Spaceflight induced changes in the human proteome

Introduction: Spaceflight is one of the most extreme conditions encountered by humans: Individuals are exposed to radiation, microgravity, hypodynamia, and will experience isolation. A better understanding of the molecular processes induced by these factors may allow us to develop personalized countermeasures to minimize risks to astronauts.

Areas covered: This review is a summary of literature searches from PubMed, NASA, Roskosmos and the authors’ research experiences and opinions. The review covers the available proteomic data on the effects of spaceflight factors on the human body, including both real space missions and ground-based model experiments.

Expert commentary: Overall, the authors believe that the present background, methodology and equipment improvements will enhance spaceflight safety and support accumulation of new knowledge on how organisms adapt to extreme conditions.     

Partial hepatectomy induced liver proteome changes in mice

Acceleration of liver regeneration could be of great clinical benefit in various liver-associated diseases. However, at present little is known about therapeutic interventions to enhance this regenerative process. Our limited understanding and the complexity of the mechanisms involved have prevented the identification of new targets for treatment. Here we propose a broad-range proteomic approach to this problem that makes possible the simultaneous study of different signaling and metabolic pathways on the liver proteome. Changes in protein expression in mouse livers (n = 5 per group) at 6 h and 12 h after partial hepatectomy and sham operation, as compared to untreated controls, were analyzed using two-dimensional gel electrophoresis, mass spectrometry (MS), and mass fingerprinting. Twelve proteins, identified by MS, were up-regulated by at least 2-fold after partial hepatectomy. These included adipose differentiation-related protein, gamma-actin, enoyl coenzyme A hydratase 1, serum amyloid A and eukaryotic translation initiation factor 3. These results indicate that liver regeneration following partial hepatectomy affects various signaling and metabolic pathways.     

Roles of hormones in the responses of excised tomato cotyledons to mannitol induced water stress

Excised tomato cotyledons were aseptically cultured in mannitol induced water stress solutions, and a number of changes were determined. Stressed cotyledons had less fresh weights than normal cotyledons throughout the experiment. Stressed cotyledons initially showed reduced proteins, RNA and chlorophyll contents as compared to normal cotyledons, but eventually showed increases which often surpassed values in normal cotyledons. Endogenous auxin and cytokinin activities were initially lower in stressed cotyledons than in normal cotyledons, but were later higher in stressed cotyledons than in normal cotyledons. Gibberellin changes were slight. Applications of exogenous kinetin were able to reverse the effects of stress in reducing fresh weights, proteins, RNA and chlorophyll contents. Applications of GA₃ only partially reverse stress Effects while IAA had no effects. Stressed cotyledons also senesced more slowly than normal cotyledons. The conclusion was reached that the cotyledons were initially injured by the stress but were able to adapt to the stress and that cytokinin activities were fundamental to the responses.     

Selective shift in the metabolic pathways of excised tomato cotyledons in response to mannitol induced water stress

Excised tomato cotyledons subjected to mannitol induced water stress had more total sugars than normal cotyledons during injury but less total sugars than normal cotyledons in the adaptive phase. During water stress injury, protein and UNA synthesis were reduced, activities of beta-fructofuranosidase and phosphofructokinase were reduced but activities of proteases, ribonuclease, hexokinase and glucose-6-phosphate dehydrogenase were increased. Additions of kinetin reversed the water stress effects on enzyme activities. It is concluded that during water stress injury, there was a fundamental reduction in cytokinin activities leading to the selective shift in the enzyme populations.     

Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.     

Gel electrophoresis patterns of proteins and peroxidases of excised tomato cotyledons subjected to mannitol induced water stress

Excised tomato cotyledonswere subjected to mannitol induced water stress solutions for three days. Extracts of proteins and the enzyme peroxidase were made and separated with polyacrylamide gel electrophoresis. The water stress injury caused increases in bigger protein molecules but decreases in smaller protein molecules. The small fastest moving peroxidase isozyme was almost completely eliminated. Applications of growth regulators to the stress solutions indicated that the protein and peroxidase changes could be explained on the basis of reduction in endogenous cytokinin activities by the water stress.     

Acute and long-term proteome changes induced by oxidative stress in the developing brain

The developing mammalian brain experiences a period of rapid growth during which various otherwise innocuous environmental factors cause widespread apoptotic neuronal death. To gain insight into developmental events influenced by a premature exposure to high oxygen levels and identify proteins engaged in neurodegenerative and reparative processes, we analyzed mouse brain proteome changes at P7, P14 and P35 caused by an exposure to hyperoxia at P6. Changes detected in the brain proteome suggested that hyperoxia leads to oxidative stress and apoptotic neuronal death. These changes were consistent with results of histological and biochemical evaluation of the brains, which revealed widespread apoptotic neuronal death and increased levels of protein carbonyls. Furthermore, we detected changes in proteins involved in synaptic function, cell proliferation and formation of neuronal connections, suggesting interference of oxidative stress with these developmental events. These effects are age-dependent, as they did not occur in mice subjected to hyperoxia in adolescence.     

