BBA - General Subjects (v.1850, #12)

Lupeol derivative mitigates Echis carinatus venom-induced tissue destruction by neutralizing venom toxins and protecting collagen and angiogenic receptors on inflammatory cells by G.D. Katkar; Rachana D. Sharma; G.J. Vishalakshi; S.K. Naveenkumar; Gaurav Madhur; R.M. Thushara; T. Narender; K.S. Girish; K. Kemparaju (2393-2409).
Echis carinatus bite is a serious threat in South-Asian countries including India, as it causes highest number of deaths and terrifying long-term tissue destruction at the bitten site. Although venom metalloproteinases and hyaluronidases are the suggested key players, studies on the effect of venom on polymorphonuclear cells, peripheral blood mononuclear cells and platelets, and their role in long-term tissue destruction are still in infancy. While, the effect of venom on collagen receptors, integrin α2β1/GP VI/DDR1 and CX3CR1 chemokine receptor present on these cells is an untouched area.Lupeol, lupeol acetate, its synthetic derivatives 28 were screened for inhibition of E. carinatus venom induced-hemorrhage in mouse model where compound 8 was found to be the most potent. Further, compound 8 efficiently neutralized venom induced hemorrhage, edema, dermonecrosis, myonecrosis, myotoxicity, pro-coagulant, oxidative stress, inflammatory cytokines and cleavage of collagen and CX3CR1 receptors on inflammatory cells in in vivo, in silico, ex vivo and in vitro studies.This study for the first time demonstrated the cleavage of collagen receptors and the receptor for angiogenesis and wound healing by the venom and its inhibition by compound 8, as these are important for firm adhesion of inflammatory cells at the damaged site to resolve inflammation and promote tissue repair.This study provides a lead in venom pharmacology, wherein, compound 8 could be a therapeutic agent for the better management of viper venom-induced long-term tissue destruction.
Keywords: E. carinatus; Venom; Local toxicity; Lupeol; Collagen receptors; Inflammatory cells;

Beyond water homeostasis: Diverse functional roles of mammalian aquaporins by Philip Kitchen; Rebecca E. Day; Mootaz M. Salman; Matthew T. Conner; Roslyn M. Bill; Alex C. Conner (2410-2421).
Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis.AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance.As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation.Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies.
Keywords: Aquaporin; Solute transport; Ion transport; Membrane trafficking; Cell volume regulation;

Endostatin's emerging roles in angiogenesis, lymphangiogenesis, disease, and clinical applications by Amit Walia; Jessica F. Yang; Yu-hui Huang; Mark I. Rosenblatt; Jin-Hong Chang; Dimitri T. Azar (2422-2438).
Angiogenesis is the process of neovascularization from pre-existing vasculature and is involved in various physiological and pathological processes. Inhibitors of angiogenesis, administered either as individual drugs or in combination with other chemotherapy, have been shown to benefit patients with various cancers. Endostatin, a 20-kDa C-terminal fragment of type XVIII collagen, is one of the most potent inhibitors of angiogenesis.We discuss the biology behind endostatin in the context of its endogenous production, the various receptors to which it binds, and the mechanisms by which it acts. We focus on its inhibitory role in angiogenesis, lymphangiogenesis, and cancer metastasis. We also present emerging clinical applications for endostatin and its potential as a therapeutic agent in the form a short peptide.The delicate balance between pro- and anti-angiogenic factors can be modulated to result in physiological wound healing or pathological tumor metastasis. Research in the last decade has emphasized an emerging clinical potential for endostatin as a biomarker and as a therapeutic short peptide. Moreover, elevated or depressed endostatin levels in diseased states may help explain the pathophysiological mechanisms of the particular disease.Endostatin was once sought after as the ‘be all and end all’ for cancer treatment; however, research throughout the last decade has made it apparent that endostatin's effects are complex and involve multiple mechanisms. A better understanding of newly discovered mechanisms and clinical applications still has the potential to lead to future advances in the use of endostatin in the clinic.
Keywords: Matricryptin; Tumor angiogenesis; Type XVIII collagen; Anti-angiogenic factor; MMP; Short endostatin peptide;

An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog by Sonia Al-Qadi; Morten Schiøtt; Steen Honoré Hansen; Peter Aadal Nielsen; Lassina Badolo (2439-2451).
ABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models.Transcriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and Drosophila melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses.A protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers.Findings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.Display Omitted
Keywords: Invertebrate in situ model; P-gp efflux transporter; Blood brain barrier; Functional characterization; Transcriptome analysis;

