Comparing the oxidative functions of neutrophil myeloperoxidase and cytochrome P450 enzymes in drug metabolism.

Drug metabolism is an essential process that chemically alters xenobiotic substrates to activate or terminate drug activity. Myeloperoxidase (MPO) is a neutrophil-derived haem-containing enzyme that is involved in killing invading pathogens, although consequentially, this same oxidative activity can produce metabolites that damage host tissue and play a role in various human pathologies. Cytochrome P450s (CYPs) are a superfamily of haem-containing enzymes that are significantly involved in the metabolism of drugs by functioning as monooxygenases and can be induced or inhibited, resulting in significant drug-drug interactions that lead to unanticipated adverse drug reactions. In this review, the functions of drug metabolism of MPO and CYPs are explored, along with their involvement and association for common enzymatic pathways by certain xenobiotics. MPO and CYPs metabolize numerous xenobiotics, although few reported studies have made a direct comparison between both enzymes. Additionally, we employed molecular docking to compare the active site and haem prosthetic group of MPO and CYPs, supporting their similar catalytic activities. Furthermore, we performed LCMS analysis and observed a shared hydroxylated mefenamic acid metabolite produced in both enzymatic systems. A proper understanding of the enzymology and mechanisms of action of MPO and CYPs is of significant importance when enhancing the beneficial functions of drugs in health and diminishing their damaging effects on diseases. Therefore, awareness of drugs and xenobiotic substrates involved in MPO and CYPs metabolism pathways will add to the knowledge base to foresee and prevent potential drug interactions and adverse events.

Tom40 in cholesterol transport.

Cholesterol initiates steroid metabolism in adrenal and gonadal mitochondria, which is essential for all mammalian survival. During stress an increased cholesterol transport rapidly increases steroidogenesis; however, the mechanism of mitochondrial cholesterol transport is unknown. Using rat testicular tissue and mouse Leydig (MA-10) cells, we report for the first time that mitochondrial translocase of outer mitochondrial membrane (OMM), Tom40, is central in cholesterol transport. Cytoplasmic cholesterol-lipids complex containing StAR protein move from the mitochondria-associated ER membrane (MAM) to the OMM, increasing cholesterol load. Tom40 interacts with StAR at the OMM increasing cholesterol transport into mitochondria. An absence of Tom40 disassembles complex formation and inhibits mitochondrial cholesterol transport and steroidogenesis. Therefore, Tom40 is essential for rapid mitochondrial cholesterol transport to initiate, maintain, and regulate activity.

Research into the Bioengineering of a Novel α-Conotoxin from the Milked Venom of Conus obscurus.

The marine cone snail produces one of the fastest prey strikes in the animal kingdom. It injects highly efficacious venom, often causing prey paralysis and death within seconds. Each snail has hundreds of conotoxins, which serve as a source for discovering and utilizing novel analgesic peptide therapeutics. In this study, we discovered, isolated, and synthesized a novel α3/5-conotoxins derived from the milked venom of Conus obscurus (α-conotoxin OI) and identified the presence of α-conotoxin SI-like sequence previously found in the venom of Conus striatus. Five synthetic analogs of the native α-conotoxin OI were generated. These analogs incorporated single residue or double residue mutations. Three synthetic post-translational modifications (PTMs) were synthetically incorporated into these analogs: N-terminal truncation, proline hydroxylation, and tryptophan bromination. The native α-conotoxin OI demonstrated nanomolar potency in Poecilia reticulata and Homosapiens muscle-type nicotinic acetylcholine receptor (nAChR) isoforms. Moreover, the synthetic α-[P9K] conotoxin OI displayed enhanced potency in both bioassays, ranging from a 2.85 (LD50) to 18.4 (IC50) fold increase in comparative bioactivity. The successful incorporation of PTMs, with retention of both potency and nAChR isoform selectivity, ultimately pushes new boundaries of peptide bioengineering and the generation of novel α-conotoxin-like sequences.

Purification and characterization of antifungal lipopeptide produced by Bacillus velezensis isolated from raw honey.

