Exploration of the Catalytic Cycle Dynamics of Vigna Radiata H+-Translocating Pyrophosphatases Through Hydrogen-Deuterium Exchange Mass Spectrometry.

Vigna radiata H+-translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.

Nutrient Supplementation Improves Contact Lens-Induced Corneal Cell Damage Based on a SIRC Cellular Model.

OBJECTIVES: The long-term use of contact lenses may damage the structure of the ocular surface and cause metabolic disorders in corneal cells. Vitamins and amino acids help maintain the physiological function of the eye. In the present study, the effects of nutrient (vitamin and amino acid) supplementation on corneal cell repair after contact lens-induced damage was investigated. METHODS: High-performance liquid chromatography was used to quantify the nutrient contents of minimum essential medium, and the MTT assay was used to measure the viability of corneal cells. A Statens Seruminstitut rabbit cornea cellular model was established to simulate contact lens-induced keratopathy and investigate the effects of vitamin and amino acid supplementations on corneal cell repair. RESULTS: The high water content lens group (78%) has a cell viability as high as 83.3%, whereas the cell viability of the low water content lens group (38%) is only 51.6%. The 32.0% difference between the two groups confirms the correlation between water content of lens and corneal viability. CONCLUSIONS: Vitamin B2, vitamin B12, asparagine, and taurine supplementation may help improve contact lens-induced damage.

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.

Simple Advanced Preparation Method for Improving the Thickness Stability of Powder Thickening Agents in Dysphagia Management.

Texture modification of foods by using thickening agents is a routine practice for assessing and treating dysphagic patients. However, a powder-thickened fluid's viscosity might change over time, and little has been proposed to overcome this inconsistency. This study aimed to evaluate variations in the thickness of a fluid thickened with a common xanthan gum-based powder and to explore the feasibility of a simple advanced preparation method for thickened liquids to improve thickness stability. Thickened fluids with concentrations of 1.0 g/100 mL, 0.7 g/100 mL, and 0.5 g/100 mL were prepared from both freshly opened and previously opened thickening powders. Fluid thickness was measured every 10 min in a series of International Dysphagia Diet Standardization Initiative flow tests. A significant time-dependent decline in thickness was observed for all three concentrations in both groups, namely those prepared with freshly opened and previously opened thickening powders, and the shortest periods to achieve a stable viscosity after liquid preparation for the two groups were 80 and 70 min, respectively. On diluting the thickened liquids from the base liquid, which was prepared at a concentration of 1.0 g/100 mL and stored at room temperature for 90 min, no significant time-dependent thickness changes were observed over the following 60 min. The simple protocol of preparing the thickest "base" liquid in advance and then diluting it to the desired thickness resulted in a consistent liquid thickness, with the prepared liquids ready to be clinically applied and consumed, with high stability within 60 min.

Applications of Hyaluronic Acid in Ophthalmology and Contact Lenses.

Hyaluronic acid (HA) is a glycosaminoglycan that was first isolated and identified from the vitreous body of a bull's eye. HA is ubiquitous in the soft connective tissues of animals and therefore has high tissue compatibility for use in medication. Because of HA's biological safety and water retention properties, it has many ophthalmology-related applications, such as in intravitreal injection, dry eye treatment, and contact lenses. Due to its broad range of applications, the identification and quantification of HA is a critical topic. This review article discusses current methods for analyzing HA. Contact lenses have become a widely used medical device, with HA commonly used as an additive to their production material, surface coating, and multipurpose solution. HA molecules on contact lenses retain moisture and increase the wearer's comfort. HA absorbed by contact lenses can also gradually release to the anterior segment of the eyes to treat dry eye. This review discusses applications of HA in ophthalmology.

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.

Unraveling cardiolipin-induced conformational change of cytochrome c through H/D exchange mass spectrometry and quartz crystal microbalance.

