Extracellular proteome changes of Deinococcus radiodurans under gamma-irradiation stress conditions

To analysis the change of Deinococcus radiodurans extracellular proteins recovering from gamma-irradiation, we examined extracellular proteome changes using two-dimensional polyacrylamide gel electrophoresis. Twenty-six spots on the gel of irradiated sample were showed significant changes compared with spots on the control gel. Using peptide mass fingerprinting via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), 21 different proteins could be distinguished. Among the identified proteins, seven are classified in transport and metabolism, and one is involved in intracellular trafficking and secretion. The other proteins are known to several functions in the cytosol. Most of the proteins have not previously been reported to be relevant to radioresistance. These results imply that the transmembrane transportation is involved in and contributes to the radioresistance in this organism.     

Altered immune proteome of Staphylococcus aureus under iron‐restricted growth conditions

Staphylococcus aureus is one of the major causative agents of severe infections, and is responsible for a high burden of morbidity and mortality. Strains of increased virulence have emerged (e.g. USA300) that can infect healthy individuals in the community and are difficult to treat. To add to the knowledge about the pathophysiology of S. aureus, the adaption to iron restriction, an important in vivo stressor, was studied and the corresponding immune response of the human host characterized. Using a combination of 1D and 2D immune proteomics, the human antibody response to the exoproteomes of S. aureus USA300Δspa grown under iron restriction or with excess iron was compared. Human antibody binding to the altered exoproteome under iron restriction showed a 2.7‐ to 6.2‐fold increase in overall signal intensity, and new antibody specificities appeared. Quantification of the secreted bacterial proteins by gel‐free proteomics showed the expected strong increase in level of proteins involved in iron acquisition during iron‐restricted growth compared to iron access. This was accompanied by decreased levels of superantigens and hemolysins. The latter was corroborated by functional peripheral blood mononuclear cell proliferation assays. The present data provide a comprehensive view of S. aureus exoproteome adaptation to iron restriction. Adults have high concentrations of serum antibodies specific for some of the newly induced proteins. We conclude that iron restriction is a common feature of the microenvironment, where S. aureus interacts with the immune system of its human host.     

Quantitative proteome profiling of C. burnetii under tetracycline stress conditions

The recommended antibiotic regimen against Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever.     

Systematic characterization of the murine mitochondrial proteome using functionally validated cardiac mitochondria

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS-based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism, and biogenesis. In addition, there are several other clusters, including proteolysis, protein folding, and reduction/oxidation signaling, which ostensibly represent previously under-appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue- and species-specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles.     

Frankia alni proteome under nitrogen-fixing and nitrogen-replete conditions

Frankia alni induces root nodules on Alnus , in which the bacterium differentiates into nitrogen (N)-fixing cells called vesicles. In culture, F. alni also undergoes major morphological changes as it alternates between N-replete and N-fixing conditions. Lack of biologically available N induces the synthesis of vesicles in which nitrogenase is protected from molecular oxygen by a thick lipid hopanoid envelope. Very little is known about the molecular basis of Frankia –host interaction as well as Frankia cell differentiation. The recent determination of the complete genome sequence of F. alni strain ACN14a has permitted us to characterize its proteome, particularly in the extracellular compartment, which could be involved in Frankia –host interaction, and in the switch from N-replete to N-fixing conditions. To that end, 126 bacterial proteins were analyzed by two-dimensional protein gel electrophoresis and identified by matrix-assisted laser desorption/ionization time of flight fingerprinting using a F. alni proteome database. Interestingly, the extracellular fraction contains some glycolytic enzymes lacking secretion signals, already reported to be extracellularly localized in some streptococci, as well as some abundant stress-resistance proteins. As expected, several proteins involved in N assimilation and oxidative defense system were upregulated in F. alni grown under N-fixing vs N-replete conditions. Furthermore, two Raf kinase inhibitor protein homologs that could play a role in cellular signaling, and a hemoglobin-like protein HbN that could be involved in detoxification of nitric oxide were also upregulated. More surprising, a succinate dehydrogenase was strongly downregulated, which could be linked to the need of pyruvate for the biosynthesis of hopanoids or to reduced oxygen diffusion in vesicles.     

Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress

Functional states of mitochondria are often reflected in characteristic mitochondrial morphology. One of the most fundamental stress conditions, hypoxia-reoxygenation has been known to cause impaired mitochondrial function accompanied by structural abnormalities, but the underlying mechanisms need further investigation. Here, we monitored bioenergetics and mitochondrial fusion-fission in real time to determine how changes in mitochondrial dynamics contribute to structural abnormalities during hypoxia-reoxygenation. Hypoxia-reoxygenation resulted in the appearance of shorter mitochondria and a decrease in fusion activity. This fusion inhibition was a result of impaired ATP synthesis rather than Opa1 cleavage. A striking feature that appeared during hypoxia in glucose-free and during reoxygenation in glucose-containing medium was the formation of donut-shaped (toroidal) mitochondria. Donut formation was triggered by opening of the permeability transition pore or K(+) channels, which in turn caused mitochondrial swelling and partial detachment from the cytoskeleton. This then favored anomalous fusion events (autofusion and fusion at several sites among 2-3 mitochondria) to produce the characteristic donuts. Donuts effectively tolerate matrix volume increases and give rise to offspring that can regain ΔΨ(m). Thus, the metabolic stress during hypoxia-reoxygenation alters mitochondrial morphology by inducing distinct patterns of mitochondrial dynamics, which includes processes that could aid mitochondrial adaptation and functional recovery.     

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.     

Altered breeding biology of the European blackbird under artificial light at night

Artificial light at night (LAN) has become a stressor of global extent. Previous work has highlighted the high potential of LAN to interfere with annual and diel rhythms of seasonal organisms as well as to affect interactions at the community level. However, our understanding how LAN induced alterations of activity and breeding cycles affect the reproductive outcome and fitness of the birds is still limited. Here, we focus on the effects of night time illumination on the breeding biology of urban European blackbirds Turdus merula. Our results indicate that blackbirds prefer illuminated nest sites and advance their date of clutch initiation by 6 d per 1 lux of night time illumination. Furthermore, daily nest survival rates increased with increasing LAN although this effect was most pronounced for the transition from dark to slightly illuminated sites. We suggest that blackbirds breeding under low artificial night light conditions benefit from the LAN‐avoidance of their major predators (nocturnal) whereas predominant predators of blackbirds nesting in the city centre are diurnal and are, thus, not affected by LAN. Hence, it seems likely that both direct effects of LAN on the timing of reproduction as well as indirect effects on interspecific interactions might contribute to the observed changes in the breeding biology of European blackbirds. This study emphasizes the diverse ecological effects of the night time illumination which are – in its complexity – still poorly understood.     

Nonezymatic formation of succinate in mitochondria under oxidative stress

The products of the reactions of mitochondrial 2-oxo acids with hydrogen peroxide and tert-butyl hydroperoxide (tert-BuOOH) were studied in a chemical system and in rat liver mitochondria. It was found by HPLC that the decarboxylation of alpha-ketoglutarate (KGL), pyruvate (PYR), and oxaloacetate (OA) by both oxidants results in the formation of succinate, acetate, and malonate, respectively. The two latter products do not metabolize in rat liver mitochondria, whereas succinate is actively oxidized, and its nonenzymatic formation from KGL may shunt the tricarboxylic acid (TCA) cycle upon inactivation of alpha-ketoglutarate dehydrogenase (KGDH) under oxidative stress, which is inherent in many diseases and aging. The occurrence of nonenzymatic oxidation of KGL in mitochondria was established by an increase in the CO(2) and succinate levels in the presence of the oxidants and inhibitors of enzymatic oxidation. H(2)O(2) and menadione as an inductor of reactive oxygen species (ROS) caused the formation of CO(2) in the presence of sodium azide and the production of succinate, fumarate, and malate in the presence of rotenone. These substrates were also formed from KGL when mitochondria were incubated with tert-BuOOH at concentrations that completely inhibit KGDH. The nonenzymatic oxidation of KGL can support the TCA cycle under oxidative stress, provided that KGL is supplied via transamination. This is supported by the finding that the strong oxidant such as tert-BuOOH did not impair respiration and its sensitivity to the transaminase inhibitor aminooxyacetate when glutamate and malate were used as substrates. The appearance of two products, KGL and fumarate, also favors the involvement of transamination. Thus, upon oxidative stress, nonenzymatic decarboxylation of KGL and transamination switch the TCA cycle to the formation and oxidation of succinate.     