Raw honey contains a diverse microbiota originating from honeybees, plants, and soil. Some gram-positive bacteria isolated from raw honey are known for their ability to produce secondary metabolites that have the potential to be exploited as antimicrobial agents. Currently, there is a high demand for natural, broad-spectrum, and eco-friendly bio-fungicides in the food industry. Naturally occurring antifungal products from food-isolated bacteria are ideal candidates for agricultural applications. To obtain novel antifungals from natural sources, we isolated bacteria from raw clover and orange blossom honey to evaluate their antifungal-producing potential. Two Bacillus velezensis isolates showed strong antifungal activity against food-isolated fungal strains. Antifungal compound production was optimized by adjusting the growth conditions of these bacterial isolates. Extracellular proteinaceous compounds were purified via ammonium sulfate precipitation, solid phase extraction, and RP-HPLC. Antifungal activity of purified products was confirmed by deferred overlay inhibition assay. Mass spectrometry (MS) was performed to determine the molecular weight of the isolated compounds. Whole genome sequencing (WGS) was conducted to predict secondary metabolite gene clusters encoded by the two antifungal-producing strains. Using MS and WGS data, we determined that the main antifungal compound produced by these two Bacillus velezensis isolates was iturin A, a lipopeptide exhibiting broad spectrum antifungal activity.

Regulation of Estradiol Synthesis by Aromatase Interacting Partner in Breast (AIPB).

Estradiol is essential for the development of female sex characteristics and fertility. Postmenopausal women and breast cancer patients have high levels of estradiol. Aromatase catalyzes estradiol synthesis; however, the factors regulating aromatase activity are unknown. We identified a new 22-kDa protein, aromatase interacting partner in breast (AIPB), from the endoplasmic reticulum of human breast tissue. AIPB expression is reduced in tumorigenic breast and further reduced in triple-negative tumors. Like that of aromatase, AIPB expression is induced by nonsteroidal estrogen. We found that AIPB and aromatase interact in nontumorigenic and tumorigenic breast tissues and cells. In tumorigenic cells, conditional AIPB overexpression decreased estradiol, and blocking AIPB availability with an AIPB-binding antibody increased estradiol. Estradiol synthesis is highly increased in AIPB knockdown cells, suggesting that the newly identified AIPB protein is important for aromatase activity and a key modulator of estradiol synthesis. Thus, a change in AIPB protein expression may represent an early event in tumorigenesis and be predictive of an increased risk of developing breast cancer.

A Novel Mitochondrial Complex of Aldosterone Synthase, Steroidogenic Acute Regulatory Protein, and Tom22 Synthesizes Aldosterone in the Rat Heart.

Aldosterone, which regulates renal salt retention, is synthesized in adrenocortical mitochondria in response to angiotensin II. Excess aldosterone causes myocardial injury and heart failure, but potential intracardiac aldosterone synthesis has been controversial. We hypothesized that the stressed heart might produce aldosterone. We used blue native gel electrophoresis, immunoblotting, protein crosslinking, coimmunoprecipitations, and mass spectrometry to assess rat cardiac aldosterone synthesis. Chronic infusion of angiotensin II increased circulating corticosterone levels 350-fold and induced cardiac fibrosis. Angiotensin II doubled and telmisartan inhibited aldosterone synthesis by heart mitochondria and cardiac production of aldosterone synthase (P450c11AS). Heart aldosterone synthesis required P450c11AS, Tom22 (a mitochondrial translocase receptor), and the intramitochondrial form of the steroidogenic acute regulatory protein (StAR); protein crosslinking and coimmunoprecipitation studies showed that these three proteins form a 110-kDa complex. In steroidogenic cells, extramitochondrial (37-kDa) StAR promotes cholesterol movement from the outer to inner mitochondrial membrane where cholesterol side-chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone, thus initiating steroidogenesis, but no function has previously been ascribed to intramitochondrial (30-kDa) StAR; our data indicate that intramitochondrial 30-kDa StAR is required for aldosterone synthesis in the heart, forming a trimolecular complex with Tom22 and P450c11AS. This is the first activity ascribed to intramitochondrial StAR, but how this promotes P450c11AS activity is unclear. The stressed heart did not express P450scc, suggesting that circulating corticosterone (rather than intracellular cholesterol) is the substrate for cardiac aldosterone synthesis. Thus, the stressed heart produced aldosterone using a previously undescribed intramitochondrial mechanism that involves P450c11AS, Tom22, and 30-kDa StAR. SIGNIFICANCE STATEMENT: Prior studies of potential cardiac aldosterone synthesis have been inconsistent. This study shows that the stressed rat heart produces aldosterone by a novel mechanism involving aldosterone synthase, Tom22, and intramitochondrial steroidogenic acute regulatory protein (StAR) apparently using circulating corticosterone as substrate. This study establishes that the stressed rat heart produces aldosterone and for the first time identifies a biological role for intramitochondrial 30-kDa StAR.

