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.

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.

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.

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.

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.

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.

Mitochondrial 3ß-hydroxysteroid dehydrogenase enzyme activity requires reversible pH-dependent conformational change at the intermembrane space.

The inner mitochondrial membrane protein 3ß-hydroxysteroid dehydrogenase 2 (3ßHSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3ßHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3ßHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3ßHSD2 retained its primarily α-helical character with a decrease in α-helical content at lower pH values, whereas the ß-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3ßHSD2 activity at pH 4.5 with ~2-fold more progesterone synthesized at pH 4.5 than at pH 3.5 and 7.4. Increasing the 3ßHSD2 concentration from 1 to 40 µg resulted in a 7-fold increase in progesterone at pH 4.5, but no change at pH 7.4. Incubation with guanidinum hydrochloride (GdmHCl) showed a three-step cooperative unfolding of 3ßHSD2 from pH 7.4 to 4.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GdmHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH 4 and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH 4, where the protein is in a pseudostable state. Based on our data, we conclude that at pH 4-5, 3ßHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.

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.

Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile.

The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-alpha and Trn-beta, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [(13)C,(15)N] Trn-alpha and [(13)C,(15)N]Trn-beta were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the alpha-carbons of the modified residues, forming three sulfur to alpha-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S'-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to alpha-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.

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.

Inner mitochondrial translocase Tim50 interacts with 3ß-hydroxysteroid dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis.

In the adrenals, testes, and ovaries, 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3ßHSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3ßHSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3ßHSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3ßHSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3ßHSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3ßHSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3ßHSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knockdown decreased 3ßHSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3ßHSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3ßHSD2 expression and activity, representing a new role for translocases in steroidogenesis.

Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein.

Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.

Structural characterization of the highly cyclized lantibiotic paenicidin A via a partial desulfurization/reduction strategy.

Lantibiotics are ribosomally synthesized antimicrobial peptides produced by bacteria that are increasingly of interest for food preservation and possible therapeutic uses. These peptides are extensively post-translationally modified, and are characterized by lanthionine and methyllanthionine thioether cross-links. Paenibacillus polymyxa NRRL B-30509 was found to produce polymyxins and tridecaptins, in addition to a novel lantibiotic termed paenicidin A. A bacteriocin termed SRCAM 602 previously reported to be produced by this organism and claimed to be responsible for inhibition of Campylobacter jejuni could not be detected either directly or by genomic analysis. The connectivities of the thioether cross-links of paenicidin A were solved using a novel partial desulfurization/reduction strategy in combination with tandem mass spectrometry. This approach overcame the limitations of NMR-based structural characterization that proved mostly unsuccessful for this peptide. Paenicidin A is a highly cyclized lantibiotic, containing six lanthionine and methyllanthionine rings, three of which are interlocking.

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.

Lipid-mediated unfolding of 3ß-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity.

For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3ß-hydroxysteroid dehydrogenase 2 (3ßHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3ßHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3ßHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3ßHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3ßHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the ß-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3ßHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and ß-sheet. As 3ßHSD2 lacks a receptor, opening the conformation may activate the protein.

Decreased cytochrome c oxidase IV expression reduces steroidogenesis.

Steroidogenic acute regulatory protein facilitates the translocation of cholesterol to the inner mitochondrial membrane, thereby initiating steroidogenesis. At the inner mitochondrial membrane, cytochrome P450 side-chain cleavage enzyme converts cholesterol to pregnenolone, an oxidative process requiring electrons from NADPH. Pregnenolone then serves as the substrate for the formation of progesterone or dehydroepiandrosterone by downstream enzymes. Studies have shown that cigarette smoke (CS) influences steroid hormone levels. To better understand the underlying mechanisms, we used a mouse model to study the effects of chronic CS exposure on steroidogenesis. Through radioimmunoassay and metabolic conversion assays, we found that CS reduced progesterone and dehydroepiandrosterone without affecting cytochrome P450 side-chain cleavage enzyme or 3ß-hydroxysteroid dehydrogenase 2 expression. However, CS did reduce expression of cytochrome c oxidase IV (COX IV), a component of the mitochondrial complex that serves as the last enzyme in the electron transport chain. Small interfering RNA-mediated COX IV knockdown indeed decreased progesterone synthesis in steroidogenic cells. In summary, COX IV likely plays a role in steroidogenesis, and passive smoking may negatively affect steroidogenesis by disrupting the electron transport chain.

4-Aminobenzoic acid hydrazide inhibition of microperoxidase-11: catalytic inhibition by reactive metabolites.

Inhibition of human peroxidase enzymes such as myeloperoxidase or eosinophil peroxidase represents a novel therapeutic area, for which there are no current clinical therapeutics. We utilized 4-aminobenzoic acid hydrazide which was reported to be a potent irreversible inhibitor of myeloperoxidase to gain insight into the role of reactive metabolites in catalytic inhibition. In order to carry out detailed studies, we used a model peroxidase, microperoxidase-11 (MP-11). We investigated the heme spectrum of MP-11 in the presence of 4-ABAH and found that heme bleaching occurred that was irreversible. This coincided with an absence of catalytic activity. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was able to significantly prevent inactivation of peroxidase activity, therefore, we performed ESR spin trapping studies and detected a carbonyl carbon-centered radical of 4-ABAH. In order to determine if the free radical metabolites became bound to MP-11, we performed high-resolution MALDI with elemental analysis to determine the change in elemental composition that occurred in these reactions. These masses were assigned to free radical metabolites of 4-ABAH and were not observed in reactions containing DMPO. We conclude that the 4-ABAH free radical metabolites which were bound to MP-11 were involved in the catalytic inhibition and were scavenged by DMPO.

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.

Chemical synthesis and biological activity of the neopetrosiamides and their analogues: revision of disulfide bond connectivity.

Neopetrosiamides A and B (2) from the marine sponge Neopetrosia sp. are two diastereomeric tricyclic peptides that inhibit tumor cell invasion associated with metastasis. The reported structures were chemically synthesized using solid-phase peptide synthesis and sequential stepwise disulfide bond formation in solution. The disulfide bond connectivity of the originally proposed structures was revised and confirmed by chemical synthesis together with a combination of HPLC analysis, disulfide mapping, and biological activity testing. This methodology was also utilized to generate analogues containing methionine or norleucine in place of the methionine sulfoxide at position 24. Compounds 4 and 6 demonstrated potent bioactivity comparable to that of the parent peptides.

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.