found in human breast cells is being generated and used as an index of human genes that are differentially expressed, both qualitatively and quantitatively. Proteins found in 15 different types of human breast cells, two from healthy tissue (from milk and reduction mammoplasty tissue) and 13 from tumor tissue, are now included in the composite map. Copies of the human breast epithelial cell protein map are available on the World Wide Web (URL: http:(/)/www.anl.gov/CMB/PMG/ projects/index_hbreast.html) with links to quantitative data and identifications for proteins found to be differentially expressed in these epithelial cells. Links to the Swiss-Prot and enzyme metabolic pathway databases are also provided. The World Wide Web presentation is designed to allow public access to the available 2-DE data together with logical connections to databases providing genome-related information.

Analysis of proteins from human breast epithelial cells using two-dimensional gel electrophoresis.

The human breast is a highly specialized, complex tissue comprised of a heterogeneous population of cells with varying functions. Interactions between the different cell types, changes in their relative abundance, state of differentiation and function in response to stimuli, as well as the alterations that lead to the aberrant growth associated with malignancy are poorly understood. Two-dimensional gel electrophoresis is being used to compare the proteins found in different breast cells in order to identify the gene products that are common or specific to particular cell types so as to provide markers that will be useful in studies of normal breast cell differentiation and the dedifferentiation or blocked differentiation characteristic of cancer. Protein patterns have been obtained from cells prepared for electrophoresis immediately after isolation from human milk, from cells cultured for fewer than ten passages after isolation from healthy breast tissue removed during reduction mammoplasty, and from cells maintained in long-term tissue culture after isolation from the pleural effusions of patients with breast carcinomas. Differential expression of cytokeratins 8, 18, and 19, shown previously to be predominantly expressed by epithelial cells in the luminal layer of breast tissue, was observed among the cells analyzed. Other non-cytokeratin proteins were also found to be differentially expressed in subsets of both the normal and tumor cells. A composite human breast cell protein pattern was created which includes all the commonly and specifically expressed proteins found in this study. This pattern will be the basis for continuing studies of proteins in the human breast.

Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.

During the analysis of liver protein expression in the offspring of male mice irradiated with fission-spectrum neutrons, one offspring displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacking detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10-formyl-THF DH; 10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6). NH2-terminal sequence analysis demonstrated that both proteins share identical sequences in the first 25 residues, and this sequence matches (96% identity) that of rat and human 10-formyl-THF DH. In addition, these proteins showed cross-reactivity to polyclonal antiserum raised against purified rat 10-formyl-THF DH. Southern (DNA) blot analysis revealed a restriction fragment length polymorphism consistent with a deletion mutation in the 10-formyl-THF DH structural gene in homozygous mice. Results of Northern (RNA) blot analysis demonstrated the absence of 10-formyl-THF DH mRNA in mice lacking 10-formyl-THF DH protein. Furthermore, liver cytosolic 10-formyl-THF DH enzymatic activity was undetectable in homozygotes. Measurement of hepatic folate pools showed that in homozygotes the total folate pool is decreased and the level of tetrahydrofolate is markedly depleted.

Altered protein expression detected in the F1 offspring of male mice exposed to fission neutrons.

Liver protein expression in F1 offspring arising from spermatogonia exposed to 60 cGy of fission spectrum neutrons from the JANUS reactor was compared to that in offspring from unexposed spermatogonia by using two-dimensional electrophoresis (2DE). Approximately 100 protein spots in 2DE patterns from 167 control offspring and 530 offspring from irradiated sires were monitored for quantitative decreases of 50%, indicative of mutation events causing the loss of one normal copy of a structural gene. Reproducible abnormalities were found only in 3 patterns, all from the offspring of neutron-irradiated sires. Two of the three patterns were from littermates (brother and sister) and both showed an approximately 70% decrease in the amount of liver protein MSN188. The third pattern was from a male mouse sired by a different male and showed an approximately 50% decrease in the abundance of protein MSN94. The decreased abundance of MSN188 and MSN94 was assumed to be due to mutation events referred to as NEUT1 and NEUT2, respectively. Sibling crosses between the 2 mice showing the NEUT1 trait produced offspring with control, decreased and undetectable levels of MSN188 in a ratio of 0.25:0.5:0.25. Test crosses between the F1 offspring expressing the NEUT2 trait back to C57BL/6JANL mice produced offspring expressing normal or decreased amounts of MSN94 in a ratio of 0.5:0.5. Inbreeding of individuals expressing decreased amounts of MSN94 produced mice expressing control, decreased amounts, or no detectable amount of that protein in a ratio of 0.25:0.5:0.25. These results indicate that the decreased abundance of MSN188 or MSN94 originally detected in the F1 offspring is due to a genetically transmissible event. Unlike the heritable protein changes observed previously in the F1 offspring of sires exposed to N-ethyl-N-nitrosourea in which a protein variant was produced, both the NEUT1 and NEUT2 mutation events appear to prevent the production of any protein product. These 2 mutations may thus represent mutation lesions other than point mutations (e.g., deletions or translocations) detectable as quantitative changes in protein expression in the F1 generation.