UV-B induced changes in antioxidant enzymes and their isoforms in cucumber (Cucumis sativus L.) cotyledons

To assess the role of antioxidant defense system on exposure to ultra-violet-B (UV-B) radiation, the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbic acid peroxidase (APX), glutathione reductase (GR) and guaiacol peroxidase (GPX), as well as the level of antioxidants ascorbic acid (AA) and alpha-tocopherol were monitored in cucumber (Cucumis sativus L. var long green) cotyledons. UV-B enhanced the activity of antioxidant enzymes as well as AA content, but decreased the level of alpha-tocopherol. Significant increase was observed in the activities of SOD and GPX. Analysis of isoforms of antioxidant enzymes by native-PAGE and activity staining revealed three isoforms of GPX in unexposed dark-grown cotyledons (control), and their intensity was enhanced by UV-B exposure. In addition, four new isoforms of GPX were observed in cotyledons after UV-B exposure. Although no new isoforms were observed for the other antioxidant enzymes, the activities of their existing isoforms were enhanced by UV-B.     

Polysaccharide and oligosaccharide changes in germinating lupin cotyledons

In germinating lupin cotyledons, there was a rapid depletion of raffinose series oligosaccharides, a temporary increase in sucrose and constant low levels of reducing monosaccharides. The major polysaccharide fraction was extracted with hot NH₄ oxalate—EDTA solution and had the constitution of intercellular/cell wall polysaccharide. GLC examination of component sugars showed that as cotyledons expanded this fraction was depleted and that there was selective hydrolysis of arabinose and galactose, so that the uronic acid proportion increased. Gel and DEAE-cellulose chromatography showed that this fraction became more heterogeneous. The neutral and acidic fractions were separated and the component sugars, viscosities, gel chromatographic behaviour and sedimentation constants of these determined. The results indicated that in the later phase of plant cell wall expansion in germinating lupin cotyledons the arabinogalactan side chains of the pectic polysaccharide fraction are selectively hydrolysed leaving a primary wall with a high uronic acid content.     

Salt-stress induced physiological and proteomic changes in tomato (Solanum lycopersicum) seedlings

Soil salinity is one of the major abiotic stress limiting crop productivity and the geographical distribution of many important crops worldwide. To gain a better understanding of the salinity stress responses at physiological and molecular level in cultivated tomato (Solanum lycopersicum. cv. Supermarmande), we carried out a comparative physiological and proteomic analysis. The tomato seedlings were cultivated using a hydroponic system in the controlled environment growth chamber. The salt stress (NaCl) was applied (0, 50, 100, 150 and 200?mM), and maintained for 14 days. Salt treatment induced a plant growth reduction estimated as fresh-dry weight. Photosynthetic pigments (chlorophyll a, b) content of NaCl-treated tomato plants was significantly decreased as the salinity level increased. Proline accumulation levels in leaf and root tissues increased significantly with increasing NaCl concentration. Relative electrolyte leakage known as an indicator of membrane damage caused by salt stress was increased proportionally according to the NaCl concentrations. Roots of control and salt-stressed plants were also sampled for phenol protein extraction. Proteins were separated by two-dimensional gel electrophoresis (2-DGE). Several proteins showed up- and downregulation during salt stress. MALDI-TOF/MS analysis and database searching of some of the identified proteins indicated that the proteins are known to be in a wide range of physiological processes, that is, energy metabolism, ROS (reactive oxygen species) scavenging and detoxification, protein translation, processing and degradation, signal transduction, hormone and amino acid metabolism, and cell wall modifications. All proteins might work cooperatively to reestablish cellular homeostasis under salt stress, water deficiency, and ionic toxicity.     

营养胁迫诱导的油菜子叶衰老过程中IAA氧化酶和细胞分裂素氧化酶活性的变化

以离体油菜子叶为材料,研究了营养胁迫诱导的子叶衰老过程中吲哚乙酸氧化酶(IAA氧化酶)和细胞分裂素氧化酶活性的变化。在光照条件下,离体子叶在不含任何无机元素的0．8％的琼脂中培养9d后,出现明显的衰老迹象(叶绿素含量下降,丙二醛含量上升),15d时完全死亡。在营养胁迫诱导的衰老过程中,IAA氧化酶和细胞分裂素氧化酶的活性表现出相似的变化趋势,在诱导处理1d时,两种酶的活性均比处理前有明显下降,之后又随着衰老进程逐渐上升。IAA氧化酶活性在诱导处理11d时达到高峰,超出处理前30％以上;比对照高出1倍以上;而细胞分裂素氧化酶活性在诱导处理13d时达到高峰,比对照高出3倍以上,也超过了处理前的水平。衰老过程中IAA氢化酶和细朐分裂素氧化酶活性的上升可能是导致内源激素含量下降的重要原因。     