Cardiolipin (CL), a crucial component in inner mitochondrial membranes, interacts with cytochrome c (cyt c) to form a peroxidase complex for the catalysis of CL oxidation. Such interaction is pivotal to the mitochondrial regulation of apoptosis and is affected by the redox state of cyt c. In the present study, the redox-dependent interaction of cyt c with CL was investigated through amide hydrogen/deuterium exchange coupled with mass spectrometry (HDXMS) and quartz crystal microbalance with dissipation monitoring (QCM-D). Ferrous cyt c exhibited a more compact conformation compared with its ferric form, which was supported by the lower number of deuterons accumulated and the greater amplitude reduction on dissipation. Upon association with CL, ferrous cyt c resulted in a moderate increase in deuteration, whereas the ferric form caused a drastic increase of deuteration, which indicated that CL-bound ferric cyt c formed an extended conformation. These results were consistent with those of the frequency (f) - dissipation (D) experiments, which revealed that ferric cyt c yielded greater values of |ΔD/Δf| within the first minute. Further fragmentation analysis based on HDXMS indicated that the effect of CL binding was considerably different on ferric and ferrous cyt c in the C-helix and the Loop 9-24. In ferric cyt c, CL binding affected Met80 and destabilized His18 interaction with heme, which was not observed with ferrous cyt c. An interaction model was proposed to explain the aforementioned results.

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.

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.

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.

Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/cytochrome P450 reductase interaction with anti-tumor activity.

BACKGROUND: Heme oxygenase (HO) catalyzes NADPH-dependent degradation of heme to liberate iron, carbon monoxide and biliverdin. The interaction between HO and cytochrome P450 reductase (CPR), an electron donor, is essential for HO activity. HO-1 is a stress-inducible isoform whereas HO-2 is constitutively expressed. HO-1 induction is commonly seen in cancers and impacts disease progression, supporting the possibility of targeting HO-1 for cancer therapy. METHODS: We employed a cell-based bioluminescence resonance energy transfer assay to screen compounds with ability to inhibit HO-1/CPR interaction. The effect of the identified compound on HO-1/CPR interaction was confirmed by pull down assay. Moreover, the anti-tumorigenic activity of the identified compound on HO-1-enhanced tumor growth and migration was assessed by trypan blue exclusion method and wound healing assay. RESULTS: Danthron was identified as an effective small molecule able to interfere with the interaction between HO-1 and CPR but not HO-2 and CPR. Additional experiments with structural analogues of danthron revealed that the positions of hydroxyl moieties significantly affected the potency of inhibition on HO-1/CPR interaction. Pull-down assay confirmed that danthron inhibited the interaction of CPR with HO-1 but not HO-2. Danthron suppressed growth and migration of HeLa cells with stable HO-1 overexpression but not mock cells. In contrast, anthrarufin, a structural analog with no ability to interfere HO-1/CPR interaction, exhibited no significant effect on HO-1-overexpressing HeLa cells. CONCLUSIONS: These findings demonstrate that danthron is an isoform-specific inhibitor for HO-1/CPR interaction and may serve as a lead compound for novel anticancer drug.

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.

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.

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.

Polyunsaturated fatty acids incorporation into cardiolipin in H9c2 cardiac myoblast.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), known as ω-3 polyunsaturated fatty acid (PUFA), are common nutrients in daily food intake and have been shown to prevent cardiovascular disease and improve cardiac functions. Cardiolipin is a mitochondrial phospholipid necessary for maintaining physiological function of mitochondria. Several studies have indicated that the cardiolipin acyl chain compositions affect the function of cardiolipin and mitochondria. Here, we investigated the structural changes of cardiolipin after DHA and EPA supplementation and compared them to arachidonic acid (AA) treatment. H9c2 cardiac myoblast was used as a cell model, and cardiolipin species was monitored and identified via LC-MS and MS/MS. Our results showed distinct mass envelopes of cardiolipin with the same carbon number but different double bonds in mass spectrum. There were 116 cardiolipin species with 36 distinct mass in 6 mass envelopes identified by MS/MS. Three days of PUFA treatment resulted in decreases of low-molecular-weight cardiolipin and increases of high-molecular-weight cardiolipin, suggesting the incorporation of exogenous DHA, EPA and AA into mitochondrial cardiolipin. PUFA incorporation was further verified by MS/MS analysis. More importantly, we found that DHA supplementation elevated the percent content of less unsaturated cardiolipin species and highly unsaturated cardiolipin species, containing ω-3 fatty acyl chains, indicating a ω-3 fatty acid incorporation mechanism with peroxidation protection. Our results indicate that PUFA supplementation differentially perturbed the fatty acyl chain compositions in the mitochondrial cardiolipin in the H9c2 cardiac myoblast, suggesting that mitochondrial membrane and the function of mitochondria are susceptible to exogenous lipid species.