Engineering degrons of yeast ornithine decarboxylase as vehicles for efficient targeted protein degradation by Rushikesh G. Joshi; Swapnali Kulkarni; C. Ratna Prabha (2452-2463).
Ornithine decarboxylase (ODC), which catalyzes the first step of polyamine biosynthesis, undergoes rapid targeted degradation (TPD) with the help of its two degron sequences, namely the N-terminal 50 residues (N50) and α/β domain (α/β) housing antizyme binding element (AzBE), in response to increased polyamine levels. Antizyme binds to AzBE of ODC and delivers it to proteasome for degradation. Entire ODC was used as a tag to demonstrate TPD of chimeric proteins.Here we fashioned three peptide sequences from yeast ODC to test their capability to act as degrons, namely N50, α/β and Nα/β (a combination of N50 and α/β), and monitored their degradation potentials in chimeric proteins. We have examined the correlation between degradation potentials and structural integrity of the peptides, to find mechanistic explanations.Nα/β with two signals in tandem is a better degron, under the regulation of antizyme. N50 like N44 reported earlier could drive chimeric proteins to degradation, while α/β could not act as an independent degron. Strong correlation was observed between functional efficacy of the peptides and their structural integrity. N50, which was believed to be unstructured, displayed propensity for helical conformation. Nα/β exhibited optimal structure, while α/β failed to adopt native like conformation.Functional efficacy of the degron Nα/β is a consequence of its structural integrity. Nα/β and N50 could target chimeric proteins to degradation. However, α/β failed in the quest. Nα/β, regulated by antizyme, is better suited than N50 for TPD to understand the function of novel proteins.Display Omitted
Keywords: Ornithine decarboxylase; Degron; Degradation determinant signal; Structure of degron; Function of degron; CD spectrum;

Tropisetron attenuated the anxiogenic effects of social isolation by modulating nitrergic system and mitochondrial function by Shayan Amiri; Hossein Amini-Khoei; Arya Haj-Mirzaian; Maryam Rahimi-Balaei; Parvaneh Naserzadeh; AhmadReza Dehpour; Shahram Ejtemaei Mehr; Mir-Jamal Hosseini (2464-2475).
Early social isolation stress (SIS) is associated with the occurrence of anxiety behaviors. It seems interaction between the nitrergic system and mitochondrial function plays a role in mediating the anxiety-like behaviors. In this study, we aimed to investigate the anxiolytic effects of tropisetron in animal model of SIS and we try to illustrate the possible role of nitrergic system and mitochondrial function.We applied early social isolation paradigm to male NMRI mice. Animals treated with various doses of tropisetron, nitric oxide agents or their combination and anxiety-like behaviors of animals were assessed using valid behavioral tests including elevated plus maze (EPM), open-field test (OFT) and hole-board test (HBT) in their adulthood. Effects of housing conditions and drug treatments on the mitochondrial function were investigated in the hippocampus by assessing the ATP, GSH, ROS and nitrite levels.Anxiogenic effects of early SIS were assessed in the EPM, OFT, and HBT. Also, SIS disrupted mitochondrial function and caused oxidative stress in the hippocampus of stressed animals. Tropisetron showed an anxiolytic effect in the stressed mice. Also, these effects were mediated by nitrergic system by affecting mitochondrial function and modulating the oxidative stress. L-arginine, a nitric oxide precursor, abolished the anxiolytic effects of tropisetron in the behavioral tasks and blocked the protective effects of it against mitochondrial and oxidative challenge.Our results demonstrated tropisetron attenuated the anxiogenic effects of SIS by mitigation of the negative effects of nitric oxide on mitochondrial function.
Keywords: Early social isolation; Tropisetron; Nitric oxide; Mitochondria; Hippocampus; Anxiety;

The free radical nitric oxide (NO) and the thiol oxidoreductase thioredoxin (Trx) play essential roles in cellular redox regulation. Recent biochemical and cellular studies have revealed a complex thiol-dependent crosstalk between NO and Trx that modulates multiple redox-dependent pathways.This review aims to discuss recent progress, as well as the remaining questions, regarding the interaction and cross regulation between NO and Trx in cellular function and dysfunction.The importance and ubiquity of NO-mediated S-nitrosylation of protein thiols as a signaling mechanism is increasingly recognized as is the central role of Trx in regulating S-nitrosylation processes. By denitrosylating diverse protein substrates, Trx plays an active role in attenuating NO signaling as well as in ameliorating nitrosative stress. Yet, at the same time, Trx can also support the activity of NO synthases, thus promoting NO production and its downstream effects. Finally, NO can reciprocally modulate the redox activity of Trx and Trx reductase.Further elucidation of the crosstalk between NO and Trx will be important for an improved understanding of the effects of reactive oxygen and nitrogen species on cellular signaling and function.Display Omitted
Keywords: Cysteine; Nitric oxide; Nitrosylation; Thioredoxin;