Sugar and starch changes in pea cotyledons during germination

Changes in starch and sugar contents in the cotyledons during germination have been compared in a smooth (cv. Alaska) and a wrinkled (cv. Progress) cultivar of the garden pea ( Pisum sativum L.). In both cultivars there was an initial accumulation of sucrose due to the hydrolysis of sucrosyl oligosaccharides, but galactose did not accumulate in the cotyledons. Starch mobilization in the Progress pea was linear with time and started before the rise in α-amylase (EC 3.2.1.1) activity in the cotyledons; sucrose was synthesized in the cotyledons, and their excision from the axis resulted in an additional accumulation of this sugar. In the Alaska pea, the onset of starch hydrolysis coincided with the rise in α-amylase activity; no accumulation of sucrose was found in excised cotyledons, whilst the sucrose content decreased continuously in attached cotyledons.
The same sugars were found in the cotyledons of both cultivars, suggesting a common pathway for starch breakdown. Maltose, maltotriose and linear malto-dextrins were not present and only trace amounts of glucose were detected, suggesting a degradation of starch by phosphorylase after an initial attack by α-amylase. α-Amylase activity in the cotyledons was higher in the presence of the axis, but was influenced by the water content of the cotyledons. Transient changes in α-amylase activity correlated well with changes in the rate of starch hydrolysis, but after 2–3 days starch mobilization was reduced in excised cotyledons probably due to the resynthesis of starch.     

In vivo pharmaco-proteomic analysis of hydroxyurea induced changes in the sickle red blood cell membrane proteome

Hydroxyurea (HU) is an effective drug for the treatment of sickle cell disease (SCD). The main clinical benefit of HU is thought to derive from its capacity to increase fetal hemoglobin (HbF) production. However, other effects leading to clinical benefit, such as improved blood rheology, have been suggested. In order to understand HU-induced changes at the proteomic level, we profiled sickle RBC membranes from of HU-treated and untreated patients. Our previous in vitro profiling studies on sickle RBC membranes identified a significant increase in predominantly anti-oxidant enzymes, protein repair and degradation components and a few RBC cytoskeletal proteins. In the present study, using 2D-DIGE (Two-Dimensional Difference In-Gel Electrophoresis) and tandem mass spectrometry, we detected 32 different proteins that significantly changed in abundance in the HU treatment group. The proteins that significantly increased in abundance were mostly membrane skeletal components involved in the regulation of RBC shape and flexibility, and those showing a significant decrease were components of the protein repair and degradation machinery. RBC palmitoylated membrane protein 55 (p55) is significantly increased in abundance at low (in vitro) and high (in vivo) concentrations of HU. Palmitoylated p55 may be an important target of HU-dependent regulation of the sickle RBC membrane, consistent with our earlier in vitro studies.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

The mixed xylem sap proteome of Fusarium oxysporum-infected tomato plants

Secreted proteins are known to play decisive roles in plant–fungus interactions. To study the molecular details of the interaction between the xylem-colonizing, plant-pathogenic fungus Fusarium oxysporum and tomato, the composition of the xylem sap proteome of infected tomato plants was investigated and compared with that of healthy plants. Two-dimensional gel separation and mass spectrometry yielded peptide masses and peptide sequences of 33 different proteins. Despite the absence of complete genome sequences of either tomato or F. oxysporum , 21 proteins were identified as tomato proteins and seven as fungal proteins. Thirteen of the tomato proteins were specific for infected plants. Sixteen tomato proteins were found in xylem sap for the first time, four of which were identified based on matches to expressed sequences only. Coding sequences for new proteins from F. oxysporum were identified through either direct matching to a database sequence, matching of peptide sequences to genome or expressed sequence tag databases of other Fusarium species, or PCR with degenerate primers on cDNA derived from infected plants followed by screening of a F. oxysporum BAC library. Together, these findings provide an excellent basis for further exploration of the interaction between xylem-colonizing pathogens and their hosts.     

The potential for predation induced somatic embryogenesis in storage cotyledons

Using seven tropical rainforest species of north Queensland, Australia, we simulated partial predation and compared the development of intact embryos and embryos from which various proportions had been removed, including in some cases the original root–shoot axis. Embryos of all species contained storage reserve cotyledons and germinate in a hypogeal manner. Species mean embryo masses ranged from 0.4–48.1 g. Partial embryo predation treatments were: ½ embryo treatment; embryos separated into component cotyledons, and ¼ embryo treatment; cotyledons separated as above and subsequently cut in half. Germination was scored as production of roots and shoots, or roots or shoots only. Embryos from all species produced seedlings in all treatments, even after the removal of up to 75 percent of the cotyledonary reserve. Moreover, the proportion germinating were not different between intact embryos and i) separated cotyledons, ii) cotyledon halves that maintained or were adjacent to the embryonic axis (except for the largest seeded species), and iii) cotyledon halves that did not include the root–shoot axis (four species). Thus, production of roots and shoots, or roots or shoots only was largely independent of the presence of the embryonic root–shoot axis – implying that somatic cells in storage cotyledons are capable of differentiating into the full range of cell types typical of shoots and roots in the absence of the root–shoot axis. The generality of this response across all seven species suggests somatic embryogenesis in storage cotyledons may be a more widespread phenomenon in tropical floras than currently considered.