Electron Transport Chain Complex II Regulates Steroid Metabolism.

The first steroidogenic enzyme, cytochrome P450-side-chain-cleavage (SCC), requires electron transport chain (ETC) complexes III and IV to initiate steroid metabolic processes for mammalian survival. ETC complex II, containing succinate dehydrogenase (quinone), acts with the TCA cycle and has no proton pumping capacity. We show that complex II is required for SCC activation through the proton pump, generating an intermediate state for addition of phosphate by succinate. Phosphate anions in the presence of succinate form a stable mitochondrial complex with higher enthalpy (-ΔH) and enhanced activity. Inhibition of succinate action prevents SCC processing at the intermediate state and ablates activity and mitochondrial protein network. This is the first report directly showing that a protein intermediate state is activated by succinate, facilitating the ETC complex II to interact with complexes III and IV for continued mitochondrial metabolic process, suggesting complex II is essential for steroid metabolism regulation.

Design, synthesis and structure of a frustrated benzoxaborole and its applications in the complexation of amines, amino acids, and protein modification.

This study describes the design and synthesis of arylboronic acid 2, the first example of a permanently open "frustrated" benzoxaborole, along with an exploration of its application in bioconjugation. An efficient and high yielding seven-step synthesis was optimized. NMR experiments confirmed that compound 2 exists in the open ortho-hydroxyalkyl arylboronic acid structure 2-I, a form that is effectively prevented to undergo a dehydrative cyclization as a result of unfavorable geometry. Compound 2-I conjugates effectively with amines to form stable hemiaminal ether structures, including a highly effective reaction with lysozyme. Complexation with cysteine induces an open structure containing a free hydroxymethyl arm, with the amino and thiol groups reacting preferentially with the formyl group to form a N,S-acetal.

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells.

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 µM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 µM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100-200 µM). In contrast, low concentrations of edaravone (1-10 µM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0-10 µM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Caution for the routine use of phenol red - It is more than just a pH indicator.

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 µM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV-visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV-vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Eosinophil peroxidase oxidizes isoniazid to form the active metabolite against M. tuberculosis, isoniazid-NAD.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD+) by the mycobacterial catalase-peroxidase enzyme, KatG, was known to be the major component of the mode of action of isoniazid (INH), an anti-tuberculosis drug. However, there are other enzymes that may catalyze this reaction. We have previously reported that neutrophil myeloperoxidase (MPO) is capable of metabolizing INH through the formation of INH-NAD+ adduct, which could be attributed to being a possible mode of action of INH. However, eosinophilic infiltration of the lungs is more pronounced and characteristic of granulomas in Mycobacterium tuberculosis-infected patients. Thus, the aim of the present study is to investigate the role of eosinophil peroxidase (EPO), a key eosinophil enzyme, during INH metabolism and the formation of its active metabolite, INH-NAD+ using purified EPO and eosinophils isolated from asthmatic donors. UV-Vis spectroscopy revealed INH oxidation by EPO led to a new product (λmax = 326 nm) in the presence of NAD+. This adduct was confirmed to be INH-NAD+ using LC-MS analysis where the intact adduct was detected (m/z = 769). Furthermore, EPO catalyzed the oxidation of INH and formed several free radical intermediates as assessed by electron paramagnetic resonance (EPR) spin-trapping; a carbon-centred radical, which is considered to be the reactive metabolite that binds with NAD+, was found when superoxide dismutase was included in the reaction. Our findings suggest that eosinophilic EPO may also play a role in the pharmacological activity of INH through the formation of INH-NAD+ adduct, and supports further evidence that human cells and enzymes are capable of producing the active metabolite involved in tuberculosis treatment.

Inner Mitochondrial Translocase Tim50 Is Central in Adrenal and Testicular Steroid Synthesis.