Tissue alkaline phosphatase is involved in lipid metabolism and gene expression and secretion of adipokines in adipocytes by Claudia Hernández-Mosqueira; Cristina Velez-delValle; Walid Kuri-Harcuch (2485-2496).
Alkaline phosphatases are dimeric hydrolytic enzymes that dephosphorylate nucleotides and proteins. AP-TNAP is found primarily in skeletal tissues were it plays a major role in the mineralization of the extracellular matrix and bone formation.In this study we found through conventional and real time PCR assays that Alpl, the gene encoding for AP-TNAP is expressed in adipose tissue and in 3 T3-F442A adipocytes. We evaluated, using RNAi its role in adipocyte metabolism, and its cytoplasmic location by immunohistochemistry.Alpl is highly expressed late in adipogenesis during adipose terminal differentiation. Knocking down Alpl increased the expression of the genes encoding for glycerophosphate dehydrogenase, and for the adipokines adiponectin, and FABP4 (aP2) but decreased that of leptin, and it also increased secretion of FABP4; these 3 proteins are important in adipocyte systemic signaling and insulin sensitivity. Inhibition of alkaline phosphatase activity in adipocytes by levamisole reduced lipolysis and the expression of various lipogenic genes. We found the enzyme intracytoplasmically, forming aggregates in close surroundings of the lipid droplets during lipolysis.We suggest that AP-TNAP activity is involved in lipid and energy metabolism of fat cells, and it might regulate glucose metabolism and insulin sensitivity via adipokine synthesis and secretion.The activity of AP-TNAP might have a critical role in the energy balance of the adipocyte, probably participating in obesity and metabolic syndrome.
Keywords: Adipogenesis; Alkaline phosphatase; AP-TNAP; 3 T3-F442A adipocytes;

A novel link between the conformations, exposure of specific epitopes, and subcellular localization of α-synuclein by Min-Kyung Nam; Ji-Hye Han; Ja-Young Jang; Si-Eun Yun; Goo-Young Kim; Seongman Kang; Hyangshuk Rhim (2497-2505).
Genetic studies and the abundance of alpha-synuclein (α-Syn) in presynaptic terminals suggest that α-Syn plays a critical role in maintaining synaptic vesicle pools. However, there are still few experimental tools for elucidating its physiological roles.Unexpectedly, we detected various cellular distribution patterns of endogenous α-Syn by immunofluorescence assays (IFAs). To provide new molecular insights into α-Syn research, we identified associations between epitopes, conformations, and subcellular localization of α-Syn and categorized them.The α-Syn exposing Y125 was found to coexist with F-actin at the edge of the cells, including the plasma membrane. α-Syn conformations exposing P128 or both F94 and K97 were partly localized to the mitochondria. These results indicate that various conformations of α-Syn are associated with specific subcellular localizations. Intriguingly, we demonstrate for the first time that the phosphorylated α-Syn at Ser129, also known as a Parkinson's disease (PD)-causing form, is targeted to the mitochondria.Our study showed that different subcellular distribution patterns of α-Syn reflect the existence of various α-Syn conformations under normal conditions.This study provides novel clues for deciphering the physiological function of α-Syn in connection with subcellular localization. Dissecting the specific α-Syn conformations may lead to useful strategies in PD therapy and diagnosis.Display Omitted
Keywords: Alpha synuclein; Conformation; Epitope; Subcellular localization;