Mouse liver protein database: a catalog of proteins detected by two-dimensional gel electrophoresis.

Alterations in the abundance or structure of mouse liver proteins are being studied using two-dimensional gel electrophoresis (2-DE) to build a database of protein changes correlating with exposure to ionizing radiation or toxic chemicals. Thus far, studies have included the analysis of proteins from the offspring of exposed parents or from the exposed individuals themselves. In order to characterize and identify proteins found altered by such exposures, sex- and strain-related differences in protein patterns have been analyzed, and the subcellular locations of a large portion of the mapped proteins have been determined. As part of these studies, data are collected and stored using a variety of computer hardware and software tools that allow the accumulation of information on the origin of samples, gel identification, experiment description, and protein similarities and differences. This accumulation of information constitutes the mouse liver protein database. Relational database software is used to tie the different facets of the database together so that the results of a variety of experiments can be compared and interrelated. The database optimizes the information obtained from 2-DE gel sets by allowing use of the data for many purposes, including monitoring of gel resolution to ensure the collection of high quality data and correlation of protein effects induced by different agents. This first edition of the Argonne National Laboratory mouse liver protein database lays the foundation for future work and communication that should elucidate the significance of observed protein effects as possible markers of exposure to toxic agents.

Biochemical characterization of a variant form of cytosolic epoxide hydrolase induced by parental exposure to N-ethyl-N-nitrosourea.

1. ENU4 mice express a protein variant originally detected in a CBF1 mouse sired by a C57BL/6 mouse exposed to N-ethyl-N-nitrosourea. It appears to be an isoelectric point variant of cytosolic epoxide hydrolase. Affinity purified cytosolic epoxide hydrolase from ENU4 mice has a pI of approximately 5.1 compared to 5.6 in other mouse strains. 2. Clofibrate induced cytosolic epoxide hydrolase to similar levels in five strains of mice. However, CBF1 and ENU4 mice were more sensitive to the induction of palmitoyl CoA oxidase activity. 3. Except for isoelectric point, the physico- and immunochemical properties of cytosolic epoxide hydrolase from ENU4 mice were similar to those of the other mouse strains. Substrate specificities for five of six substrates tested were also similar.

Evidence for regulatory genes on mouse chromosome 7 that affect the quantitative expression of proteins in the fetal and newborn liver.

A series of deletions around the albino locus on mouse chromosome 7 is believed to include one or more regulatory genes that control the activities of a cluster of liver enzymes. To further characterize the functions of this region of the mouse genome, we have used quantitative two-dimensional electrophoresis to analyze the effects of two of these deletions, c3H and c14CoS, on the expression of liver proteins. More than 400 distinct protein gene products were quantitated in livers from fetal and newborn wild-type homozygous (cch/cch), heterozygous (cch/c3H or cch/c14CoS), and deletion homozygous (c3H/c3H or c14CoS/c14CoS) mice. Livers of fetal and newborn c3H heterozygotes and homozygous wild-type littermates produced qualitatively identical protein patterns after two-dimensional electrophoresis. In livers of c3H homozygous fetuses, however, abnormal amounts (either increased or decreased relative to homozygous wild-type and heterozygous littermates) of 29 proteins were found. Twenty-eight of these 29 protein anomalies were also found in livers of newborn c3H homozygotes. Livers of fetal and newborn mice homozygous for the c14CoS deletion, which overlaps the c3H deletion and produces a similar phenotype, expressed normal amounts of these proteins. One of the 29 proteins (MSN807) has an amino-terminal sequence similar to a 23-kDa translationally controlled protein abundant in mouse erythroleukemia and sarcoma-180 cells. These results suggest that normal chromosome 7 contains genes, located within the region of the c3H but not the c14CoS deletion, that regulate the abundance of specific proteins in the liver. These proteins cannot be related to the phenotypic alterations shared by the c3H and c14CoS deletions.