Cell cycle arrest and cell survival induce reverse trends of cardiolipin remodeling.

Cell survival from the arrested state can be a cause of the cancer recurrence. Transition from the arrest state to the growth state is highly regulated by mitochondrial activity, which is related to the lipid compositions of the mitochondrial membrane. Cardiolipin is a critical phospholipid for the mitochondrial integrity and functions. We examined the changes of cardiolipin species by LC-MS in the transition between cell cycle arrest and cell reviving in HT1080 fibrosarcoma cells. We have identified 41 cardiolipin species by MS/MS and semi-quantitated them to analyze the detailed changes of cardiolipin species. The mass spectra of cardiolipin with the same carbon number form an envelope, and the C64, C66, C68, C70 C72 and C74 envelopes in HT1080 cells show a normal distribution in the full scan mass spectrum. The cardiolipin quantity in a cell decreases while entering the cell cycle arrest, but maintains at a similar level through cell survival. While cells awakening from the arrested state and preparing itself for replication, the groups with short acyl chains, such as C64, C66 and C68 show a decrease of cardiolipin percentage, but the groups with long acyl chains, such as C70 and C72 display an increase of cardiolipin percentage. Interestingly, the trends of the cardiolipin species changes during the arresting state are completely opposite to cell growing state. Our results indicate that the cardiolipin species shift from the short chain to long chain cardiolipin during the transition from cell cycle arrest to cell progression.

Biomolecular logic gate for analysis of the New Delhi metallo-ß-lactamase (NDM)-coding gene with concurrent determination of its drug resistance-encoding fragments.

We herein exploit a newly schemed logic gate to superiorly facilitate analysis of long and highly structured nucleic acids. This strategy uniquely enables the identification of NDM-specific genes and concurrent screening of two active site-encoded fragments, which is promising for evaluating microbial drug resistance.

Insertion of the Ca²âº-independent phospholipase A2 into a phospholipid bilayer via coarse-grained and atomistic molecular dynamics simulations.

Group VI Ca²âº-independent phospholipase A2 (iPLA2) is a water-soluble enzyme that is active when associated with phospholipid membranes. Despite its clear pharmaceutical relevance, no X-ray or NMR structural information is currently available for the iPLA2 or its membrane complex. In this paper, we combine homology modeling with coarse-grained (CG) and all-atom (AA) molecular dynamics (MD) simulations to build structural models of iPLA2 in association with a phospholipid bilayer. CG-MD simulations of the membrane insertion process were employed to provide a starting point for an atomistic description. Six AA-MD simulations were then conducted for 60 ns, starting from different initial CG structures, to refine the membrane complex. The resulting structures are shown to be consistent with each other and with deuterium exchange mass spectrometry (DXMS) experiments, suggesting that our approach is suitable for the modeling of iPLA2 at the membrane surface. The models show that an anchoring region (residues 710-724) forms an amphipathic helix that is stabilized by the membrane. In future studies, the proposed iPLA2 models should provide a structural basis for understanding the mechanisms of lipid extraction and drug-inhibition. In addition, the dual-resolution approach discussed here should provide the means for the future exploration of the impact of lipid diversity and sequence mutations on the activity of iPLA2 and related enzymes.

Assessing phospholipase A2 activity toward cardiolipin by mass spectrometry.

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and iPLA2, but not for Lp-PLA2. Our results also show that none of these PLA2s have significant PLA1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA2, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA2 assays. The sPLA2s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA2 and cPLA2, and activates sPLA2 at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA2 activity and the ability to assay the various forms of PLA2 is important in understanding its function.