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.

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.

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.

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.

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.

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.

The subtherapeutic dose of valproic acid induces the activity of cardiolipin-dependent proteins.

A mood-stabilizing anticonvulsant valproic acid (VPA) is a drug with a pleiotropic effect on cells. Here, we describe the impact of VPA on the metabolic function of human HAP1 cells. We show that VPA altered the biosynthetic pathway of cardiolipin (CL) and affected the activities of mitochondrial enzymes such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and NADH dehydrogenase. We demonstrate that a therapeutic dose of VPA (0.6 mM) has a harmful effect on cell growth and increases the production of reactive oxygen species and superoxides. On the contrary, less concentrated VPA (0.06 mM) increased the activities of CL-dependent enzymes leading to an increased level of oxidative phosphorylation and ATP production. The effect of VPA was also tested on the Barth syndrome model, which is characterized by a reduced amount of CL and an increased level of monolyso-CL. In this model, VPA treatment slightly attenuated the mitochondrial defects by altering the activities of CL-dependent enzymes. However, the presence of CL was essential for the increase in ATP production by VPA. Our findings highlight the potential therapeutic role of VPA in normalizing mitochondrial function in BTHS and shed light on the intricate interplay between lipid metabolism and mitochondrial physiology in health and disease. SUMMARY: This study investigates the dose-dependent effect of valproate, a mood-stabilizing drug, on mitochondrial function. The therapeutic concentration reduced overall cellular metabolic activity, while a subtherapeutic concentration notably improved the function of cardiolipin-dependent proteins within mitochondria. These findings shed light on novel aspects of valproate's effect and suggest potential practical applications for its use. By elucidating the differential effects of valproate doses on mitochondrial activity, this research underscores the drug's multifaceted role in cellular metabolism and highlights avenues for further exploration in therapeutic interventions.

Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), specifically Group VIIA PLA(2), is a member of the phospholipase A(2) superfamily and is found mainly associated with LDL and HDL in human plasma. Lp-PLA(2) is considered as a risk factor, a potential biomarker, a target for therapy in the treatment of cardiovascular disease, and evidence suggests that the level of Lp-PLA(2) in plasma is associated with the risk of future cardiovascular and stroke events. The differential location of the enzyme in LDL/HDL lipoproteins has been suggested to affect Lp-PLA(2) function and/or its physiological role and an abnormal distribution of the enzyme may correlate with diseases. Although a mutagenesis study suggested that a surface helix (residues 362-369) mediates the association between Lp-PLA(2) and HDL, the molecular details and mechanism of association has remained unknown. We have now employed hydrogen deuterium exchange mass spectrometry to characterize the interaction between recombinant human Lp-PLA(2) and human HDL. We have found that specific residues 113-120, 192-204, and 360-368 likely mediate HDL binding. In a previous study, we showed that residues 113-120 are important for Lp-PLA(2)-liposome interactions. We now find that residues 192-204 show a decreased deuteration level when Lp-PLA(2) is exposed to apoA-I, but not apoA-II, the most abundant apoproteins in HDL, and additionally, residues 360-368 are only affected by HDL.The results suggest that apoA-I and phospholipid membranes play crucial roles in Lp-PLA(2) localization to HDL.

Essential role of ELOVL4 protein in very long chain fatty acid synthesis and retinal function.

Very long chain polyunsaturated fatty acid (VLC-PUFA)-containing glycerophospholipids are highly enriched in the retina; however, details regarding the specific synthesis and function of these highly unusual retinal glycerophospholipids are lacking. Elongation of very long chain fatty acids-4 (ELOVL4) has been identified as a fatty acid elongase protein involved in the synthesis of VLC-PUFAs. Mutations in ELOVL4 have also been implicated in an autosomal dominant form of Stargardt disease (STGD3), a type of juvenile macular degeneration. We have generated photoreceptor-specific conditional knock-out mice and used high performance liquid chromatography-mass spectrometry (HPLC-MS) to examine and analyze the fatty acid composition of retinal membrane glycerophosphatidylcholine and glycerophosphatidylethanolamine species. We also used immunofluorescent staining and histology coupled with electrophysiological data to assess retinal morphology and visual response. The conditional knock-out mice showed a significant decrease in retinal glycerophospholipids containing VLC-PUFAs, specifically contained in the sn-1 position of glycerophosphatidylcholine, implicating the role of Elovl4 in their synthesis. Conditional knock-out mice were also found to have abnormal accumulation of lipid droplets and lipofuscin-like granules while demonstrating photoreceptor-specific abnormalities in visual response, indicating the critical role of Elovl4 for proper rod or cone photoreceptor function. Altogether, this study demonstrates the essential role of ELOVL4 in VLC-PUFA synthesis and retinal function.

Fluoroketone inhibition of Ca(2+)-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics.

The mechanism of inhibition of group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA(2) were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA(2) should greatly improve our ability to design new inhibitors with higher potency and selectivity.

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.

Lipoprotein-associated phospholipase A(2) interacts with phospholipid vesicles via a surface-disposed hydrophobic α-helix.

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis, and sepsis. Lp-PLA(2) also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA(2) plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically determined structure of the enzyme is known, the enzyme's mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA(2) with dimyristoylphosphatidylcholine (DMPC) vesicles and found that specific residues 113-120 in one of the enzyme's surface-disposed hydrophobic α-helices likely mediate liposome binding.

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.

2-Oxoamide inhibitors of phospholipase A2 activity and cellular arachidonate release based on dipeptides and pseudodipeptides.

A series of 2-oxoamides based on dipeptides and pseudodipeptides were synthesized and their activities towards two human intracellular phospholipases A(2) (GIVA cPLA(2) and GVIA iPLA(2)) and one human secretory phospholipase A(2) (GV sPLA(2)) were evaluated. Derivatives containing a free carboxyl group are selective GIVA cPLA(2) inhibitors. A derivative based on the ethyl ester of an ether pseudodipeptide is the first 2-oxoamide, which preferentially inhibits GVIA iPLA(2). The effect of 2-oxoamides on the generation of arachidonic acid from RAW 264.7 macrophages was also studied and it was found that selective GIVA cPLA(2) inhibitors preferentially inhibited cellular arachidonic acid release; one pseudodipeptide gave an IC(50) value of 2muM.