The S-nitrosylation status of PCNA localized in cytosol impacts the apoptotic pathway in a Parkinson's disease paradigm.

It is generally accepted that nitric oxide (NO) or its derivatives, reactive nitrogen species (RNS), are involved in the development of Parkinson's disease (PD). Recently, emerging evidence in the study of PD has indicated that protein S-nitrosylation triggers the signaling changes in neurons. In this study, SH-SY5Y cells treated with rotenone were used as a model of neuronal death in PD. The treated cells underwent significant apoptosis, which was accompanied by an increase in intracellular NO in a rotenone dose-dependent manner. The CyDye switch approach was employed to screen for changes in S-nitrosylated (SNO) proteins in response to the rotenone treatment. Seven proteins with increased S-nitrosylation were identified in the treated SH-SY5Y cells, which included proliferating cell nuclear antigen (PCNA). Although PCNA is generally located in the nucleus and participates in DNA replication and repair, significant PCNA was identified in the SH-SY5Y cytosol. Using immunoprecipitation and pull-down approaches, PCNA was found to interact with caspase-9; using mass spectrometry, the two cysteine residues PCNA-Cys81 and -Cys162 were identified as candidate S-nitrosylated residues. In addition, the evidence obtained from in vitro and the cell model studies indicated that the S-nitrosylation of PCNA-Cys81 affected the interaction between PCNA and caspase-9. Furthermore, the interaction of PCNA and caspase-9 partially blocked caspase-9 activation, indicating that the S-nitrosylation of cytosolic PCNA may be a mediator of the apoptotic pathway.

Quantitative evaluation of the mitochondrial proteomes of Drosophila melanogaster adapted to extreme oxygen conditions.

Mitochondria are the primary organelles that consume oxygen and provide energy for cellular activities. To investigate the mitochondrial mechanisms underlying adaptation to extreme oxygen conditions, we generated Drosophila strains that could survive in low- or high-oxygen environments (LOF or HOF, respectively), examined their mitochondria at the ultrastructural level via transmission electron microscopy, studied the activity of their respiratory chain complexes, and quantitatively analyzed the protein abundance responses of the mitochondrial proteomes using Isobaric tag for relative and absolute quantitation (iTRAQ). A total of 718 proteins were identified with high confidence, and 55 and 75 mitochondrial proteins displayed significant differences in abundance in LOF and HOF, respectively, compared with the control flies. Importantly, these differentially expressed mitochondrial proteins are primarily involved in respiration, calcium regulation, the oxidative response, and mitochondrial protein translation. A correlation analysis of the changes in the levels of the mRNAs corresponding to differentially regulated mitochondrial proteins revealed two sets of proteins with different modes of regulation (transcriptional vs. post-transcriptional) in both LOF and HOF. We believe that these findings will not only enhance our understanding of the mechanisms underlying adaptation to extreme oxygen conditions in Drosophila but also provide a clue in studying human disease induced by altered oxygen tension in tissues and cells.

Ultrastructural modifications in the mitochondria of hypoxia-adapted Drosophila melanogaster.

Chronic hypoxia (CH) occurs under certain physiological or pathological conditions, including in people who reside at high altitude or suffer chronic cardiovascular or pulmonary diseases. As mitochondria are the predominant oxygen-consuming organelles to generate ATP through oxidative phosphorylation in cells, their responses, through structural or molecular modifications, to limited oxygen supply play an important role in the overall functional adaptation to hypoxia. Here, we report the adaptive mitochondrial ultrastructural modifications and the functional impacts in a recently generated hypoxia-adapted Drosophila melanogaster strain that survives severe, otherwise lethal, hypoxic conditions. Using electron tomography, we discovered increased mitochondrial volume density and cristae abundance, yet also cristae fragmentation and a unique honeycomb-like structure in the mitochondria of hypoxia-adapted flies. The homeostatic levels of adenylate and energy charge were similar between hypoxia-adapted and naïve control flies and the hypoxia-adapted flies remained active under severe hypoxia as quantified by negative geotaxis behavior. The equilibrium ATP level was lower in hypoxia-adapted flies than those of the naïve controls tested under severe hypoxia that inhibited the motion of control flies. Our results suggest that the structural rearrangement in the mitochondria of hypoxia-adapted flies may be an important adaptive mechanism that plays a critical role in preserving adenylate homeostasis and metabolism as well as muscle function under chronic hypoxic conditions.

Proteomic analysis of glutathione S-transferase isoforms in mouse liver mitochondria.

