Daila S. Gridley1,2, Michael J. Pecaut1,2, Lora M. Green1, E. Clifford Herrmann2, Brandon Bianski1, James M. Slater1, Louis S. Stodieck4,
Virginia L. Ferguson4 and Lawrence B. Sandberg2,3*
1 Department of Radiation Medicine, Loma Linda University and Medical Center, Loma Linda, CA 92354, USA
2 Department of Basic Sciences, Loma Linda University and Medical Center, Loma Linda, CA 92354, USA
3 Department of Rheumatology, Pathology and Human Anatomy, Loma Linda University and Medical Center, Loma Linda, CA 92354, USA
4 Department of Aerospace Engineering, Bioserve Space Technologies, University of Colorado, Boulder, CO 80309, USA
*Corresponding author: Lawrence B. Sandberg, Department of Basic Sciences,
Loma Linda University, MT 231, 11085 Campus Street, Loma Linda, California
92354, USA, Tel: 909-558-4527; Fax: 909-558-4887; E-mail: lsandberg@llu.edu
Received October 05, 2012; Accepted October 27, 2012; Published October 30,
2012
Citation: Gridley DS, Pecaut MJ, Green LM, Herrmann EC, Bianski B, et al.
(2012) Effects of Space Flight on the Expression of Liver Proteins in the Mouse. J
Proteomics Bioinform 5: 256-261. doi:10.4172/jpb.1000246
Abstract
Raw data derived from mass spectroscopic (MS) analyses of formalin-fixed paraffin-embedded (FFPE) tissue sections of the essential metabolic organ, liver, allocated by the provider (Amgen) from mice subjected to 13 days of microgravity on NASA Flight STS-118 were analyzed by two different search engines, using shotgun proteomics. With the eight statistically significant readouts in hand, Ingenuity Pathway Analysis (IPA) was employed to visualize probable biologic pathway relationships among proteins that might be associated with alterations in liver biochemistry due to space flight. Most noteworthy was the finding of up-regulation of the first urea cycle enzyme carbamoylphosphate synthetase, consistent with increased amino acid catabolism resulting from gravitational changes, and/ or other stress associated with missions in space. Down-regulation of fructose-bisphosphate aldolase B, regucalcin, ribonuclease UK114, alpha enolase, glycine N-methyltransferase and S-adenosyl methionine synthetase isoform type-1 was observed. 60 kDa heat shock protein was elevated.
Introduction
Spaceflight has been shown to affect a number of mammalian body systems. This report provides experimental data on liver proteins of mice that were part of the Commercial Biomedical Test Module-2 flown on the space shuttle Endeavour (STS-118), a 13-day mission
to the International Space Station in August, 2007. Several studies on these mice have now been published [1-7]. In earlier studies on humans, loss of Lean Body Mass (LBM), fat and water, as well as loss of strength and changes in plasma protein and amino acid levels were reported. An increase in urinary interleukin-6 (IL-6) on the first day of flight in the Columbia space shuttle indicated an acute-phase response from the liver [8]. Using a 15N-glycine tracer technique to study protein turnover in four Russian cosmonauts and two U.S. astronauts residing long term aboard the MIR orbital station, investigators found a nearly 50% decrease in protein synthesis [9]. In a review of the literature, Stein and Wade [10] noted that the shift towards increased activity of glycolytic enzymes associated with muscle atrophy, a major concern during spaceflight, is accompanied by increased gluconeogenesis in the liver. Leach et al. [11] showed that there are increased levels of
3-methyl histidine, creatinine and sarcosine due to muscle breakdown, and decreased levels of plasma albumin and transferrin inferring inadequate dietary protein intake on the early Soviet and Skylab missions. The protein depletion seen in astronauts upon landing after missions is followed by a post-flight anabolic phase so that muscles regain normal protein levels, a process that apparently can affect protein status in other body compartments [12]. These above studies, done with hippuric acid (15N glycine) administration and plasma sampling showed that the fractional synthetic rates of fibrinogen, complement C-3, ceruloplasmin, and haptoglobin were low on day 6 after landing compared to pre-flight measurements. These findings were consistent with limited amino acid availability due to substrate competition between muscles and other tissues.