Ferritin heavy chain mediates the protective effect of heme oxygenase-1 against oxidative stress by Hui-Teng Cheng; Chung-Jen Yen; Chen-Chih Chang; Kuo-Tong Huang; Kuo-Hsuan Chen; Rui-Yang Zhang; Ping-Yi Lee; Shi-Chuen Miaw; Jenq-Wen Huang; Chih-Kang Chiang; Kwan-Dun Wu; Kuan-Yu Hung (2506-2517).
The phenomenon that heme oxygenase-1 (HO-1) protects cell from injury yet its enzymatic product, iron, may facilitate generation of free radical has been long puzzling. Here we establish a functional connection between ferritin heavy chain (FHC) and HO-1. In human lupus nephritis HO-1 and FHC are colocalized within the glomeruli. In rodent anti-Thy1 (thymocyte antigen 1) induced glomerulonephritis, heme oxygenase blockade lowers the expression of FHC and accelerates mesangial cell death. Stimulation of heme oxygenase in cultured rat mesangial cell enhances its resistance to hydrogen peroxide, whereas FHC knockdown by RNA interference compromises this salutary effect. RNA interference of HO-1 makes the cell more susceptible to hydrogen peroxide, which can be rescued by forced expression of wild-type FHC but not mutants that lose the capacity of iron storage and ferroxidase activity. Phosphorylation of JunD was not sustained in these cells. Microarray analysis identifies four candidate transcriptional factors that may regulate the HO-1-induced transcription of FHC. Our results support the role of FHC in neutralizing the iron toxicity as well as mediating the protective effect of HO-1 in response to oxidative stress.
Keywords: Heme oxygenase-1; Ferritin heavy chain; Oxidative stress; Inflammation;

Modulation and pre-amplification of PAR1 signaling by ADP acting via the P2Y12 receptor during platelet subpopulation formation by S.S. Shakhidzhanov; V.I. Shaturny; M.A. Panteleev; A.N. Sveshnikova (2518-2529).
Two major soluble blood platelet activators are thrombin and ADP. Of these two, only thrombin can induce mitochondrial collapse and programmed cell death leading to phosphatidylserine (PS) exposure required for blood clotting reactions acceleration. Thrombin can also greatly potentiate collagen-induced PS exposure. However, ADP acting through the P2Y12 receptor was shown to increase the PS-exposing (PS+) platelets fraction produced by thrombin or thrombin-plus-collagen via an unknown mechanism.We developed a comprehensive multicompartmental computational model of platelet PAR1-and-P2Y12 calcium signal transduction that included cytoplasmic signaling, dense tubular system and mitochondria. To test model predictions, flow cytometry experiments with washed, annexin V-labeled platelets were performed.Stimulation of thrombin receptor PAR1 in the model induced cytoplasmic calcium oscillations, calcium uptake by mitochondria, opening of the permeability transition pore and collapse of the mitochondrial membrane potential. ADP stimulation of P2Y12 led to cAMP decrease that, in turn, caused changes in phospholipase C phosphorylation by protein kinase A, increase in cytoplasmic calcium level and, consequently, PS+ platelet formation. ADP addition before stimulation of PAR1 produced much greater increase of the PS+ fraction because cAMP concentration had time to go down prior to calcium oscillations; this prediction was also tested and confirmed experimentally.These results suggest a mechanism of ADP-dependent PS exposure regulation and show a likely mode of action that could be important for the PS exposure regulation in thrombi, where ADP is released before thrombin formation.
Keywords: Calcium signaling; Differential equations; Adenylyl cyclase; Cyclic AMP; Phospholipase C; Mitochondria;

MicroRNA expression and protein acetylation pattern in respiratory and limb muscles of Parp-1−/− and Parp-2−/− mice with lung cancer cachexia by Alba Chacon-Cabrera; Clara Fermoselle; Ida Salmela; Jose Yelamos; Esther Barreiro (2530-2543).
Current treatment options for cachexia, which impairs disease prognosis, are limited. Muscle-enriched microRNAs and protein acetylation are involved in muscle wasting including lung cancer (LC) cachexia. Poly(ADP-ribose) polymerases (PARP) are involved in muscle metabolism. We hypothesized that muscle-enriched microRNA, protein hyperacetylation, and expression levels of myogenic transcription factors (MTFs) and downstream targets, muscle loss and function improve in LC cachectic Parp-1−/− and Parp-2−/− mice.Body and muscle weights, grip strength, muscle phenotype, muscle-enriched microRNAs (miR-1, -133, -206, and -486), protein acetylation, acetylated levels of FoxO1, FoxO3, and PGC-1α, histone deacetylases (HDACs) including SIRT1, MTFs, and downstream targets (α-actin, PGC-1α, and creatine kinase) were evaluated in diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) wild type (WT), Parp-1−/− and Parp-2−/− mice.Compared to WT cachectic animals, in both respiratory and limb muscles of Parp-1−/− and Parp-2−/− cachectic mice: downregulation of muscle-specific microRNAs was counterbalanced especially in gastrocnemius of Parp-1−/− mice; increased protein acetylation was attenuated (improvement in HDAC3, SIRT-1, and acetylated FoxO3 levels in both muscles, acetylated FoxO1 levels in the diaphragm); reduced MTFs and creatine kinase levels were mitigated; body and muscle weights, strength, and muscle fiber sizes improved, while tumor weight and growth decreased.These molecular findings may explain the improvements seen in body and muscle weights, limb muscle force and fiber sizes in both Parp-1−/− and Parp-2−/− cachectic mice.PARP-1 and -2 play a role in cancer-induced cachexia, thus selective pharmacological inhibition of PARP-1 and -2 may be of interest in clinical settings.Display Omitted
Keywords: Cancer-induced cachexia; Muscle-enriched microRNAs; Protein hyperacetylation; Myogenic transcription factors; Muscle structure and function; Parp-1−/− and Parp-2−/− mice;