Quantitation of human leukocyte proteins after silver staining: a study with two-dimensional electrophoresis.

The quantitative attributes of human leukocyte proteins detected by silver staining two-dimensional electrophoresis (2-DE) gels were studied by using computer-assisted data analysis. Experiments included (a) analysis of replicate patterns of the same sample, (b) analysis of different dilutions of the same sample, and (c) analysis of samples from different individuals. Over 200 proteins were observed to have coefficients of variation (CV) less than or equal to 15% when data from replicate patterns were analyzed. In contrast, 8 proteins had CV values of less than or equal to 15% when data from different samples were analyzed. The dilution experiment showed that a majority of the proteins detected with some consistency (i.e., observed in at least 80% of the patterns) have a linear relationship between the amount of protein loaded onto a 2-DE gel and the spot volume in the final 2-DE pattern. The slope of the curves and the deviation from linearity were found to be quite protein-specific. These results indicate that optimization of sample purity and minimization of staining protocol variables are required to limit the background quantitative variability between and within 2-DE runs to a level that will allow detection of quantitative changes indicative of biological responses.

A comparative study of the effects of clofibrate, ciprofibrate, WY-14,643, and di-(2-ethylhexyl)-phthalate on liver protein expression in mice.

Chemical, physical, or mechanical insults to a cellular system cause changes in the rates of protein synthesis or degradation. To define patterns of quantitative protein changes and identify the biochemical basis of those changes, we are studying fluctuations in mouse liver protein abundance in response to factors such as stages of maturation, changes in diet, and exposure to chemicals. Two-dimensional gel electrophoresis (2DE) together with computer-assisted data analysis is used to collect quantitative data from groups of 2DE patterns. Our analysis of liver protein expression in mice treated with four different chemicals known to cause peroxisome proliferation serves as an example of the changes in protein expression that can be monitored by using this approach. The database we are building indicates that changes specific to a particular treatment as well as generalized effects can be identified, providing the opportunity to identify patterns of protein expression that are indicators of biological response to different types of cellular insults.

The analysis of recessive lethal mutations in mice by using two-dimensional gel electrophoresis of liver proteins.

We have used two-dimensional gel electrophoresis (2DE) coupled with computer-assisted data analysis to analyze liver-protein expression in mice known to be heterozygous carriers of recessive lethal mutations induced in In(1)1Rk or In(7)13Rk inversion stocks by exposure to either triethylene melamine or ionizing radiation. Carriers of 8 different mutations and corresponding littermate controls (average of 17 individuals in each group) were screened for liver-protein differences. Both qualitative and quantitative protein differences were detected that correlated with unique pedigrees among the mouse stocks analyzed. Such strain-specific differences demonstrated that quantitative differences (either increases or decreases) in protein abundance of greater than 25% can be readily detected by using this 2DE system. Thus the 50% reduction in expression of a protein expected in the event of a structural gene deletion is well within the level of detection. No significant quantitative decreases in protein expression that correlated with the recessive lethal mutations were detected, however.

Heritable protein variants induced by exposure to ethylnitrosourea: heritability, subcellular location, and tissue distribution.