Adrenal and gonadal mitochondrial metabolic activity requires electrons from cofactors, cholesterol, and a substrate for rapid steroid synthesis, an essential requirement for mammalian survival. Substrate activity depends on its environment, which is regulated by chaperones and mitochondrial translocases. Cytochrome P450 side-chain cleavage enzyme (SCC or CYP11A1) catalyzes cholesterol to pregnenolone conversion, although its mechanism of action is not well understood. We find that SCC is directly imported into the mitochondrial matrix, where its N-terminal sequence is cleaved sequentially, after which it becomes activated following the second cleavage, which is dependent on the folding of the protein. Following integration of the SCC C terminus into the TIM23 complex, amino acids 141 to 146 interact with the intermembrane-exposed Tim50 protein, forming a large complex. The absence of Tim50 or its mutation reduced enzymatic activity. For the first time, we report that a protein activated at the matrix remains mostly unfolded and is transported back to the IMS to integrate with the TIM23 translocase complex and align with the Tim50 protein. Amino acid changes that suppress the association of Tim50 with SCC ablate metabolic activity. Thus, the TIM23 complex is the central regulator of metabolism guided by Tim50.

Mitochondrial metabolic regulation by GRP78.

Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity.

Endoplasmic Reticulum Stress Enhances Mitochondrial Metabolic Activity in Mammalian Adrenals and Gonads.

The acute response to stress consists of a series of physiological programs to promote survival by generating glucocorticoids and activating stress response genes that increase the synthesis of many chaperone proteins specific to individual organelles. In the endoplasmic reticulum (ER), short-term stress triggers activation of the unfolded protein response (UPR) module that either leads to neutralization of the initial stress or adaptation to it; chronic stress favors cell death. UPR induces expression of the transcription factor, C/EBP homology protein (CHOP), and its deletion protects against the lethal consequences of prolonged UPR. Here, we show that stress-induced CHOP expression coincides with increased metabolic activity. During stress, the ER and mitochondria come close to each other, resulting in the formation of a complex consisting of the mitochondrial translocase, translocase of outer mitochondrial membrane 22 (Tom22), steroidogenic acute regulatory protein (StAR), and 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2) via its intermembrane space (IMS)-exposed charged unstructured loop region. Stress increased the circulation of phosphates, which elevated pregnenolone synthesis by 2-fold by increasing the stability of 3ßHSD2 and its association with the mitochondrion-associated ER membrane (MAM) and mitochondrial proteins. In summary, cytoplasmic CHOP plays a central role in coordinating the interaction of MAM proteins with the outer mitochondrial membrane translocase, Tom22, to activate metabolic activity in the IMS by enhanced phosphate circulation.

Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.

An Outer Mitochondrial Translocase, Tom22, Is Crucial for Inner Mitochondrial Steroidogenic Regulation in Adrenal and Gonadal Tissues.

After cholesterol is transported into the mitochondria of steroidogenic tissues, the first steroid, pregnenolone, is synthesized in adrenal and gonadal tissues to initiate steroid synthesis by catalyzing the conversion of pregnenolone to progesterone, which is mediated by the inner mitochondrial enzyme 3ß-hydroxysteroid dehydrogenase 2 (3ßHSD2). We report that the mitochondrial translocase Tom22 is essential for metabolic conversion, as its knockdown by small interfering RNA (siRNA) completely ablated progesterone conversion in both steroidogenic mouse Leydig MA-10 and human adrenal NCI cells. Tom22 forms a 500-kDa complex with mitochondrial proteins associated with 3ßHSD2. Although the absence of Tom22 did not inhibit mitochondrial import of cytochrome P450scc (cytochrome P450 side chain cleavage enzyme) and aldosterone synthase, it did inhibit 3ßHSD2 expression. Electron microscopy showed that Tom22 is localized at the outer mitochondrial membrane (OMM), while 3ßHSD2 is localized at the inner mitochondrial space (IMS), where it interacts through a specific region with Tom22 with its C-terminal amino acids and a small amino acid segment of Tom22 exposed to the IMS. Therefore, Tom22 is a critical regulator of steroidogenesis, and thus, it is essential for mammalian survival.

Phenylbutazone Oxidation via Cu,Zn-SOD Peroxidase Activity: An EPR Study.

