Double bonds of unsaturated fatty acids differentially regulate mitochondrial cardiolipin remodeling.

BACKGROUND: Supplemented fatty acids can incorporate into cardiolipin (CL) and affect its remodeling. The change in CL species may alter the mitochondrial membrane composition, potentially disturbing the mitochondrial structure and function during inflammation. METHOD: To investigate the effect of the unsaturation of fatty acids on CL, we supplemented macrophage-like RAW264.7 cells with 18-carbon unsaturated fatty acids including oleic acid (OA, 18:1), linoleic acid (LA, 18:2), α-linolenic acid (ALA, 18:3), γ-linolenic acid (GLA, 18:3), and stearidonic acid (SDA, 18:4). Mitochondrial changes in CL were measured through mass spectrometry. RESULT: Our data indicated that OA(18:1) was the most efficient fatty acid that incorporated into CL, forming symmetrical CL without fatty acid elongation and desaturation. In addition, LA(18:2) and ALA(18:3) were further elongated before incorporation, significantly increasing the number of double bonds and the chain length of CL. GLA and SDA were not optimal substrates for remodeling enzymes. The findings of RT-qPCR experiments revealed that none of these changes in CL occurred through the regulation of CL remodeling- or synthesis-related genes. The fatty acid desaturase and transportation genes-Fads2 and Cpt1a, respectively-were differentially regulated by the supplementation of five unsaturated 18-carbon fatty acids. CONCLUSIONS: The process of fatty acid incorporation to CL was regulated by the fatty acid desaturation and transportation into mitochondria in macrophage. The double bonds of fatty acids significantly affect the incorporation process and preference. Intact OA(18:1) was incorporated to CL; LA(18:2) and ALA(18:3) were desaturated and elongated to long chain fatty acid before the incorporation; GLA(18:3) and SDA(18:4) were unfavorable for the CL incorporation.

Omega-3 and omega-6 fatty acid differentially impact cardiolipin remodeling in activated macrophage.

BACKGROUND: The macrophage plays an important role in innate immunity to induce immune responses. Lipid replacement therapy has been shown to change the lipid compositions of mitochondria and potentially becomes an alternative to reduce the inflammatory response. METHODS: We examined the effects of omega-6 arachidonic acid (AA), omega-3 eicosapentaenoic acid (EPA), and omega-3 docosahexaenoic acid (DHA) supplementation on the activated the macrophage cell line RAW264.7 via KdO2-lipid A (KLA). The mitochondrial cardiolipin (CL) and monolysocardiolipin (MLCL) were analyzed by LC-MS. RESULTS: After macrophage activation by KLA, CL shifted to saturated species, but did not affect the quantity of CL. Inhibition of delta 6 desaturase also resulted in the same trend of CL species shift. We further examined the changes in CL and MLCL species induced by polyunsaturated fatty acid supplementation during inflammation. After supplementation of AA, EPA and DHA, the MLCL/CL ratio increased significantly in all treatments. The percentages of the long-chain species highly elevated and those of short-chain species reduced in both CL and MLCL. CONCLUSIONS: Comparisons of AA, EPA and DHA supplementation revealed that the 20-carbon EPA (20:5) and AA (20:4) triggered higher incorporation and CL remodeling efficiency than 22-carbon DHA (22:6). EPA supplementation not only efficiently extended the chain length of CL but also increased the unsaturation of CL.

Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry.

Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization.

Elucidation of tRNA-cytochrome c interactions through hydrogen/deuterium exchange mass spectrometry.

Cytochrome c (cyt c) is a mitochondrial protein responsible for transferring electrons between electron transport chain complexes III and IV. The release of cyt c from the mitochondria has been considered as a commitment step in intrinsic apoptosis. Transfer RNA (tRNA) has recently been found to interact with the released cyt c to prevent the formation of the apoptosome complex, thus preventing cell apoptosis. To understand the molecular basis of tRNA-cyt c interactions, we applied hydrogen/deuterium exchange mass spectrometry (HDXMS) to analyze the interactions between tRNA and cyt c. tRNAPhe binding to cyt c reduced the deuteration level of cyt c in all analyzed regions, indicating that tRNA binding blocks the solvent-accessible regions and results in the formation of a more compact conformation. Substitution of the tRNAPhe with the total tRNA from brewer's yeast in the HDXMS experiment significantly reduced deuteration in the N-terminus and the region 18-32 residue of cyt c, where all tRNAs are bound. To clarify the cause of binding, we used synthesized single-stranded oligonucleotides of 12-mer dA and dT to form complexes with cyt c. The exchange of the nucleotide bases between adenine and thymine did not affect the deuteration level of cyt c. However, the regions 1-10 and 65-82 showed minor decreases after unstructured dA or dT DNA binding. Collectively, these results reveal that cyt c maintains its globular structure to interact with tRNA. The region 18-32 selectively interacts with tRNA, and N-terminal 1-10 interacts with oligonucleotides electrostatically.