The heritability, subcellular location, and tissue distribution of liver protein alterations found in the two-dimensional electrophoresis patterns of 4 offspring from male mice treated with N-ethyl-N-nitrosourea (ENU) were studied. Mice homozygous for each of the 4 ENU-induced protein variants were found to be viable and fertile, although the number of homozygous offspring from crosses between heterozygous carriers of one variant (ENU 2) was less than that expected for a nondetrimental trait. Two of the proteins altered by ENU-induced mutations were associated with the crude mitochondrial fraction, another was found predominantly in the microsomal fraction, and the fourth was associated with the mitochondrial, microsomal, and cytosol fractions. All of the ENU-induced mutations affected proteins that were not liver-specific; i.e., the proteins were found in other tissues in addition to the liver.

A heritable variant of mouse liver ornithine aminotransferase (EC 2.6.1.13) induced by ethylnitrosourea.

A variant of ornithine aminotransferase (OAT, EC 2.6.1.13) has been detected in an offspring of a male mouse treated with ethylnitrosourea. The evidence presented to support the identification of the protein variant (ENU 2) as altered OAT includes (a) a corresponding 50% decrease in the abundance of a protein, located one charge unit basic to the variant, which comigrates on two-dimensional gel patterns with purified mouse liver OAT; (b) the binding of anti-rat-OAT antibody to the variant; (c) the increased abundance of the variant protein in the livers of mice fed a high protein diet (85% casein); and (d) purification of the variant through an OAT purification protocol.

Comparison of rat and mouse ornithine aminotransferase with respect to molecular properties and regulation of synthesis.

A comparative study of the synthesis patterns and molecular properties of mouse and rat ornithine aminotransferase (OAT) was conducted. The two enzymes were found to be very similar with respect to catalytic properties, two-dimensional electrophoresis patterns of tryptic digests, amino acid compositions, and antibody cross-reactivity. In vitro translation assays for OAT synthesis on free polysomes isolated from livers at different times of day showed similar circadian fluctuations in OAT synthesis for both species. However, hybridization measurements revealed no circadian changes in the levels of hybridizable OAT mRNA in these livers. These results demonstrate that the circadian cycling of OAT synthesis is regulated at the level of translation in both the rat and the mouse.

Detection of heritable mutations as quantitative changes in protein expression.

A computerized search for the appearance of heritable mutations (as indicated by changes in protein expression) was conducted on three sets of mice, whose sires had been either untreated, exposed to 3 gray units of gamma radiation, or treated with 150 mg/kg ethylnitrosourea. Proteins from the livers of approximately 800 mice were separated by two-dimensional electrophoresis, and abundances were measured by using image analysis techniques. Heritable mutations were detected by the appearance of new proteins or by the quantitative decrease in abundance of normally occurring proteins. Measurements of the variability of the protein abundance indicate that at least 48 proteins are consistent enough to be used in searches when mutations are expected to result in a 50% reduction in the normal amount of protein. New proteins were found in four offspring from ethylnitrosourea-treated mice, and in each case a nearby spot was found to be significantly diminished. These mutations were confirmed in subsequent generations. A computer-assisted search detected three of these mutations on the basis of the abundance decrease alone. These results indicate that two-dimensional electrophoresis can be used to detect mutations reflected as quantitative changes in protein expression, provided that the proteins to be monitored are quantitatively stable when samples from different individuals are compared.

Effects of toxic agents at the protein level: quantitative measurement of 213 mouse liver proteins following xenobiotic treatment.

By analyzing two-dimensional electrophoretic patterns of mouse liver proteins with a computerized image analysis system, we have observed quantitative changes in the abundance of more than 70 proteins in mice treated with various agents. Aroclor 1254, a mixture of polychlorinated biphenyls known to induce a broad spectrum of microsomal activity, induces the largest group of changes (60 proteins altered at p less than 0.001 significance). Phenobarbital produces a small set of characteristic changes that forms part of the much larger Aroclor 1254 effect. Ibuprofen treatment produces a phenobarbital-like pattern of change, with the addition of at least one protein change not observed with any of the other treatments. Cycloheximide and carbon tetrachloride each induces a different characteristic pattern of protein alteration. We have assigned most of the mouse liver proteins to a specific subcellular fraction, and it appears that the predominant class of proteins altered by each compound is present in the soluble phase, rather than in the microsomal fraction. The ability to survey large numbers of tissue proteins for involvement in pharmacologic and toxic effects may allow a more comprehensive understanding of the mechanisms of action in vivo and provide new markers of tissue damage.