Cardiolipin interacts with beta-2-glycoprotein I and forms an open conformation-Mechanisms analyzed using hydrogen/deuterium exchange.

Beta-2-glycoprotein I (ß2 GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of ß2 GPI can be recognized by the anti-ß2 GPI antibody. Here, we prepared the anionic di-oleoyl-phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the ß2 GPI. The conformational changes of ß2 GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21-27 significantly increased after ß2 GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21-27 from the ring conformation. After ß2 GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1-20, 21-27, 196-205, 273-279 and 297-306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70-86, 153-162, 191-198, 196-205 and 273-279 indicated ß2 GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of ß2 GPI at sequences 21-27 and forms an intermediate conformation after 10 min of interaction. After a complete protein-lipid interaction, high negatively charged CL membrane induced a major conformation transition of ß2 GPI.

Chlorella diet alters mitochondrial cardiolipin contents differentially in organs of Danio rerio analyzed by a lipidomics approach.

The zebrafish (Danio rerio) is an important and widely used vertebrate model organism for the study of human diseases which include disorders caused by dysfunctional mitochondria. Mitochondria play an essential role in both energy metabolism and apoptosis, which are mediated through a mitochondrial phospholipid cardiolipin (CL). In order to examine the cardiolipin profile in the zebrafish model, we developed a CL analysis platform by using liquid chromatography-mass spectrometry (LC-MS). Meanwhile, we tested whether chlorella diet would alter the CL profile in the larval fish, and in various organs of the adult fish. The results showed that chlorella diet increased the chain length of CL in larval fish. In the adult zebrafish, the distribution patterns of CL species were similar between the adult brain and eye tissues, and between the heart and muscles. Interestingly, monolyso-cardiolipin (MLCL) was not detected in brain and eyes but found in other examined tissues, indicating a different remodeling mechanism to maintain the CL integrity. While the adult zebrafish were fed with chlorella for four weeks, the CL distribution showed an increase of the species of saturated acyl chains in the brain and eyes, but a decrease in the other organs. Moreover, chlorella diet led to a decrease of MLCL percentage in organs except the non-MLCL-containing brain and eyes. The CL analysis in the zebrafish provides an important tool for studying the mechanism of mitochondria diseases, and may also be useful for testing medical regimens targeting against the Barth Syndrome.

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.