The hypothesis of this present study was that there will be distinct changes in the profile of major liver proteins of the mouse that are introduced by conditions associated with spaceflight. This profile may occur in humans, as well after a mission in space.
Materials and Methods
Animals and liver sample collection C57BL/6NTac female mice (n=10; Taconic Farms, Inc.,
Germantown, NY) were shipped at about 7 weeks of age to the National Aeronautics and Space Administration (NASA) Space Life Sciences Laboratory (SLSL) at the Kennedy Space Center. For both flight (FLT) and ground control (GRD) mice, similarity of housing conditions in animal enclosure modules (AEM) and acclimatization procedures prior to take off have been described [1]. The FLT mice flew onboard the Space Shuttle Endeavour (STS-118) for 13 days.
By using telemetry from the shuttle, the AEM-housed GRD were exposed to environmental conditions comparable to FLT animals (i.e. temperature, humidity, CO2) on a 48-h delay. The FLT and GRD mice housed in the AEMs were equipped with solid food bars and a water dispenser so that nourishment was available continually even after landing. Within 3-6 h after landing of the space shuttle, FLT and GRD mice were evaluated for muscle strength and scanned with nuclear
magnetic resonance imaging to assess lean and fat mass composition (performed by Amgen investigators). Mice were then euthanized with 100% CO2. The NASA, Amgen, Inc., Loma Linda University and University of Colorado Institutional Animal Care and Use Committees approved this study. A Material Transfer Agreement was also obtained for the transfer of mouse tissues. Liver tissues were alloted to the Loma Linda NASA Laboratory by Amgen. Protein analysis FFPE tissue blocks were prepared from the livers harvested from FLT and GRD mice (5 in each group, 10 animals total). The latter served as the basis for comparisons in this study. Sections were cut
at 10 μm thickness and de-paraffinized, but not cover-slipped. Serial sections for protein sampling remained unstained and were stored briefly in water. Areas of the FLT and GRD liver sections selected for uniformity were removed from the slides with a 3 mm punch. These punched tissue samples, each containing approximately 30,000 cells, were placed in 20 μL of Expression Pathology digestion buffer (www.expressionpathology.com). (Expression Pathology Inc. Rockville, MD). The Expression Pathology protocol for digest preparation and analysis was followed precisely for all the samples. This consisted of first heating the punched tissue samples in the proprietary digestion buffer at 95°C for 1 h. This was followed by digestion with sequencing grade trypsin (www.promega.com) (Promega Corp. Madison WI), overnight at 37°C. Small aliquots were analyzed for protein content using a Micro BCA Protein Assay Kit (www.piercenet.com) (Thermo Fisher Scientific, Rockford, IL), prior to subjecting the remaining digest to dithiothreitol reduction. Peptide samples 1.5 μg by protein assay from each digest were then evaluated by LC/MS/MS on a Thermo-Electron LCQ Deca XP mass spectrometer (www.thermo.com) (Thermo Fisher Scientific), using nano-electrospray equipment produced by New Objective, Woburn, MA (www.newobjective.com). This consisted of reverse-phase collection of peptides on a 2 cm x 75 μm capture column followed by separation of peptides on a 10 cm x 75 μm vented analytical
column [13], using Micro Magic RP-18AQ resin (www.michrom.com) (Michrom Bioresources, Inc., Auburn, CA). MS analyses were accomplished with a 5-part protocol cycle that consisted of one full MS survey scan (from 400 to 1700 m/z), followed by acquisition of collisional-induced dissociation tandem mass spectra of the four most intense ions in the survey scan. The nanoflow solvent gradient (linear 2-60% acetonitrile) extended over 3 h, using solvent B (95% acetonitrile with 0.1% formic acid) developed against solvent A (2% acetonitrile with 0.1% formic acid), at a flow rate of 250-300 nL/min. Samples were run as groups of five followed by water blank, preceeded and followed by a Michrom BSA trypsin-digested control followed by a water blank.