In conventional in vitro encapsulation of molecular cargo, the multi-subunit ferritin protein cages are disassembled in extremely acidic pH and re-assembled in the presence of highly concentrated cargo materials, which results in poor yields due to the low-pH treatment. In contrast, Archaeoglobus fulgidus open-pore ferritin (AfFtn) and its closed-pore mutant (AfFtn-AA) are present as dimeric species in neutral buffers that self-assemble into cage-like structure upon addition of metal ions.To understand the iron-mediated self-assembly and ascorbate-mediated disassembly properties, we studied the iron binding and release profile of the AfFtn and AfFtn-AA, and the corresponding oligomerization of their subunits.Fe2+ binding and conversion to Fe3+ triggered the self-assembly of cage-like structures from dimeric species of AfFtn and AfFtn-AA subunits, while disassembly was induced by dissolving the iron core with reducing agents. The closed-pore AfFtn-AA has identical iron binding kinetics but lower iron release rates when compared to AfFtn. While the iron binding rate is proportional to Fe2+ concentration, the iron release rate can be controlled by varying ascorbate concentrations.The AfFtn and AfFtn-AA cages formed by iron mineralization could be disassembled by dissolving the iron core. The open-pores of AfFtn contribute to enhanced reductive iron release while the small channels located at the 3-fold symmetry axis (3-fold channels) are used for iron uptake.The iron-mediated self-assembly/disassembly property of AfFtn offers a new set of molecular trigger for formation and dissociation of the protein cage, which can potentially regulate uptake and release of molecular cargo from protein cages.
Keywords: Protein cage; Self-assembly; Dissociation; Molecular release; Binding kinetics; Release kinetics;

Cidec promotes the differentiation of human adipocytes by degradation of AMPKα through ubiquitin-proteasome pathway by Yuqiao Xu; Yu Gu; Gang Liu; Feng Zhang; Jie Li; Fang Liu; Zhiwen Zhang; Jing Ye; Qing Li (2552-2562).
We previously showed that Cidec was localized on the surface of lipid droplets and could promote the differentiation of human adipocytes, but the molecular mechanism was still unknown.In this study, we first sought to identify proteins that interact with Cidec using yeast two-hybrid system. The results revealed that Cidec could directly interact with AMPKα1 subunit. We further showed that AMPKα levels decreased while Cidec increased during the adipogenic differentiation of human adipocytes. Meanwhile, we observed that the increased Cidec could reduce AMPKα level in adipocytes, and the downregulation of AMPKα could help to promote the differentiation of adipocytes. The results of co-immunoprecipitation and immunofluorescent proved that Cidec biochemically interacted and co-localized with AMPKα1, which meant Cidec was a regulator for AMPKα stability through an ubiquitin-proteasome pathway.Our data suggested that Cidec could interact with and down-regulate AMPKα through an ubiquitin-proteasome degradation pathway, which provided a possible mechanism of Cidec in promoting human adipocytes differentiation.Our work proposed a new possible mechanism for human adipogenesis, and also provided a potential role of AMPKα as a target in treating obesity or obesity-related diseases.
Keywords: Cidec; AMPKα; Adipocyte; Differentiation; Degradation;