Phosphatidylglycerol Supplementation Alters Mitochondrial Morphology and Cardiolipin Composition.

The pathogenic variant of the TAZ gene is directly associated with Barth syndrome. Because tafazzin in the mitochondria is responsible for cardiolipin (CL) remodeling, all molecules related to the metabolism of CL can affect or be affected by TAZ mutation. In this study, we intend to recover the distortion of the mitochondrial lipid composition, especially CL, for Barth syndrome treatment. The genetically edited TAZ knockout HAP1 cells were demonstrated to be a suitable cellular model, where CL desaturation occurred and monolyso-CL (MLCL) was accumulated. From the species analysis by mass spectrometry, phosphatidylethanolamine showed changed species content after TAZ knockout. TAZ knockout also caused genetic down-regulation of PGS gene and up-regulation of PNPLA8 gene, which may decrease the biosynthesis of CLs and increase the hydrolysis product MLCL. Supplemented phosphatidylglycerol(18:1)2 (PG(18:1)2) was successfully biosynthesized to mature symmetrical CL and drastically decrease the concentration of MLCL to recover the morphology of mitochondria and the cristae shape of inner mitochondria. Newly synthesized mature CL may induce the down-regulation of PLA2G6 and PNPLA8 genes to potentially decrease MLCL production. The excess supplemented PG was further metabolized into phosphatidylcholine and phosphatidylethanolamine.

Phosphatidylglycerol Incorporates into Cardiolipin to Improve Mitochondrial Activity and Inhibits Inflammation.

Chronic inflammation and concomitant oxidative stress can induce mitochondrial dysfunction due to cardiolipin (CL) abnormalities in the mitochondrial inner membrane. To examine the responses of mitochondria to inflammation, macrophage-like RAW264.7 cells were activated by Kdo2-Lipid A (KLA) in our inflammation model, and then the mitochondrial CL profile, mitochondrial activity, and the mRNA expression of CL metabolism-related genes were examined. The results demonstrated that KLA activation caused CL desaturation and the partial loss of mitochondrial activity. KLA activation also induced the gene upregulation of cyclooxygenase (COX)-2 and phospholipid scramblase 3, and the gene downregulation of COX-1, lipoxygenase 5, and Δ-6 desaturase. We further examined the phophatidylglycerol (PG) inhibition effects on inflammation. PG supplementation resulted in a 358-fold inhibition of COX-2 mRNA expression. PG(18:1)2 and PG(18:2)2 were incorporated into CLs to considerably alter the CL profile. The decreased CL and increased monolysocardiolipin (MLCL) quantity resulted in a reduced CL/MLCL ratio. KLA-activated macrophages responded differentially to PG(18:1)2 and PG(18:2)2 supplementation. Specifically, PG(18:1)2 induced less changes in the CL/MLCL ratio than did PG(18:2)2, which resulted in a 50% reduction in the CL/MLCL ratio. However, both PG types rescued 20-30% of the mitochondrial activity that had been affected by KLA activation.

Chemotherapy Drug Induced Discoordination of Mitochondrial Life Cycle Detected by Cardiolipin Fluctuation.

Chemotherapy drugs have been prescribed for the systemic treatment of cancer. We selected three chemotherapy drugs, including methotrexate, mitomycine C and vincristine to inhibit the proliferation of HT1080 human fibrosarcoma cells in S, G2 and M phases of the cell cycle respectively. These chemotherapy drugs showed significant toxicity and growth inhibition to the cancer cells measured by MTT assay. After treated with a 50% inhibitory dosage for 48 hours, these cancer cells showed significant accumulation of cardiolipin (CL), which was a reverse trend of the nutritional deficiency induced arrest at G1 phase. The quantity of each CL species was further semi-quantitated by HPLC-ion trap mass spectrometer. Methotraxate treatment caused unique increases of acyl chain length on CL, which were the opposite of the serum starvation, mitomycine C and vincristine treatments. Although mitomycine C and vincristine have different mechanisms to induce cell cycle arrest, these two drugs displayed similar effects on decreasing chain length of CL. Continuation of CL synthesis during cell cycle arrest indicated the chemotherapy drugs resulting in the discoordination of the mitochondrial life cycle from the cell cycle and thus caused the accumulation of CL. These finding reveals that the pre-remodeling nascent CL accumulates during the methotraxate induced arrest; however, the post-remodeling mature CL accumulates during the mitomycine C and vincristine induced arrest after the synthesis phase.