We investigated the effect of Cu,Zn-superoxide dismutase (Cu,Zn-SOD)-peroxidase activity on the oxidation of the nonsteroidal anti-inflammatory drug phenylbutazone (PBZ). We utilized electron paramagnetic resonance (EPR) spectroscopy to detect free radical intermediates of PBZ, UV-vis spectrophotometry to monitor PBZ oxidation, oxygen analysis to determine the involvement of C-centered radicals, and LC/MS to determine the resulting metabolites. Using EPR spectroscopy and spin-trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we found that the spin adduct of CO3(•-) (DMPO/(•)OH) was attenuated with increasing PBZ concentrations. The resulting PBZ radical, which was assigned as a carbon-centered radical based on computer simulation of hyperfine splitting constants, was trapped by both DMPO and MNP spin traps. Similar to Cu,Zn-SOD-peroxidase activity, an identical PBZ carbon-centered radical was also detected with the presence of both myeloperoxidase (MPO/H2O2) and horseradish peroxidase (HRP/H2O2). Oxygen analysis revealed depletion in oxygen levels when PBZ was oxidized by SOD peroxidase-activity, further supporting carbon radical formation. In addition, UV-vis spectra showed that the λmax for PBZ (λ = 260 nm) declined in intensity and shifted to a new peak that was similar to the spectrum for 4-hydroxy-PBZ when oxidized by Cu,Zn-SOD-peroxidase activity. LC/MS evidence supported the formation of 4-hydroxy-PBZ when compared to that of a standard, and 4-hydroperoxy-PBZ was also detected in significant yield. These findings together indicate that the carbonate radical, a product of SOD peroxidase activity, appears to play a role in PBZ metabolism. Interestingly, these results are similar to findings from heme peroxidase enzymes, and the context of this metabolic pathway is discussed in terms of a mechanism for PBZ-induced toxicity.

Mitochondria-associated endoplasmic reticulum membrane (MAM) regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltage-dependent anion channel 2 (VDAC2) interaction.

Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221-229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis.

Cholesterol-mediated conformational changes in the steroidogenic acute regulatory protein are essential for steroidogenesis.

Although the mechanism by which the steroidogenic acute regulatory protein (StAR) promotes steroidogenesis has been studied extensively, it remains incompletely characterized. Because structural analysis has revealed a hydrophobic sterol-binding pocket (SBP) within StAR, this study sought to examine the regulatory role of cholesterol concentrations on protein folding and mitochondrial import. Stopped-flow analyses revealed that at low concentrations, cholesterol promotes StAR folding. With increasing cholesterol concentrations, an intermediate state is reached followed by StAR unfolding. With 5 µg/mL cholesterol, the apparent binding was 0.011 s(-1), and the unfolding time (t1/2) was 63 s. The apparent binding increased from 0.036 to 0.049 s(-1) when the cholesterol concentration was increased from 50 µg/mL to 100 µg/mL while t1/2 decreased from 19 to 14 s. These cholesterol-induced conformational changes were not mediated by chemical chaperones. Protein fingerprinting analysis of StAR in the absence and presence of cholesterol by mass spectrometry revealed that the cholesterol binding region, comprising amino acids 132-188, is protected from proteolysis. In the absence of cholesterol, a longer region of amino acids from position 62 to 188 was protected, which is suggestive of organization into smaller, tightly folded regions with cholesterol. In addition, rapid cholesterol metabolism was required for the import of StAR into the mitochondria, suggesting that the mitochondria have a limited capacity for import and processing of steroidogenic proteins, which is dependent on cholesterol storage. Thus, cholesterol regulates StAR conformation, activating it to an intermediate flexible state for mitochondrial import and its enhanced cholesterol transfer capacity.

Generation of free oligosaccharides from bacterial protein N-linked glycosylation systems.

All Campylobacter species are capable of N-glycosylating their proteins and releasing the same oligosaccharides into the periplasm as free oligosaccharides (fOS). Previously, analysis of fOS production in Campylobacter required fOS derivatization or large culture volumes and several chromatography steps prior to fOS analysis. In this study, label-free fOS extraction and purification methods were developed and coupled with quantitative analysis techniques. Our method follows three simple steps: (1) fOS extraction from the periplasmic space, (2) fOS purification using silica gel chromatography followed by porous graphitized carbon purification and (3) fOS analysis and accurate quantitation using a combination of thin-layer chromatography, mass spectrometry, NMR, and high performance anion exchange chromatography with pulsed amperometric detection. We applied our techniques to analyze fOS from C. jejuni, C. lari, C. rectus, and C. fetus fetus that produce different fOS structures. We accurately quantified fOS in Campylobacter species that ranged from 7.80 (±0.84) to 49.82 (±0.46) nmoles per gram of wet cell pellet and determined that the C. jejuni fOS comprises 2.5% of the dry cell weight. In addition, a novel di-phosphorylated fOS species was identified in C. lari. This method provides a sensitive and quantitative method to investigate the genesis, biology and breakdown of fOS in the bacterial N-glycosylation systems.