The S100 proteins in epidermis: Topology and function by Wiesława Leśniak; Agnieszka Graczyk-Jarzynka (2563-2572).
S100 proteins are small calcium binding proteins encoded by genes located in the epidermal differentiation complex (EDC). Differently to other proteins encoded by EDC genes, which are indispensable for normal epidermal differentiation, the role of S100 proteins in the epidermis remains largely unknown.Particular S100 proteins differ in their distribution in epidermal layers, skin appendages, melanocytes and Langerhans cells. Taking into account that each epidermal component consists of specialized cells with well-defined functions, such differential distribution may be indicative of the function of a given S100 protein. We used this criterion together with the survey of the current experimental data pertinent to epidermis to provide a fairly comprehensive view on the possible function of individual S100 proteins in this tissue.S100 proteins are differently expressed and, despite extensive structural homology, perform diverse functions in the epidermis. Certain S100 proteins probably ensure constant epidermal renewal and support wound healing while others act in epidermal differentiation or have a protective role. As their expression is differently affected in various skin pathologies, particular S100 proteins could be valuable diagnostic markers.S100 proteins seem to be important although not yet fully recognized epidermal constituents. Better understanding of their role in the epidermis might be helpful in designing therapies to various skin diseases.
Keywords: S100 proteins; Epidermal differentiation complex (EDC); Epidermis; Keratinocyte; Melanocytes; Langerhans cells;

Comprehensive assessment of Cytochrome P450 reactions: A multiplex approach using real-time ESI-MS by Therese Burkhardt; Thomas Letzel; Jörg E. Drewes; Johanna Grassmann (2573-2581).
The detailed analysis of Cytochrome P450 (CYP) catalyzed reactions is of great interest, since those are of importance for biotechnical applications, drug interaction studies and environmental research. Often cocktail approaches are carried out in order to monitor several CYP activities in a single experiment. Commonly in these approaches product formation is detected and IC50 values are determined.In the present work, the reactions of two different CYP isoforms were monitored using real-time electrospray ionization mass spectrometry. Multiplex experiments using the highly specific CYP2A6 with its corresponding substrate coumarin as well as the highly promiscuous CYP3A4 with testosterone were conducted. Product formation and substrate depletion were simultaneously monitored and compared to the single CYP experiments. The diffusion-controlled rate of reaction and conversion rates that are used as parameters to assess the enzymatic activity were calculated for all measurements conducted.Differences in conversion rates and the theoretical rate of reaction that were observed for single CYP and multiplex experiments, respectively, reveal the complexity of the underlying mechanisms. Findings of this study imply that there might be distinct deviations between product formation and substrate degradation when mixtures are used.Detailed results indicate that for a comprehensive assessment of these enzymatic reactions both product and substrate should be considered.The direct hyphenation of enzymatic reactions to mass spectrometry allows for a comprehensive assessment of enzymatic behavior. Due to the benefits of this technique, the entire system which includes substrate, product and intermediates can be investigated. Thus, besides IC50 values further information regarding the enzymatic behavior offers the opportunity for a more detailed insight.Display Omitted
Keywords: Cytochrome P450; Diffusion-controlled rate of reaction; Substrate degradation rate; Product formation rate; Real-time mass spectrometry; Multiplex;

Production of 4-hydroxybutyrate from succinate semialdehyde in butyrate biosynthesis in Porphyromonas gingivalis by Yasuo Yoshida; Mitsunari Sato; Keiji Nagano; Yoshiaki Hasegawa; Takashi Okamoto; Fuminobu Yoshimura (2582-2591).
Despite evidence demonstrating the importance of butyrate-producing bacteria in host health and disease, the characterization of enzymes responsible for butyrate production has not been fully elucidated in the periodontopathogen, Porphyromonas gingivalis.LC-MS/MS and colorimetric analyses were employed to enzymatically characterize recombinant PGN_0724 in P. gingivalis as a succinate semialdehyde reductase. The concentration of short chain fatty acids in the culture supernatant of the wild-type bacteria and a mutant strain lacking the PGN_0724 gene were quantified using GC-MS.Incubation of recombinant PGN_0724 with succinate semialdehyde and NADH resulted in the production of 4-hydroxybutyrate as well as consumption of succinate semialdehyde. Double reciprocal plots showed that the reaction catalyzed by the PGN_0724 protein was associated with a ternary complex mechanism. The growth speed and final turbidity of the mutant strain were much lower than those of the wild-type cells. The capacity of the mutant strain to produce butyrate, isobutyrate, and isovalerate was 30%, 15%, and 45%, respectively, of that of the wild-type strain, while the mutant strain produced approximately 3.9-fold more propionate than the wild type.The pathway responsible for butyrate production is important for the growth of P. gingivalis and appears to be associated with production of the other short chain fatty acids.The aim of this study was to delineate the mechanisms involved in the production of 4-hydroxybutyrate, which is an intermediate in the biosynthetic pathway for production of butyrate, which is a virulence factor in P. gingivalis.
Keywords: Porphyromonas gingivalis; Butyrate; Succinate semialdehyde; Short chain fatty acid; 4-hydroxybutyrate;