Isocitrate oxidation in dog heart mitochondria under anoxic conditions

The roles of NAD- Specific Isocitrate Dehydrogenase (NAD- IDH) (EC 1.1.1.4), NADP- Specific Isocitrate Dehydrogenase (NADP- IDH) (EC 1.1.1.42) and Piridin Nucleotide Transhydrogenase (Transhydrogenase) (EC 1.6.1.1) in the mitochondrial oxidation of isocitrate through the respiratory chain in conditions of normal and increased energy requirements have been studied in submitochondrial particles isolated from healthy and ischemic dog hearts. The activities of both, NAD- IDH and NADP- IDH were increased in conditions of anoxia, while Transhydrogenase remained unchanged. The results obtained showed that the mitochondrial oxidation of isocitrate in dog myocardium occurs mainly through the NAD- IDH pathway in normoxic and anoxic conditions.     

Altered localization of amyloid precursor protein under endoplasmic reticulum stress

Recent reports have shown that the endoplasmic reticulum (ER) stress is relevant to the pathogenesis of Alzheimer disease. Following the amyloid cascade hypothesis, we therefore attempted to investigate the effects of ER stress on amyloid-beta peptide (Abeta) generation. In this study, we found that ER stress altered the localization of amyloid precursor protein (APP) from late compartments to early compartments of the secretory pathway, and decreased the level of Abeta 40 and Abeta 42 release by beta- and gamma-cutting. Transient transfection with BiP/GRP78 also caused a shift of APP and a reduction in Abeta secretion. It was revealed that the ER stress response facilitated binding of BiP/GRP78 to APP, thereby causing it to be retained in the early compartments apart from a location suitable for the cleavages of Abeta. These findings suggest that induction of BiP/GRP78 during ER stress may be one of the regulatory mechanisms of Abeta generation.     

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.     

Symbiotic nitrogen fixation under stress conditions

Summary An outline is given of the possibility selecting Rhizobium strains capable of performing a relatively good symbiosis with leguminous plants, growing under stress conditions. A Rhizobium strain capable of nodulating pea plants in acid soils is described. A pea cultivar, resistant to the majority of Rhizobium strains when growing at 20°C, is found to require a short period of a higher temperature for successful nodulation. The effect of non-photosynthetic light and its possible significance under natural conditions is discussed.     

Defining the protein complex proteome of plant mitochondria

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.     

Deterioration of rat liver mitochondria under conditions of metabolite deprivation.

In a previous study [Parce, Cunningham & Waite (1978) Biochemistry 17, 1634-1639] changes in mitochondrial phospholipid metabolism and energy-linked functions were monitored as coupled mitochondria were aged in iso-osmotic sucrose solution at 18 degrees C. The sequence of events that occur in mitochondrial deterioration under the above conditions have been established more completely. Total adenine nucleotides are depleted early in the aging process, and their loss parallels the decline in respiratory control. Related to the loss of total adenine nucleotides is a dramatic decrease in ADP and ATP translocation (uptake). The decline of respiratory control is due primarily to a decrease in State-3 respiration; loss of this respiratory activity can be related to the decline in ADP translocation. Mitochondrial ATPase activity does not increase significantly until State-4 respiration has increased appreciably. At the time of loss of respiratory control the ATPase activity increases to equal the uncoupler-stimulated activity. The H+/O ratio and P/O ratios do not decrease appreciably until respiratory control is lost. Similarly, permeability of the membrane to the passive diffusion of protons increases only after respiratory control is lost. There observations reinforce our earlier conclusion that there are two main phases in mitochondrial aging. The first phase is characterized by loss of the ability to translocate adenine nucleotides. The second phase is characterized by a decline in the ability of the mitochondrion to conserve energy (i.e. maintain a respiration-driven proton gradient) and to synthesize ATP.