Minimal carryover was detected in the water blanks. Data analysis Runs were first evaluated by the Thermo Bioworks software generating .dta files, then Perl .mgf files (www.perl.com) for evaluation
by the Mascot search engine (www.matrixscience.com), using an International Protein Index (IPI) mouse FASTA library (www.ebi.ac.uk/IPI), modified to a concatenated format so as to detect false
discoveries. These results were then moved into Scaffold (version 3.3.3)(www.proteomesoftware.com). Merged resultant files from Mascot and X!-Tandem (www.thegpm.com) within Scaffold gave a final readout of the analysis, using the spectral quantitative value display option with filter settings of: Min Protein 95%, Min # Peptides 2, min Peptide 95%. This gave 67 proteins identified for this dataset. The aim to obtain highly reliable but not necessarily comprehensive quantitative data was attained in positive and negative format by expression as log2 fold changes (FLT/GRD). Thus IPA, which utilizes fold change data as well as P-values for reliability, gave good pathway analyses with this format. By combining FLT and GRD data within Scaffold and entering each analysis as a separate biosample, a T-test analysis was generated between the FLT and GRD groups, and also a log2 fold change for each from FLT AVG / GRD AVG calculations was created. The Quantitative Value display option with Total Ion Current (TIC) was chosen as the quantitative method.
Statistical and pathway analyses
For this type of data, the T-test method of analysis has been shown to be the best [14]. These two groups (FLT and GRD) of data were then analyzed through the use of Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA) (www.ingenuity.com) to generate significant pathway networks, and for comparison with canonical pathway networks within the IPA databases. Data was thus entered into IPA using the IPI accession numbers, the P-values and
log2 fold change values, the latter allowed demonstration of positive (up-regulated) and negative (down-regulated) values for the flight group within IPA (Table 1). The entire contents of the Scaffold analysis dataset, table 1 (67 proteins) was fed into IPA because even proteins of lower significance (P-value>0.050) have an advantageous influence on the outcome of the pathway analysis (Figure 1).
Results and Discussion
In our present MS study comparing FLT versus GRD mice, measurement of statistically significant (P<0.05) changes of eight proteins was achieved in a specific mouse organ, the liver, via an innovative methodology that allows analysis of FFPE tissue for total tissue protein composition. The Expression Pathology reagents and procedure provided a simple method for obtaining these data from FFPE tissues (without this statistical data, no one could have accomplished this study). Scaffold yielded a spectral quantitative value and total ion current for each peptide identified within each analysis of the MS/MS data. Of the 8 proteins, only carbamoyl-phosphate synthetase and 60
kDa heat shock protein, a chaperonin, gave significant positive log2 fold change values. Carbamoyl-phosphate synthetase was the protein present in highest concentration. This enzyme provides the entry point of ammonia into the urea cycle, and is found primarily in the liver [15]. The urea cycle is the major mechanism for ridding the organism of catabolic ammonia. The other enzymes of the urea cycle (ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase and arginase-1) were also apparent in the MS spectra, but their levels were not significantly altered between FLT mice and GRD controls. These are also present in table 1. Their inter-relationship is
shown in figure 2. Because it is up-regulated, carbamoyl-phosphate synthetase appears to be the major urea cycle regulatory enzyme in these mice.
Ammonia in excess of that transformed into urea is toxic to cells via the glutamate dehydrogenase (GDH) catalyzed reaction, even though nontoxic glutamate is produced. The toxicity is due to the
concomitant depletion of alpha keto glutarate, by the GDH-catalyzed reaction and consequently, the other Krebs cycle intermediates [15]. Thus, a metabolic result of detoxifying excess ammonia by GDH activity lowers the amount of oxaloacetate, the substrate required for entry of acetyl coenzyme A into the Krebs cycle; with the result that energy metabolism is inhibited. Up-regulation of GDH is indicated in FLT liver samples, although not statistically significant (Table 1). Even
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