Analysis Of Mitochondrial Proteomes To The Thoracic Muscle Of Hypoxia And Hyperoxia Flies

Hypoxia or hyperoxia stress is closely associated with many pathological and physiological processes such as myocardial infarction, ischemic stroke, oxygen therapy and ischemia-reperfusion. As mitochondria regulate energy metabolism and control oxygen consumption in cells, the organelle plays essential role in dealing with such stress. The relevant mechanisms how mitochondria cope with the harsh circumstance have yet to be explored. Drosophila is a mature model in genetic and functional studies. In addition, most Drosophila tissues are directly exposed to environmental oxygen because air is directly diffused into tissues through a tracheal system. This unique feature makes Drosophila a convenient model for studying the molecular responses of mitochondria to oxygen fluctuations.We have collaborated with our colleagues in University of California, Sandiego, and generated hypoxia and hyperoxia tolerant flies (LOF and HOF) through controlling oxygen pressure in the chamber for long term. We isolated thoracic muscle from LOF and HOF followed by preparation of mitochondria from the thoracic muscle through differential centrifugation. Upon examination of the mitochondrial ultrastructure through transmission electron microscope, we found the mitochondrial structures in LOF and HOF changed significantly as compared with control wild type, in which LOF was observed in honeycomb-patterned mitochondria, the large amplitude swelling and the volume fraction of the mitochondria increased, while HOF was found in regional distortion and the relatively normal volume fraction of the mitochondria. We selected specific substrate for each respiratory chain complexes and measured the activities of each complex. The results of respiration activity demonstrated that the activity of complex I was only decreased in HOF, of complex III and IV were only increased in LOF, while of complex II was decreased both in LOF and HOF, indicating that the morphological and functional changes of the mitochondria in LOF and HOF during adaptation in extreme oxygen condition. We then raised a question what is the molecular basis for such functional changes.We adopted an updated approach of quantitative proteome, iTRAQ, and a strictly statistic evaluation aiming at identification of the comprehensively differential proteins in LOF and HOF and exploration of the mechanisms related with fly mitochondria under extreme oxygen stress. Using a high accuracy mass spectrometry, a total of 718 proteins from 5426 unique peptides corresponding to 40020 MS/MS spectra (FDR<0.01) were identified. The Pearson correlation analysis showed the correlation coefficients (R2) of the duplications were all above 0.9, suggesting the quantitative data with high confidence. Comparing with wild fly,55 and 75 proteins were identified in significantly changed abundances in LOF and HOF, respectively. Based on analysis of Cluster of Orthologous Groups (COG) to the differential proteins, we summarized that most proteins involved in calcium regulation, oxidative related, chaperons and mitochondrial translational system were down-regulated in LOF, but were up-regulated in HOF, while the respiratory chain complex proteins were significantly down-regulated in HOF, but were not so much change in LOF. We analyzed the abundance distribution of differential proteins, and found the abundant proteins with relatively higher percentile. Of the differential proteins with higher abundances, HOF had more respiratory chain complex proteins, whereas LOF possessed more membrane associate proteins. Additionally, we overall looked at the correlation of the changes of protein and mRNA abundances in the two fly models. The results revealed that the typical mitochondrial proteins, such as respiratory chain complex proteins, mitochondrial translation related proteins, carbohydrate/lipid metabolism related proteins and chaperons, exhibited a good correlation of quantitative proteins and mRNAs, while these non-typical mitochondrial proteins, such as calcium regulating proteins, membrane associate proteins and peptide/amino acid metabolism related proteins, presented a poor correlation of the two sets of the quantitative data. Obviously, the transcriptional and translational elements in both LOF and HOF are likely involved in regulation of protein abundances and functions during adaptive process under extreme oxygen stress.In LOF thoracic muscle mitochondria, the enzyme activity of respiratory chain complexes was detected in significant change, whereas the quantitative proteomics upon iTRAQ suggested majority of respiratory chain complexes relatively constant in protein abundance. We further sought an answer to the conflict observation. We conducted Blue Native and Western Blot to specifically check the abundances of mitochondrial proteins, and achieved the similar conclusion, implying that as compared with wild type the abundances of the LOF mitochondrial proteins remained constant. Through HPLC analysis, we measured the energy charge in the muscle samples and found the corresponding values no significant changes as well in LOF. On the other hand, the image analysis to mitochondrial 2DE displayed several differential spots between LOF and wild. Importantly, these spots with high percentile appeared in string pattern, in which the spots shared with similar molecular mass but located at different pls. The phenomenon provides a clue that some mitochondrial proteins are modified under extreme oxygen stress. To test the hypothesis, we selected a protein with string spots on the 2DE, Succinly CoA synthetase (SCS), and further examined it possible modification and functional changes in LOF. With antibody against SCS on SDS- PAGE/Western blot, we found no difference of the total of SCS abundance between LOF and wild, however, upon 2DE/Western blot, the spots of SCS in LOF were found obvious shift to acidic pI. We monitored the SCS activity in LOF and wild fly, and perceived increased activity of SCS in LOF. We came to a deduction, therefore, that the activity changes of respiratory chain complexes in LOF may be not directly resulted from the complex component abundance but from the modification status of respiratory proteins.