AIM: To survey glutathione (GSH) S-transferase (GST) isoforms in mitochondria and to reveal the isoforms' biological significance in diabetic mice. METHODS: The presence of GSTs in mouse liver mitochondria was systematically screened by two proteomic approaches, namely, GSH affinity chromatography/two dimensional electrophoresis (2DE/MALDI TOF/TOF MS) and SDS-PAGE/LC ESI MS/MS. The proteomic results were further confirmed by Western blotting using monoclonal antibodies against GSTs. To evaluate the liver mitochondrial GSTs quantitatively, calibration curves were generated by the loading amounts of individual recombinant GST protein vs the relative intensities elicited from the Western blotting. An extensive comparison of the liver mitochondrial GSTs was conducted between normal and db/db diabetic mice. Student's t test was adopted for the estimation of regression and significant difference. RESULTS: Using GSH affinity/2DE/MALDI TOF/TOF MS, three GSTs, namely, alpha3, mu1 and pi1, were identified; whereas five GSTs, alpha3, mu1, pi1, kappa1 and zeta1, were detected in mouse liver mitochondria using SDS-PAGE/LC ESI MS/MS, of these GSTs, GST kappa1 was reported as a specific mitochondrial GST. The R² values of regression ranged between values of about 0.86 and 0.98, which were acceptable for the quantification. Based on the measurement of the GST abundances in liver mitochondria of normal and diabetic mice, the four GSTs, alpha3, kappa1, mu1 and zeta1, were found to be almost comparable between the two sets of animals, whereas, lower GST pi1 was detected in the diabetic mice compared with normal ones, the signal of Western blotting in control and db/db diabetic mice liver mitochondria is 134.61 ± 53.84 vs 99.74 ± 46.2, with P < 0.05. CONCLUSION: Our results indicate that GSTs exist widely in mitochondria and its abundances of mitochondrial GSTs might be tissue-dependent and disease-related.

A micropreparation of mitochondria from cells using magnetic beads with immunoaffinity.

Mitochondrial preparation is a key technique in the study of mitochondria. Growing evidence has demonstrated that mitochondrial proteins are tissue or cell type dependent. Locating the proteins in the global presence of mitochondrial membranes is a primary consideration in adopting antibodies for affinity enrichment of mitochondria on a micro scale. Two proteins located on the outer membrane of mitochondria, cytochrome b5 type B (CYB5B) and synaptojanin-2-binding protein (SYNJ2BP), were selected as candidates based on a survey of databases and the literature. The polyclonal antibodies against the truncated CYB5B and SYNJ2BP exhibited specific recognition to mitochondria and wider sensitivity to several tested mouse tissues and cell lines, whereas the antibody 22-kDa translocase of the outer mitochondrial membrane (TOM22) nearly missed detection of mitochondria in the liver and responded minimally to mitochondria from H9C2 and L-02 cells. Through the affinity enrichment for cellular mitochondria using magnetic beads coated with anti-CYB5B or anti-SYNJ2BP, we found that the anti-CYB5B beads could enrich mitochondria more efficiently even on a scale of 10,000 cultured cells. For the integrity and protein components, the enriched mitochondria on anti-CYB5B were carefully examined and were accepted in further functional study. We propose that an anti-CYB5B immunomagnetic approach is feasible in the micropreparation of mitochondria from cultured cells.

Monitoring succinyl-CoA:3-oxoacid CoA transferase nitration in mitochondria using monoclonal antibodies.

Two tyrosine residues (Tyr(4) and Tyr(76)) of succinyl-CoA:3-oxoacid CoA transferase (SCOT) are sensitive to nitric oxide (NO) stress, as assessed by mass spectrometry and site-direct mutagenesis. However, monitoring the SCOT nitration in tissue or cells is challenging. Herein, we describe the development of an assay to detect nitrated SCOT directly using site-specific antibodies; the monoclonal antibodies were generated and screened against nitrated peptides of SCOT. After stringent filtration, two antibodies, anti-SCOT4N and anti-SCOT76N, which specifically recognise Tyr(4) or Tyr(76) of SCOT, respectively, were successfully selected. In a cell model over-expressing iNOS in the mitochondria, nitrated SCOT was significantly increased compared with control cells. In addition, in a mouse model of diabetes, nitrated Tyr(4) and Tyr(76) in the heart and kidney were higher compared to the control animals. Our results using monoclonal antibodies against nitrated SCOT peptides are in good agreement with the proteomic data.

Proteomic survey towards the tissue-specific proteins of mouse mitochondria.

Mitochondrion plays the key functions in mammalian cells. It is believed that mitochondrion exerts the common biologic functions in many tissues, but also performs some specific functions correspondent with tissues where it is localized. To identify the tissue-specific mitochondrial proteins, we carried out a systematic survey towards mitochondrial proteins in the tissues of C57BL/6J mouse, such as liver, kidney and heart. The mitochondrial proteins were separated by 2DE and identified by MALDI-TOF/TOF MS. Total of 87 unique proteins were identified as the tissue-specific ones, and some representatives were further verified through ICPL quantification and Western blot. Because these issue-specific proteins are coded from nuclear genes, real-time PCR was employed to examine the mRNA status of six typical genes found in the tissues.With combining of the expression data and the co-localization images obtained from confocal microscope, we came to the conclusion that the tissue-specifically mitochondrial proteins were widely distributed among the mouse tissues. Our investigation, therefore, indeed provides a solid base to further explore the biological significance of the mitochondrial proteins with tissue-orientation.